SESSION II - BREEDING AND GERMPLASM RELEASE

ORAL COMMUNICATION

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Oral Communication Abstract - S2a

BREEDING AND GERMPLASM RELEASE IN ITALY

N. DI FONZO

Istituto Sperimentale per la Cerealicoltura, Sezione di Foggia

The aim of this overview is to synthesize the main aspects of the Italian studies and research in plant breeding. Genetic improvement of crops in Italian agriculture has been achieved by means of all traditional breeding methodologies. After that an increasing utilization of advanced techniques were performed and remarkable results have been obtained. Information related to different topics will be given included plant breeder’s right protection, seed multiplication, dynamic of germplasm release, economic and political aspects.The species considered in the presentation are cereals (wheat, maize, barley, rice), tomato, potato, sugar beet, grain legumes and fodder crops. These cover most of the cultivated area. Italy is characterised by different environments from North to South with many constraints that limit the yield. Studies are considering genotype x location interaction effects, selection environments, yield stability targets, yield components, quantity and quality of yield, genetic dissection of the target traits, abiotic and biotic stresses, etc.. As an example, emphasis should be placed on resistance to drought and high temperature and to major pests and pathogenes.In addition to conventional approaches to the breeding, some advanced technologies have been considered. On the basis of these many genes related to qualitative and quantitative charactes have been identified, localised in map and markers are now available for plant breeding using the MAS technique. Moreover, in addition to conventional approaches to the breeding, transformation techniques offer the possibility of genetically engineering improved agronomic and quality traits. Genetic modification (GM) is used to down-regulate or silence the expression of native genes, thus producing “targeted mutants”, to insert additional copies of native genes and it may be used to insert and express foreign genes, from either related or distant sources. Actually, in wheat, studies are considering manipulation of gluten protein trafficking, lipoxygenase and pasta colour. Today there is considerable disparity in the extent to which GM technology has been applied in breeding programmes and the production of commercial varieties. However, progress will ultimately be determined by two major factors: the level of public sector investment and acceptability of GM products to regulatory authorities and consumers.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Oral Communication Abstract - S2b

TRACING FUNCTIONAL POLYMORPHISMS THROUGH BARLEY BREEDING HISTORY

P. DONINI, E. CHIAPPARINO, D. O’SULLIVAN

NIAB, Huntingdon Road, CB3 0LE, Cambridge - UK

SNP, association mapping, functional polymorphisms, beta-amilase, barley

We have initiated a study on trends in genetic diversity in recent European barley breeding programmes using a sample of 500 cultivated barley varieties representing a large proportion of the acreage sown to barley in the EU over the last 60 years.

As a contrast to neutral marker studies, we decided to make a survey of functional polymorphisms in genes known to have been the target of selection in breeding programmes. We will present a case study of haplotype diversity in the endosperm-specific Beta-amylase 1 gene of barley on chromosome 4H, encoding a starch-degrading enzyme crucial to the malting quality characteristics of the barley. SNP genotyping allowed us to detect novel haplotypes which were confirmed by resequencing the relevant fragments of the coding sequence. The predicted functional significance of the observed haplotypic diversity will be discussed, as will the transmission of particular alleles through known pedigrees.

This approach has considerable power to give a retrospective analysis of barley breeding prior to the molecular marker era. 

Finally, we examine the feasibility of using phenotypically well characterised cultivated varieties for association mapping of traits for which the genetic basis is as yet unknown by augmenting the few known functional polymorphisms with an appropriate density of genome-wide random polymorphisms.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Oral Communication Abstract - S2c

BREEDING FOR HIGH FORAGE PRODUCTION AND SEED RETENTION IN COMMON VETCH (VICIA SATIVA L.)

F. CALCAGNO, T. SOARE, A. BOZZINI, G. SOARE, F. CALCAGNO

EUROGEN 

common vetch, pod shattering, seed production, breeding

Pod shattering is a common feature in wild legumes, assuring seed dispersal and plant diffusion.

In all domesticated legume crops the "non shattering" is the first and most important trait to be selected by humans, since it assures the possibility of an easy seed harvesting. This character is essential in food legumes, but not always present in many legume crops cultivated for forage production.

Common Vetch represents in Italy and in the Mediterranean basin one of the most important and high quality forage crops, cultivated both alone or in mixture with a cereal (normally oats). 

However, all the common vetch cultivars certified in Italy present the wild "shattering pod" character, wich reduces quantity and quality seed production. Moreover, pod shattering is also making difficult mechanical harvesting and is causing weed problems in subsequent (often cereal ) crops. 

In 1999 we started a breeding program in order to transfer the "non shattering pod" character, present in a line obtained from ICARDA, but not agronomically adapted to our ecological conditions, into two cultivars: "Francesca" and "Marianna", the result of a previous breeding programme, wich are now widely cultivated in Italy and Spain.

In our material we confirmed the full penetrance of the character, its monogenic and recessive inheritance.

By repeated backcrosses we are now recovering the basic high agronomical performance of the two released cultivars, in addition with the absence of seed dispersal, making the seed production of common vetch more profitable for seed producers and traders and less expensive for farmers.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Oral Communication Abstract - S2d

MARKER-ASSISTED SELECTION FOR VIRUS AND LOW TEMPERATURE TOLERANCE IN BARLEY

N. PECCHIONI**, E. FRANCIA*, D. BARABASCHI*, G. LAIDÓ*, F. REGGIANI*, B. TOTH***, V. SIP****, F. RIZZA*, G. DELOGU*

*) Istituto Sperimentale per la Cerealicoltura, Sezione di Fiorenzuola, Via San Protaso 302, 29017 Fiorenzuola d'Arda (PC)
**) Università degli Studi di Modena e Reggio Emilia, Facoltà di Agraria, Via J.F. Kennedy 17, 42100 Reggio Emilia
***) Agricultural Research Institute of the Hungarian Academy of Sciences, 2462 Martonvásár, Hungary
****) Research Institute of Crop Production, Prague-Ruzyne, Czech Republic

BaYMV, BYDV, MAS, barley, frost tolerance, STS markers

The soil-borne barley yellow mosaic virus (BaYMV), the aphid-borne barley yellow dwarf virus (BYDV) and the seed-borne fungus Pyrenophora graminea (leaf stripe) are the most serious diseases for the barley crop in Southern Europe environments, where mildew and other foliar diseases have a relatively small or not significant effect on yields. The development of barley cultivars resistant to the three diseases is thus an urgent objective of barley breeding for the area, in a context of increasing organic farming for cereals. Nevertheless, breeding for multiple diseases encounters several economical and technical problems, and cultivars resistant to the former diseases are not yet available. The bottlenecks of barley breeding for resistance to BYDV are the availability of sources of resistance, mainly based on semi-dominant Yd2 gene, and the labour-intensive inoculation test made with infected aphids. Availability of resistant germplasm and inoculation test are easier for BaYMV and, in relative terms, also for leaf stripe resistance. In this view, two schemes of marker-assisted selection (MAS) have been followed to develop high yielding advanced lines: an assisted pedigree by introducing the two virus resistances, and a "gene pyramiding" scheme to introduce four loci of resistance - two to leaf stripe, and one each to BYDV and BaYMV. STS and SSR markers have been selected for the aim and applied to segregating progenies together with phenotypic selection for agronomic traits.

Results of the two MAS processes are here presented, including yield performances of the advanced breeding lines.

Also frost tolerance is an important trait for winter barley breeding. Field selection for this trait is not always efficient since, especially in Southern Europe, severe winter frost is an erratic event. Recent advances in terms of cloned genes and of molecular markers in barley can give to molecular breeders the necessary informations for the development of new, simple PCR-based molecular markers that can be important tools to select quickly the frost tolerant genotypes. We report here the development of three PCR-based markers, two STS markers, derived from the WG644 and PSR637 RFLP probes, and one RAPD, OPA17, chosen for their location on chromosome 5H, where most important loci for frost tolerance had been previously mapped. In the 'Nure' x 'Tremois' barley map, based on a segregating population of doubled haploid lines, the PSR637, the WG644 STSs and the OPA17 RAPD were mapped on the long arm of chromosome 5H. The developed markers were also validated on a set of winter and spring barley genotypes with different levels of frost tolerance. The efficiency of the developed markers to separate the frost tolerant and susceptible genotypes was clear.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Oral Communication Abstract - S2e

TWO ROWED AND SIX ROWED VARIETIES RESISTANT TO BARLEY LEAF STRIPE DEVELOPED USING CONVENTIONAL AND NON CONVENTIONAL (MAS) SELECTION METHODS

G. DELOGU, N. FACCINI, R. ALBERICI, GP. VALÉ

Istituto Sperimentale per la Cerealicoltura, Sezione di Fiorenzuola d’Arda, via S. Protaso 302, 29017 Fiorenzuola d’Arda (PC)

Pyrenophora graminea resistance, selection methods, new varieties

Pyrenophora graminea Ito and Kuribayashi (anamorph Drechslera graminea) is a seed-borne pathogen causing barley leaf stripe. The disease is widely distributed in most barley growing areas, where it causes serious damages and yield losses. At present disease control is focused mainly on seed dressing with a systemic fungicide, but the risk of naturally occurring resistance within the pathogen populations, together with increasing legal limitations to the use of fungicides and the development of organic farming, requires the development of new strategies for disease control. Several sources of resistance to P. graminea were reported in the literature and based on polygenic partial resistance genes or on single gene factors conferring complete resistance. To this aim we are currently applying breeding procedures to develop new varieties resistant to barley leaf stripe.

The programs started in 1987 using as source of complete resistance to P. graminea the cultivars Alpha, Tipper (two rowed type) (unknown gene) and Thibaut (six rowed type) and later the cultivar Rebelle (six rowed type). The last two genotypes both carrying Rdg2 gene. These cultivars have been crossed with susceptible advanced lines and high yielding varieties. 

Since 1989 selection in F2 generation had been carried out using pedigree methods integrated by artificial infection in the field with spreader of natural population of P. graminea (cvs  Perga and Express). After identifications of the ipervirulent isolate Dg2, the evaluation of resistance in the pedigree scheme, in 1990, has been modified introducing the screening in F3 generation with isolate Dg2 (Sandwich test). Recently the identification of a based marker linked the Rdg2 gene, called MWG2018, offer the possibility for the evaluation of the F2 plant visual selected in the field for agronomic characteristics. The use of different methodologies of selection applied starting from different F2 populations are presented. Practical results have been reached by integrating conventional and non conventional breeding procedure to incorporate single resistant gene as well as poligenic resistant genes.

Aiace, Auriga and Vela, are the varieties recently released for the seed companies.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Oral Communication Abstract - S2f

IMMUNOMODULATION OF CMV (CUCUMBER MOSAIC VIRUS) IN TOMATO BY INTRACELLULAR SINGLE CHAIN ANTIBODY FRAGMENTS

M. E. VILLANI, E. BENVENUTO, R. FRANCONI 

ENEA, BIOTEC GEN, CR Casaccia, Via Anguillarese 301, 00060 Roma

ntrabody, scFv, immunotherapy, CMV

Immunomodulation by using recombinant antibodies is an emerging tool for manipulating animal as well as plant metabolism. 

In order to isolate recombinant single-chain variable fragment (scFv) antibodies specific for the cucumber mosaic virus (CMV), a pandemic plant pathogen, we used a phage display library composed of intrinsically stable scFvs, able to correctly fold in the reducing environment of the cytoplasm ('F8 library'). Several phage clones were obtained after four rounds of  'biopanning’ on the immobilised virus. Two clones were analysed in details. Affinity measurements revealed affinities for CMV in the range of nanomolarity, suggesting that these scFv fragments could represent a valuable tool for inexpensive diagnosis and potential inhibitors of virus multiplication. 

In order to protect the plants from virus infection, we constructed transgenic microtomato plants expressing the scFv in the cytoplasm, the place in which virus replication occurs. Several transgenic lines were selected for scFv protein expression. Challenge of the T1 generations, performed utilizing CMV-infected N. benthamiana plant extracts, led to the identification of at least two transgenic lines protected against CMV infection. The T2 generation of these plants was challenged using a viral load extimated to be in the range of 20 µg/ml. After 15 days post inoculation, these plants showed no evident systemic symptoms, while wild-type microtomatoes were all severe affected. Among the transgenic plants, some of them showed delayed mild symptoms after 1 months p.i. Analysis of  scFv expression revealed a correlation between the level of resistance and the quantity of the protein.

These data represent the first demonstration that recombinant antibodies derived from the 'F8 library' can act as effective intrabodies in vivo.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Oral Communication Abstract - S2g

POTATO BREEDING IN CHILE

J. C. KALAZICH*, P. MALAGAMBA**, B. SAGREDO*

*) Centro Regional de Investigación Remehue, Instituto de Investigaciones Agropecuarias (INIA), Casilla 24-O, Osorno, Chile
**) Adjunct Scientist from the Internacional Potato Center (CIP), INIA, Fidel Oteiza 1256, Piso 12, Providencia, Santiago, Chile

potato breeding, South America, Chile, disease resistance, molecular markers

The Potato is a very important crop in Chile being third in area (80,000 ha) and first in production (1.3 Million Tons). The crop is planted from 30ºS.L. to 54ºS.L. INIA, created in 1964, started by introducing varieties mainly from Holland some of which still dominates. These introductions were also accompanied by seed later stopped after the seed program in Chile consolidated. In early 70’s INIA started its own breeding program releasing so far 7 varieties, the first in 1983 (Yagana-INIA) and the last in 2002 (Karu-INIA). The general objective of this program is to develop varieties suitable for different uses and production systems, both for Chilean and foreign markets. The specific objectives regarding uses consider fresh and processing, mainly french fries and chips.  With regard to disease and pest resistance the breeding work focuses on viruses (PVY, PVX and PLRV); golden nematode (GN) and insects including Potato Tuber Moth (PTM) and Leaf Miner Fly (LMF). The size of the program is around 65,000 genotypes a year. Crosses and first breeding steps are done in South Chile (40ºS.L.) because of good environment for seed production and maintenance. From year four on the material is tested routinely under three environments from 30º to 41ºS.L. For most traits conventional breeding techniques are used, however lately efforts to shorten the breeding period are being made so novel techniques have been introduced. A PCR marker from a RFLP TG689 for GN H1 gene recently obtained at Cornell University and a SCAR marker for PVY (Ryadg) are already in use in the program. Markers for PVX and to PLRV the latter through a collaboration with CIP are being studied. For resistance to insect pests the program uses S. tuberosum-S. berthaultii derivatives introduced from Cornell University as true seed in a project financed by McKnight Foundation and in collaboration also with U. of Idaho and Embrapa, Brazil. High resistance to PTM in tubers has been obtained and maintained after crossing with INIA’s advanced clones getting near-variety genotypes.  SSR and AFLP markers to gain efficiency in screening for resistance to PTM and candidate chemicals that may be involved in tuber resistance are under study. Through an agreement with U. of Naples and Italpatate Chilean advanced clones-varieties are being tested under Italian conditions. As part of this work Pukara-INIA (1993) was registered in Italy by Italpatate in 2002.  

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Oral Communication Abstract - S2h

IL MIGLIORAMENTO GENETICO DELLE PIANTE DI INTERESSE AGRARIO PRESSO LE ISTITUZIONI PUBBLICHE DI RICERCA DOPO LA LEGGE 383/2001

C. FIDEGHELLI

Istituto Sperimentale per la Frutticoltura, Via Fioranello 52, 00134 Roma

La legge n. 383 del 2001 ha radicalmente cambiato la legislazione precedente in materia di brevetti per quanto riguarda la posizione dei ricercatori pubblici nei confronti delle amministrazioni di appartenenza.

E’ noto che i brevetti vegetali sono assimilati ai brevetti industriali e, fino al 2001, erano disciplinati dal regio decreto n. 1127 del 29 giugno 1939 e, per quanto riguarda gli impiegati civili delle stato, dall’art. 34 del testo unico delle disposizioni concernenti lo statuto discendente dal decreto del Presidente della Repubblica n. 3 del 10 gennaio 1957.

L’art. 23 del r.d. del 1939 stabiliva che i diritti derivanti dall’invenzione appartengono al datore di lavoro, salvo il diritto spettante all’inventore di essere riconosciuto autore, nei casi in cui l’attività inventiva è prevista come oggetto del rapporto di lavoro.

Anche quando non ricorreva questa condizione, la Pubblica Amministrazione aveva comunque il diritto di prelazione per l’acquisto del brevetto, dedotti i contributi che l’inventore aveva ricevuto per ottenere l’invenzione.

L’art. 34 dello statuto degli impiegati statali prevedeva la corresponsione di un equo premio all’inventore, proporzionato all’ importanza dell’innovazione.

La legge 383/01 cambia tutto e , all’art. 7, stabilisce che il ricercatore di una università o di una istituzione pubblica di ricerca “è titolare esclusivo dei diritti derivanti dall’invenzione brevettabile di cui è autore. In caso di più autori, i diritti derivanti dall’invenzione appartengono a tutti in parti uguali, salvo diversa pattuizione”.

L’inventore presenta la domanda di brevetto e ne dà comunicazione all’amministrazione che stabilisce l’importo massimo spettante alla stessa amministrazione di quanto è ricavabile dalla vendita del brevetto o del suo sfruttamento.

In ogni caso l’inventore ha diritto a non meno del 50% di proventi.

L’inventore ha tempo 5 anni dal brevetto per poterlo sfruttare, dopo di che l’università o la pubblica amministrazione acquisisce il diritto gratuito di sfruttamento dell’invenzione.

La legge ha l’evidente scopo di stimolare i ricercatori pubblici a trovare innovazioni brevettabili e che abbiano un impatto economico immediato sui vari comparti produttivi.

Se la legge può essere condivisa in questo obiettivo di dare maggior concretezza alla ricerca pubblica di quanto non avvenga normalmente, non fa più la distinzione esistente nella legge precedente tra le ricerche che hanno per obiettivo la creazione di innovazioni brevettabili e le ricerche che hanno obiettivi meno finalizzati.

Al primo caso appartiene l’attività di miglioramento genetico dei vegetali che ha come conseguenza obbligata la costituzione di nuove varietà che, come tali, sono brevettabili. Diverso è il caso del ricercatore che nel portare avanti i propri studi ha l’intuizione e la capacità di mettere a punto una innovazione applicabile ad un processo produttivo e sfruttabile economicamente.

In questo caso sembra giusto riconoscere al ricercatore una capacità in più rispetto alla semplice conduzione di una ricerca e farlo partecipe dei benefici economici che la sua innovazione ha portato, nel caso di attività di miglioramento genetico non riesco a capire come lo stato, dopo aver pagato stipendio e costi della ricerca, possa rinunciare alla gestione dell’ innovazione mettendo il ricercatore pubblico in una posizione di grande vantaggio rispetto ai “breeder” privati che devono rischiare in proprio.

Una indagine condotta in tutti i più importanti paesi frutticoli del mondo ha evidenziato come la situazione italiana sia assolutamente unica: nei casi più favorevoli al ricercatore, la pubblica amministrazione riconosce il diritto alla partecipazione agli utili provenienti dall’innovazione (variabile dal 10 al 30-35%) ma mai rinuncia alla titolarità del brevetto.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Oral Communication Abstract - S2i

ADVANCED HIGH YIELDING LINES OF HULLESS WINTER BARLEY FOR THE DEVELOPMENT OF FUNCTIONAL FOODS

D. BARABASCHI*, N. PECCHIONI**, E. FRANCIA*, M. BARONCHELLI*, F. FINOCCHIARO*, B. FERRARI*, A. GIANINETTI*

*) Istituto Sperimentale per la Cerealicoltura, Sezione di Fiorenzuola, Via San Protaso 302, 29017 Fiorenzuola d'Arda (PC)
**) Università degli Studi di Modena e Reggio Emilia, Facoltà di Agraria, Via J.F. Kennedy 17, 42100 Reggio Emilia

hulless barley, waxy, beta-glucans, functional food

Barley is interesting from a nutritional point of view for the typical grain composition that makes it an ideal cereal for the production of "functional foods". Such components are mainly eta-glucans (soluble fiber) and tocols (vitamin E).

The eta-glucans are an heterogeneous family of polysaccharides, typical of barley and oats, that constitute the cellular walls of the seed endosperm, and that, introduced into diet, can contribute to the lowering of hematic cholesterol and of hematic glucose content. Therefore, the interest of researchers is for the production of baked foods containing barley flour, then rich in dietary fiber, i.e. eta-glucans. Also by using specific techniques for the enrichment of flour in eta-glucans (e.g. turboseparation), it has been possible to produce high eta-glucan bread and bakery products made of mixed wheat and enriched barley flour with organolectic characteristics not too far from those made of pure wheat flour.

Hulless barley, lacking of glumelets, gives a grain of superior quality for food respect to hulled barley, although it yields generally less, for both the absence of the glumelets and the lower efforts on the improvement of the hulless germplasm.

In the Istituto Sperimentale per la Cerealicoltura it had been active a breeding program for winter hulless barley, with the main objective of yield stability, and based on pedigree and backcross schemes that lead to the release of cultivar 'Zacinto' (2000). Inside the frame of the programme, a SCAR marker has been developed for the recognition of heterozyigous genotypes in hulled x hulless backcrosses, and this marker has been used for the acceleration of such breeding schemes.

In a second phase, the resistance to the soil-borne mosaic virus complex (BaMMV-BaYMV), and to the yellow dwarf virus (BYDV) have been introduced in the programme, by means of molecular Marker-Assisted Selection.

A specific replicated yield trial has been recently set up in which the advanced high yielding hulless lines, together with hulled and hulless checks have been evaluated in different locations in Italy for the average production of eta-glucans and tocols, and for the evaluation of the effects of the genotype x environment interaction on the production of such compounds.

In the last years, the virus-resistant advanced lines have been also crossed to Canadian and Australian hulless waxy lines with the aim to develop high yielding, virus-resistant and high eta-glucan winter barleys. In fact it is well-known that waxy barley genotypes contain consistently higher-eta-glucans respect to the ones with normal starch.

The most recent results of the hulless barley breeding programme for the production of functional foods at the Istituto Sperimentale per la Cerealicoltura are here presented.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Oral Communication Abstract - S2l

BREEDING TRITORDEUM FOR HIGH CAROTENOID CONTENT

S.G. ATIENZA*,**,***, P. HERNÁNDEZ**, J.B. ÁLVAREZ****, C. M. RAMÍREZ**, J. BALLESTEROS**, L.M. MARTÍN****, A. MARTÍN**

*) Departamento de Agronomía y Mejora Genética, IAS-CSIC, Córdoba, España
**) C.I.F.A. Departamento de Mejora y Agronomía, Apdo. 3092, 14080 Córdoba, España
***) Present address: Istituto Sperimentale per la Cerealicoltura. Sezione de Fiorenzuola d’Arda, Via S. Protaso 302, I-29017 Fiorenzuola d‘Arda (PC), Italy 
****) Departamento de Genética. Escuela Técnica Superior de Ingenieros Agrónomos y de Montes. Universidad de Córdoba, Apdo. 3048, E-14080 Córdoba, Spain

breeding, tritordeum, QTL mapping, carotenoids, end-use

The hexaploid tritordeum (AABBHchHch) is the amphiploid obtained after chromosome doubling of hybrids between Hordeum chilense Roem. et Schultz. and durum wheat Triticum turgidum ssp. durum (Desf.) Husn. This amphiploid is the subject of a breeding programme as a new crop or as bridge to transfer useful genes from H. chilense to wheat (Martín and Cubero 1981, Martín et al. 1998). Breeding for end-use quality is one of the most important tasks within cereals. In this work we present the breeding for high carotenoid content in tritordeum. Carotenoids are responsible of the yellow colour that is of prime importance for pasta and Chinese alkaline noodle productions. Tritordeums show a higher carotenoid content than their respective wheat parents (Alvarez et al. 1999). Consequently, the genes responsible for this difference are inherited from H. chilense. We have used suitable material for the study of the genetics of the high carotenoid content in tritordeum. In this way, first we used a set of chromosome addition lines for the entries H1 and H7 of H. chilense in durum wheat developed by (Miller et al. 1981). Using this set of addition lines, Alvarez et al. (1998) found that the genes for carotenoid content are located in the alpha-arm of the chromosome 7 of H. chilense. This work constituted the first step for the localization of the genes controlling the carotenoid content in H. chilense. In a second step we have developed a genetic linkage map derived from the H1 and H7 lines, contrasting for the carotenoid content (Hernández et al. 2001). Using this map we have detected a new QTL located on chromosome 2 explaining about the 14,8% of the phenotypic variance and named Carot1. This result constitutes a first step to develop a MAS program for carotenoid content in tritordeum. This will be very useful in both tritordeum breeding and to transfer the genes for high carotenoid content from tritordeum to bread wheat.

As conclusions,

-Tritordeum shows a higher carotenoid content than both bread and durum wheat.
-The alpha-arm of the chromosome 7 of H. chilense is responsible for high carotenoid content as showed using chromosome addition lines.
-The chromosome 2 of H. chilense contains a QTL responsible for high carotenoid content. 
-These results are helpful towards the development of molecular markers suitable for a MAS program for tritordeum breeding and for introgressing these genes into bread wheat.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Oral Communication Abstract - S2m

IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES INVOLVED IN HORDEUM VULGARE-PYRENOPHORA GRAMINEA INTERACTION

V. BONARDI*, A. HAEGI**, G. VALÈ***, E. DALLAGLIO***, A. PORTA-PUGLIA**, M. DELLEDONNE*

*) Università degli Studi di Verona,  Strada Le Grazie 15,  Cà Vignal 1, 37134 Verona
**) Istituto Sperimentale per la Patologia vegetale, via C.G. Bertero 22, 00156 Roma
***) Istituto Sperimentale per la Cerealicoltura , via S. Protaso 302, 29017 Fiorenzuola d’Arda, Piacenza

AFLP-TP, Hordeum vulgare, Pyrenophora graminea, barley leaf stripe

Pyrenophora graminea is the agent of barley leaf stripe, a disease occuring in almost all barley growing areas, characterized by chlorotic stripes that gradually extend to the full length of the leaf and finally become necrotic. The fungus is unable to cause any secondary infection through leaf-to-leaf transmission, so it could be considered a true seed-borne pathogen. The mechanisms of pathogenicity/resistance of barley-Pyrenophora graminea interaction are not well understood. A variety of sources of resistance to the fungus have been reported but these are often contradictory. Moreover, mechanisms of resistance controlled by one, two, three or more genes have been proposed.

Understanding the molecular mechanisms underlying plant-pathogen interaction will clearly benefit the agricultural industry, leading to informed strategies for reducing crop losses caused by the disease. As a matter of fact the fungus is supposed to establish an intimate association with the plant and such interaction involves complex recognition events between the two partners, leading to signaling cascades and regulation of countless genes required for, or associated with, the interaction. Identifying the battery of genes modulated during the infection will help, from one hand, to develop a detailed picture about seed’s pathology and pathogenetic variability in fungi in order to restrain the disease, and, from the other, to get a better knowledge on the interaction of Pyrenophora graminea with barley in order to develop molecular markers useful in the selection of resistant plants. 

AFLP-TP technique is a sensitive and reproducible fragment-based technology that has a number of advantages over other methods for genome-wide expression analysis: it does not require prior sequence information, it allows the identification of novel genes, and it provides quantitative expression profiles.

To elucidate the nature of the molecular “conversation” between barley and Pyrenophora graminea, the AFLP-TP patterns of the fungus itself and of resistant and susceptible cultivars, alone and in their interaction with the fungus, have been compared. The interaction has been studied at three times of infection (7, 10 and 15 days). From the analysis of the entire transcriptional profile, 1,162 genes were found to be differentially regulated; in particular 76.7 % of those were up-regulated and the remaining 23.3 % down-regulated with respect to the uninfected susceptible cultivar. A comprehensive collection of infection-modulated genes has been built. Hierarchical clustering of the expression profiles resulted in a variety of groups which may reveal a putative functional classification of the transcript tags.

500 AFLP fragments have been already cloned in a T-vector and they are being sequenced in order to get a clear picture of the dynamic exchange of signals that occurs between barley and Pyrenophora graminea  to result in resistance or susceptibility. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Oral Communication Abstract - S2n

COMPARATIVE ANALYSIS OF GENETIC LINKAGE MAPS AND QTL INFORMATION PRODUCED FOR GRAPE (VITIS SPP.)

A. MADINI*, F. SEVINI*, L. COSTANTINI*, R. CHAABANE*,**, F. MOREIRA*,***, R. MARINO*, M. S. GRANDO*

*) Lab. of Molecular Genetics, Istituto Agrario di San Michele all’Adige, via E. Mach 1, 38010 San Michele a/A (TN)
**) DIBCA Genetics and Plant Breeding Section, University of Bari, Via Amendola 165/A, 70126 Bari
***) Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPG), Brasil

linkage maps, QTL, vitis, grape

Grapevine is one of the most important crops in Europe based on cultivated surface, economic and cultural criteria. Despite these considerations, few is known on the genetic determinism of characters of agronomic interest and on the gene sequences which, either alone or in combination, are required for each specific trait. Since recently detailed genetic maps of polymorphic markers started to be available, the interest to optimise the strategies for breeding of new high quality grapevine varieties resistant to pathogens is rising. 

Today, the development of DNA sequences and the increasing number of mapping projects are exploiting the potential of codominant molecular markers to generate a common reference genetic map for grape. A set of microsatellite markers enables the identification of all 19 homologous linkage groups and enhances the direct comparison of information derived from different crosses. Moreover, several experiments are being performed to assess QTL stability across different genetic backgrounds and environments.  

We discuss here the comparative analysis and utility of existing molecular maps of grape that were based on both intra-vinifera crossings (i.e. Italia x Big Perlon, Riesling r. x Cabernet Sauvignon) and F1 hybrid progenies (i.e. V. vinifera Moscato b. x V. riparia, V. rupestris x Muscadinia rotundifolia). First evaluation of QTL detected for the traits ‘muscat flavour’  and ‘resistance to Downy mildew’ are reported as well from mapping experiments designed to investigate the genetic control of very important traits for table grape and wine production.

Research supported by Projects Advanced Biology (Fondazione delle Casse di Risparmio di Trento e Rovereto) and INCO-DEV Marker Assisted Selection for TablE gRape MASTER (European Commission).

POSTER

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.01

DOUBLE CYCLE IN SUNFLOWER (HELIANTHUS ANNUUS L.) BREEDING

D. LAURETI, A. DEL GATTO

Istituto Sperimentale per le Colture Industriali, Sezione operativa periferica di Osimo, via Torri 26, 60025 Osimo (AN)

sunflower, double cycle, seed moisture, germination, earliness

Intensive breeding of sunflower hybrids involves reaching the use of the lines as soon as possible from the start of the program. It is known that using green houses, in vitro culture and climatic chambers, it is possible to obtain from three to six generations per year according the use of immature embryos, etc. (Marin, 2000). In the absence of the above facilities, until know, only a traditional cycle x year has been done. However the relatively short sunflower cycle and the potential use of immature embryos could allow at least two cycles per year, To verify this possibility, in 2002, an experiment was conducted to test the possibility of obtaining two generations from three hybrids of different earliness (Floralie, early, Gloriasol, medium and Select, late), at three sowing times and four harvestings and consequently sowings of the second cycle. Sowings were done the 4; 15 and 26th March, harvesting after 12; 24 and 32 days after end flowering and at maturity.

Seed moisture in the first cycle was influenced by cultivar earliness and harvesting time. It was lower in the earliest hybrid and higher in the latest and was proportionally lower in relation to the harvestings (57 vs. 37; 16 and 11%, respectively from 12 days to maturity).

Thousand seed weight was influenced by cultivar, but on the average 74% of the mature weight (37; 47; 49 and 51 g respectively for each harvest) was already reached 12 days from the end of flowering. Seed yield after 12 days was 66% of that at maturity.

Seed germination in the first cycle was influenced by cultivar (9; 33 and 23%) and harvesting time (12; 21; 31; and 26%) and was higher in the latest, even if, at maturity, it was not significantly different from the third one.

Seeds of the second cycle gave plants that reached maturity in all harvestings for all hybrids, but seed germination in the second cycle was 62; 60; 41 and 12% in relationship to the four harvestings of the first cycle and consequently it replantings, showing that in all the combinations tested it was possible to obtain two sunflower cycles per year. Only with the first two harvestings of the first cycle it was possible to have seed germination agronomically acceptable for commercial use certainly due to good weather conditions during maturity.

Marin, I.V. 2000. Helia, 23, 33, 129-134

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.02

ARTICHOKE HYBRID PRODUCTION BY USE OF MALESTERILE CLONES

C. STAMIGNA*, G. PANDOZY**, F. MICOZZI***, P. CRINÒ****, F.  SACCARDO***

*) ENEA C.R. Trisaia (Mt) e C.R. Casaccia (Roma), UTS Biotecnologie, Protezione della Salute e degli Ecosistemi 
**) ARSIAL, Roma 
***)Università degli Studi della Tuscia, Dipartimento Produzione Vegetale, Viterbo

Cynara scolymus, F1 hybrids, malesterility

After identification and characterization of 5 malesterile (MS) plants within an INRA F2 population, artichoke breeding activities have been addressed towards their exploitation in producing hybrids capable of reducing the cultivation costs and the disease widespread. Foreign hybrids up to now available are not well adapted to our pedoclimatic conditions as well as to changes of our production calendar. 

The different malesterile parents, maintained by both shoots in the field and micropropagation in the laboratory, have been crossed either with selected clones of “romanesco” type or with cardoon to transfer interesting traits. F1 and F2 offsprings as well as synthetic populations have been realized within French clones selected for their high ability to produce seed and genetic stability. Phenotypic analyses on plants and cytological studies on malesterility trait that, in our F2 populations, confirmed a segregation ratio corresponding to the involvement of two recessive genes, have been carried out. Among the hybrids realized, the cross combination between a malesterile clone and Italian cv. Grato 1 evidenced a good genetic uniformity of the plants and heads which belonged to the “romanesco” typology. The use of molecular markers is allowing to better characterize the hybrids obtained. The offsprings from the cross combination MS x cardoon showed a great genetic variability and potentialities to select plants resistant to Verticillium dalhiae.

The production of F1 hybrids offers new possibilities to artichoke breeding with new chances of producing interesting genotypes well adapted to our pedoclimatic conditions and also characterized by a wide earliness range and adaptability to mechanical harvesting. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.03

MICROPROPAGATION OF GLOBE ARTICHOKE (CYNARA SCOLYMUS L.) FOR BIODIVERSITY CONSERVATION AND EXPLOITATION 

G. ANCORA, L. CUOZZO, R. TAVAZZA

ENEA, UTS BIOTEC, C.R. Casaccia, C.P. 2400, Rome, Italy

globe artichoke, micropropagation,biodiversity

In the last years the production of in vitro propagated material of globe artichoke has given rise to the distribution to the farmers of millions of plantlets of clone “C3” of the cultivar “Romanesco”, which is preferred for his earliness and, thus, the greater income in respect to the previous cultivated populations. However, the use of this clone by most farmers has determined a drastic reduction of the existing variability.

In the framework of a project financed by the Ministry of University and Research aimed at the exploitation of ecotypes of different plant species a search of ecotypes of “Romanesco” cultivars of globe artichoke has been undertaken in order to rescue and characterise ecotypes present in the Lazio region.

To this purpose, from plants present in small fields in the north and in the south of the Roman area, offshoots have been collected and utilised to isolate shoot apices for in vitro culture and propagation. 

Different culture conditions have been developed for the different phases of in vitro culture. In particular, a new basal medium has been developed in which, in respect to MS medium, NH4NO3 has been reduced to 800 mg/l, while 1000 mg/l Ca(NO3)2 x 4H2O have been added.

For shoot development the basal medium has been supplemented with  0.8 mg/l  BAP and 0.2 mg/l  IBA, while for proliferation the basal medium containing 2 mg/l kinetin and 0.2 mg/l  IBA was used. 

Under this conditions, generally, a satisfactory rate of shoot proliferation was obtained  accompanied by a good shoot quality, even though differences in the rate of proliferation was observed among the different clones, possibly due to their genetic diversity. To this respect experiments to improve the proliferation of some low proliferating clones are undergoing.

The shoots, 4-5 cm in length, rooted on a medium containing 10 mg/l IAA for a week followed by the transfer on hormon-free medium. 

Plantlets of different clones (about 25) were successfully transferred in soil and will be utilised for the morphological and productive analysis.

This work was supported  by a grant from the Ministry of University and Research  (MIUR), Settore Agrobiotecnologie, “Progetto  SCRIGNO.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.04

AFLP markers DISCLOSE diversity in a collection of artichoke germplasm

G. SONNANTE*, M. IPPEDICO*, A. DE PAOLIS**, D. PIGNONE*

*) CNR, Intitute of Plant Genetics, Research Section of Bari 
**) CNR, Institute of Plant Production Science, Section of Lecce

AFLP, artichoke, germplasm, wild progenitor, genetic similarity

Artichoke, Cynara cardunculus L. var. scolymus (L.) Fiori is a diploid outcrossing species, originated in the Mediterranean basin, which has been much appreciated since ancient times for both its tasty heads and pharmaceutical properties. Artichoke is traditionally cultivated all over the Mediterranean Basin and is generally vegetatively propagated; the highest density of cultivation is found in Italy, where the largest germplasm diversity is also observed. Wild cardoons are also widespread in Central and Southern Italy.

The primary gene pool of artichoke includes wild (C. cardunculus L. var. sylvestris Lam.) and cultivated (C. cardunculus L. var. altilis DC) cardoons. These latter subspecies are genetically cohesive with the cultivated artichokes. The wild cardoons are considered to be the progenitors of cultivated Cynara. 

In order to assess genetic variation and relationships among artichoke varieties and between these and cardoons, AFLP markers were developed and used in genetic analysis. Forty artichoke varieties and/or ecotypes were selected from the world-wide artichoke living collection held at the Institute of Plant Genetics in Bari according to their morpho-productive characteristics and geographical origin; moreover, one genotype of cultivated cardoon and two genotypes of wild cardoon, were included in the analysis pool. AFLP analysis was performed using fluorescence labelled selective primers and samples were run in an automated sequencer. Nine primer combinations were selected for further analysis out of the 24 initially tested. A total of 650 fragments were detected, 88.46% of which were polymorphic. The data were scored as presence or absence of a given fragment and similarity among samples was calculated on the basis of Jaccard's index. The similarity matrix so obtained was used to perform cluster analyses using UPGMA method and principal coordinate analysis (PCO). Both the dendrogram and PCO plot evidenced a clear separation of the cultivated and wild cardoon from the cultivated artichoke. Within the cultivated artichokes, genotypes were clustered mostly on the basis of the morphological characteristics of capitula, which allow to distinguish four main groups in this crop: "Spinosi", "Violetti", "Catanesi" and "Romaneschi".

It is worth noticing that AFLP markers, thanks to their high level of polymorphism in this gene pool, proved particularly useful in distinguishing between closely related genotypes and between morpho-goups. Their usefulness in managing artichoke living collections is discussed in detail.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.05

JANA: A NEW COCKSFOOT VARIETY FOR MEDITERRANEAN ENVIRONMENTS

E. PIANO, M. ROMANI, A.M. CARRONI, L. PECETTI

Istituto Sperimentale per le Colture Foraggere, Lodi

cocksfoot, Dactylis glomerata, drought stress, Mediterranean environment, summer irrigation 

In Mediterranean environments, the persistence of perennial grasses, such as cocksfoot (Dactylis glomerata L.), is heavily limited by long periods of drought in summer. The most effective plant strategy of survival is summer dormancy, which represents therefore a main selection criterion for varieties to be grown under rainfed conditions. However, development of varieties able to combine effective summer dormancy under stress with responsiveness to moisture avilability, in case of prolonged rainfall season or when supplementary irrigation in summer is provided, is also required. A new variety has been released by our Institute, which aims at meeting the above-mentioned requirements. This variety, named ‘Jana’, derives from materials singled out within both a wild population collected at Bonorva, Sardinia, and the variety ‘Currie’ of Mediterranean origin. The selection process of Jana was based on two consecutive phases, both contemplating contrasting irrigation treatments in summer: I) a preliminary phenotypic selection; and II) a subsequent selection based on a half-sib progeny test, which narrowed the previously selected plants. In the present investigation, ‘Jana’ was compared with one variety selected from Mediterranean germplasm and known to be summer dormant (‘K2M’), three varieties adapted to temperate conditions (‘Cambria’, ‘Dora’ and ‘Porto’), and the two parental populations (‘Bonorva’ and ‘Currie’). The trial, carried out in south Sardinia, included three consecutive evaluation phases: I) under rainfed conditions from autumn to spring; II) under contrasting moisture conditions in the following summer; and III) under rainfed conditions in the subsequent autumn-spring season to assess the effect of the previous summer treatments. ‘Jana’ showed the highest dry-matter yield under both rainfed conditions and summer irrigation, although not significantly different from the best temperate varieties, while it did outyield significantly ‘Bonorva’ and ‘Currie’ from which it derives, then evidencing a remarkable selection response. Owing to summer dormancy, its survival under the summer stress treatment was close to 90%, whereas all the summer-active, temperate varieties did not survive the drought. However, such a dormancy was facultative, as indicated by the yield response under irrigation. Preventing summer dormancy by irrigation had no detrimental effect on the subsequent regrowth of Mediterranean varieties in autumn-spring. At the same time, the mechanism of drought tolerance also did not affect the subsequent performance of ‘Jana’, as it outyielded significantly all varieties, including the other Mediterranean germplasm, in the autumn-spring period following the summer stress treatment. ‘Jana’ appears therefore to possess the wished feature of combining an effective drought tolerance (summer dormancy) under stressful conditions with an appreciable responsiveness to moisture availability. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.06

FROM THE WILD TO CULTIVATION: NATIVE BERMUDAGRASS GERMPLASM UNDER ADVANCED SELECTION FOR TURFS

E. PIANO, M. ROMANI, A.M. CARRONI, L. PECETTI

Istituto Sperimentale per le Colture Foraggere, Lodi

bermudagrass, Cynodon dactylon, genetic resources, selection, turfgrasses

Bermudagrass is a warm-season grass well adapted to tropical and warm temperate climates, and very suitable for high-quality turfs, owing to its good density, texture and wear-tolerance. Despite its potential interest for the Mediterranean areas of Italy, until recent years the research on bermudagrass in this country has been scanty, and all the varieties avaialble in the market are of foreign origin. In the framework of a novel national research programme on turfgrasses, great attention has been paid to the collection, evaluation and valorisation of native genetic resources of turfgrass species, to enhance the knowledge on the local germplasm, and favour the development of varieties originating from this germplasm. Altogether, 57 bermudagrass accessions were collected, mostly in Mediterranean environments. In this study, information is reported on 25 accessions (20 from the island of Sardinia, 4 from the coastal strip of Liguria, and one from Emilia in northern Italy), that were commonly evaluated for a two-year period at two contrasting locations (viz., one with Mediterranean climate in Sardinia and one with sub-continental climate in northern Italy) for overall turf quality, leaf texture, turf density, and duration of turf green colour (from spring greenup to the onset of the cold season). At both locations of evaluation, a group of six populations, collected in Sardinia, outstandingly emerged from the native germplasm, and also with respect to a set of commercial varieties used as references. The six populations showed very high turf quality, high density, fine texture, and longer duration of the turf green colour. The duration of the green appearance was greatly influenced by the environment, but at both locations the six populations were green for about 7-10 days longer than the best commercial cultivars. These populations obviously represent material of great interest for the breeding activity. Further to this evaluation, the level of ploidy of the six populations has been checked, to exclude that the positive turf attributes were accounted for by their diploid or triploid (hybrid) nature, as our objectives are tetraploid, seed-propagated cultivars. All six populations have shown 2n=4x=36 chromosomes, insuring that this germplasm represents ‘common’ Cynodon dactylon. A preliminary selection of clones within these populations (for frost tolerance and turf quality) has been carried out, and an evaluation of promising clones under dense-sward conditions is in progress. Investigations on seed yield (to assess the possible presence of intra-population mechanisms of self-sterility/self-incompatibility) have been arranged.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.07

MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF SAINFON ECOTYPES COMES FROM ABRUZZO REGION

R. TORRICELLI, S. ANCILLOTTI, D.D. SILVERI 

Department of Plant Biology and Agroenvironmental Biotechnologies, University of Perugia Borgo XX Giugno 74, 06100 Perugia (Italy)

sainfon, landraces, AFLP

Sainfon (Onobrychis viciaefolia Scop) belongs to Leguminosae family, tribe Hedysareae. The genus is formed by 125 species. O. viciaefolia (2n=4x=28) is a forage crop useful in marginal lands of Central Italy, in particular in a region such as Abruzzo largely characterized by poor and hilly lands. The cultivation of sainfoin in Italy is based on few varieties and local ecotypes which are very interesting resources for varietal development. 

The Agricultural Development Agency of the Abruzzo Region is developing a project devoted to the collection, conservation and characterization of sainfoin ecotypes. The aim of this project is to investigate on the genetic material comes from the farmers of Abruzzo region and development new varieties adapted at the climate of this Region. 

Sixteen sainfon populations (13 landraces and 3 commercial cultivars) were seeded in 2001 spring in three experimental fields (two in Abruzzo and one in Umbria region) under a randomized complete block design with four replications. Morphological variables measured during 2002-2003 were: flowering time, dry matter yield, plant height and after cut regrowth. 

Furthermore, to characterize DNA polymorphisms among the collected populations, samples from 10 genotypes of each accession were used to carry out amplified fragment length polymorphisms (AFLPs) analysis. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.08

MORPHO-AGRONOMIC DIVERSITY IN A GROUP OF RECENT ELITE GRASSPEA (LATHYRUS SATIVUS L.) LINES

G. B. POLIGNANO*, P. UGGENTI*, V. ALBA*, V. BISIGNANO*, C. DELLA GATTA** 

*) Istituto di Genetica Vegetale, C.N.R., Bari 
**) Dipartimento di Biologia e Chimica Agroforestale ed Ambientale, Università di Bari, Bari 

Lathyrus sativus L., breeding lines, evaluation, multivariate analyses

Field studies to evaluate the Bari collection of grass pea (Lathyrus spp.) were initiated in 1999 on 332 entries (Polignano et. al., 2001). Additional details of this study can be found in Granati et al. (2003), Bisignano et al. (2002) and Polignano et al. (2002). More recently a core collection for the Bari Lathyrus sativus germplasm collection was developed as a list of entries that will have priority for attention in grass pea genetic studies and breeding work (Polignano et al., 2003). From 332 total entries, core representing 12% of the collection was developed. Actually, the general objective of our evaluation program is to identify a core of improved lines with adequate diversity to fit the targeted needs of both breeders and farmers. According to this utilizing morpho-agronomic descriptors a representative survey on the diversity in a group of recent elite grasspea lines was performed. Using a randomized experimental design with two replications, forty-seven progenies of single plants (pure lines) characterized by white large seed, high yield and high biomass, that were selected from 25 entries belonging to the Bari grasspea core collection were evaluated. This kind of white large and yielding germplasm deserves special attention since it is high appreciated for consumers and farmers in Italy. The evaluation was carried out in a field trial in Matera (Basilicata region) during the 2001-2002. Seeds of each progeny were sown in a single row plot placed at a distance of 30 cm between each other on the row. A distance of 1.50 m was used between the rows. 5 plants were randomly identified in each plot and used to measure 16 morpho-agronomic and seed quality traits. Univariate and multivariate analyses were used to estimate differences among the tested progenies. In particular, canonical variables were calculated based on all 9 quantitative traits and each qualitative trait as the classification variable in order to find out whether specific qualitative trait correspond to certain agronomical proprieties. In addition a hierarchical procedure it has been followed to define groups and to establish similarity and dissimilarity among progenies. The results from this evaluation show that the most promising lines could be used directly for production as new varieties and/or hybridization in the grasspea breeding program.  Yield of these lines and their relative ranking or consistency in performance form the basis for recommendations to users.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.09

VARIATION AMONG ITALIAN ALFALFA ECOTYPES BY MEANS OF BIO-AGRONOMIC CHARACTERS AND AMPLIFIED FRAGMENTS LENGTH POLYMORPHISM ANALYSES

M. CARELLI*, F. PUPILLI**, A. BUSTI**, M. ZACCARDELLI***, G. GNOCCHI*, P. GAUDENZI*, C. SCOTTI*

*) Istituto Sperimentale Colture Foraggere, Lodi, Italy
**) Istituto di Genetica Vegetale, CNR, Perugia, Italy
***) Istituto Sperimentale Colture Industriali, Battipaglia, Italy.

alfalfa, ecotypes, AFLP markers

The 14 Italian alfalfa ecotypes, each represented by a variable number (1-21) of accessions corresponding to different ‘foundation’ farms, were evaluated in a cold greenhouse at Lodi for 3-years culture together with check cultivars. Plant material (480 plants/ accession) was organised in a randomized block design with 5 blocks; irrigation was non-limiting. Four cuts were made in the sowing year, eight and three cuts respectively in the 1st and 2nd productive year. Dry matter, mortality, stem height and earliness were recorded at plot basis. For this study, the highest yielding accession was chosen to represent each of the different ecotypes; besides, two varieties with different genetic origin were included as well. AFLP analyses were performed on 3 independent bulks/accession, each bulk deriving from mixing 100 plantlets (one trifoliate leaf/plantlet).

The variation among ecotypes for vigour (DMY), estimated on 15 cuttings, was highly significant and mainly related to the production in summer cuttings and to the response to autumn conditions. Final mortality was correlated with the total production (r=0.75 **) and showed a highly significant variation among ecotypes. In both cases, however, the variation appeared continuous, with a group of ecotypes from the central-eastern Po Valley showing the highest productivity and the lowest mortality values. AFLP analyses, performed with different selective primers, generated 20 and 61 polymorphic bands that, once combined, produced a unique pattern per ecotype. The genetic distances among ecotypes, estimated on AFLP data, ranged from 0.0154 to 0.2269; UPGMA dendrogram indicated a relative genetic similarity value of about 0.84 for the clusters including all the ecotypes and the check cultivar Lodi. The molecular data seem then to confirm an important common  background for the Italian alfalfa ecotypes with the possibility, however, of distinguishing them on the basis of the different frequencies of  specific fragments.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.10

FOUR AND EIGHT-CONSTITUENTS FREE-HYBRIDS IN ALFALFA. DRY MATTER YIELD FROM DIALLEL CROSSES: SOWING YEAR RESULTS

P. ROTILI*, C. SCOTTI*, S. GNOCCHI*, D. KERTIKOVA**, G. GNOCCHI*

*) Istituto Sperimentale Colture Foraggere, Lodi, Italy
**) Institute of Forage Crops, Pleven, Bulgaria

alfalfa, diallel cross, free-hybrid, pod fertility

General objectives: a) Construction of free-hybrids in alfalfa (Medicago sativa L.),using partially inbred (S2) parental plants (Rotili et al., 1999). b) Experimental evaluation of free-hybrids and synthetics derived from the same parental plants (Table 1).

    	Table 1. Free-hybrid program
	4 - constituents	8 - constituents	
	A B C D	E F G H I L M N	
1997	Diallel cross	Diallel cross	
1998	6 2S2Syn1 (SH)	4S2Syn1 	16 2S2Syn1 (SH)	8S2Syn1	
1999	6 2S2Syn2 	4S2Syn2	8 S2DHF1	8S2Syn2	
2000	6 2S2Syn3 	4S2Syn3	8 S2DHF2	8S2Syn3	
2001	Diallel cross		Diallel cross		
	15 S2DH	4S2Syn4	28 S2OH	8S2Syn4	
2002-03	Agronomic Test	
 	SH: Simple Hybrid; DH: Double Hybrid; OH: Octuple Hybrid

4-constituents program: partly inbred (S2) constituents originating each from different sources (groups A and B). Diallel crossing among 6S2Syn3 parents to obtain Double Hybrids (DH).

8-constituents program: partly inbred (S2) constituents originating each from different sources (group C), each from two different sources (group E) and each from a single cultivar (group D). Diallel crossing among 8 S2DHF2 to obtain Octuple Hybrids (OH).

The trial was planted in a cold greenhouse, in concrete boxes 25x80x65 cm containing two rows of plants (final density of 250 plants/m2), in a randomized block design with 5 blocks. Data were collected on the 15 central plants of each row, each population being then represented by  150 plants; irrigation was not limiting. Five cuttings were made during the sowing year; dry matter yield, earliness and mortality were recorded at plot basis.

At the 4-constituents level, only one hybrid significantly outyielded for the total year production the corresponding synthetic 4S2Syn4 (+ 29%), although 8 and 2 hybrids respectively in the two diallelic groups showed dry matter production 10-23% higher than the corresponding synthetic. At the 8-constituents level, the hybrids were not different or significantly less yielding than the corresponding 8S2Syn4.

As for the free-hybrids, 4-constituents model resulted significantly more performing than 8-constituents one; the synthetics at 4 and 8 constituents did not differ significantly in Syn4 generation while in Syn3 the 4-constituents model showed higher DMY than 8-constituents one.

Both G.C.A. and S.C.A. variances were highly significant in all the diallelic groups. However, S.C.A. resulted more important than G.C.A. in the 4-constituents crosses while the opposite was true for the 8-constituents crossings. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.11

NEW SPECIES OF FODDER ADAPTED TO DRY - WARM ENVIRONMENTS : GRAN VELIERO AND VELOX TWO NARBON VETCH (VICIA NARBONENSIS L.) VARIETIES

G. VENORA, S. BLANGIFORTI

Stazione Sperimentale di Granicoltura per la Sicilia, Caltagirone 

Vicia narbonensis L., fodder, dry - warm environments, vetch, biodiversity

The results of a selection project conducted through a period of five years are reported, two new varieties have been selected; they are adapted to dry - warm climate, useful in rotation or to break down the wheat monoculture in Mediterranean environments. The selection of more adapted species was carried out growing the more spread wild Mediterranean species of genus Vicia. 

On selected species the useful grow techniques to improve grain and hay yield, were studied.

Two varieties of Vicia narbonensis L. were relised as Gran Veliero e Velox. in 2003.

The two varieties mainly differ for the biological cycle : Velox is more praecox than 10 days.

In the carried out experiments Vicia narbonensis L. distinguished itself among the other vetch species for the following advantages that can offer to Mediterranean agriculture :
-	Resistance to the bird damages during the sowing period.
-	Cold tolerance 
-	Drought tolerance 
-	Erect habit 
-	Re-growing after grazing
-	Pods un-shattering at maturation
-	Good leaves persistence in the hay
-	Good grain yield 
-	High hay production
-	High protein content in the seeds and straw 
-	Optimal digestion for polygastric
-	Full crop mechanisation.
-	
From the obtained results we can say that the new two varieties of Vicia narbonensis L., are suited to Mediterranean climate for their more good performance, quality and yield than the other common cropped vetch.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.12

NEW METHODS FOR THE CLASSIFICATION OF POPLAR CLONES BY MEANS OF MORPHOLOGICAL DESCRIPTORS

F.M.STEFANINI**, A. GIORCELLI***, F.PICCO*** and A.CAMUSSI*

*) Dipartimento di Biotecnologie agrarie and 
**) Dipartimento di Statistica Università di Firenze 
***) Istituto di Sperimentazione per la Pioppicoltura, Casale Monferrato (AL)

poplar clones, classification methods, descriptors, Random Forest

Tests for distinctness, homogeneity and stability of Poplar clones are still based on the use of proper descriptors of the main morphological and phenetic characteristics of the plant. Although the importance of a precise identification of clones is widely acknowledged, no sound technique has yet come into wide use. Many descriptors related to economic and productivity traits show a reduced repeatability or within clone variability. The poor discriminant power of single traits can be counterbalanced by a multivariate approach. 

In the frame of a research project to develop models for the joint analysis of descriptors with different statistical properties, we have analysed experimental data related to 30 Poplar clones, representative of the germplasm collection. The experimental design consists in ten replications in two localities (Casale Monferrato and Mantova). In each replication 18 descriptors (both qualitatitive and quantitative) were recorded on two plants. The individual discriminant power of descriptors were estimated by usual statistical techniques (linear model and logistic model).

A multivariate approach by means of parametric procedure is ineffective due to the joint presence of variables with different sampling properties.

We have applied some new numerical techniques based on computer simulation approaches to overcome difficulties due to the probability distribution of different traits. Among others, the procedure known as Random Forest  was particularly suitable for clones discrimination. Random Forest, proposed by Leo Breiman (University of California, Berkeley,USA), is based on the building of a large set (Forest) of classification trees, generated at random, which are allowed to evolve generation by generation on the basis of computer simulations. Some internal estimates are produced and they are useful to describe the classification process and the relative importance of single traits. The procedure can include data of different distributive properties (quantitative traits, categorical variables and so on).

With our data, we received a good classification performance with a mean misclassification rate of 0,13 and with 22 clones with a rate under this value.
The procedure allowed to individuate the best variables according to classification ability. Final aim of the work is to individuate simple rules that can be easily applied in the typical condition of nursery practice.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.13

NUCLEOTIDE AND HAPLOTYPE  DIVERSITY IN WINE CULTIVARS OF GRAPE (VITIS VINIFERA L.)

G. PRETE, F. CATTONARO, M. MORGANTE

Dipartimento di Produzione Vegetale e Tecnologie Agrarie, Università degli Studi di Udine, via delle Scienze 208, 33100 Udine

grape, SNP, variation, haplotypes, bottleneck

Developments in sequencing technology made it possible to detect DNA polimorphisms at the single nucleotide level. SNPs (Single Nucleotide Polimorphism) and haplotypes derived from them are both a tool to investigate the evolutionary history of genes and species as well as a novel tool to genetically map traits through association approaches that rely on linkage disequilibrium and existing populations. 

Pursuing this aim, 13 genes in 16 cultivars of grape used for wine production have been examined. The 13 loci have been chosen from GenBank sequences and primers for PCR have been designed targeting whenever available UTRs and introns. SNPs and Indels have been identified by comparing sequences (chromatograms and text format) from all individuals by using Phred/Phrap/Consed (Ewing et al. 1998). As grapevine is an outcrossing and heterozygous species, the exact sequence of haplotypes at each locus was ambiguous whenever an individual was heterozygous at any locus. To resolve these ambiguities, amplicons from some individuals at each locus have been cloned and the haplotype data coming from cloned loci and homozygous loci have been exploited to infer the remaining haplotypes by using the Haplotyper software (Niu et al. 2003).

All loci investigated were polymorphic. The observed frequency of nucleotide changes is high, on average 1 SNP every  69 bp in coding regions and 1 every 48 bp in non- coding regions; frequency of Indels, found only in non- coding region, is 1 every 714 bp. A mean of 7 haplotypes per gene has been found in the sample of 32 chromosomes we analysed. Linkage disequilibrium as measured by both D’ and r2 did not decay  within 700 bp distances.

The analysis of sequence diversity by means of the Tajima’s D test shows a significant deficiency of rare variants and an excess of common ones across most loci. This is an indication of a recent bottleneck which may be the result of the breeding and selection process. The high levels of SNP variation we detected in transcribed regions of elite material as well as the presence of significant LD levels at least within short distances make association mapping approaches very attractive for wine grape, especially when the biological difficulties related to the creation of segregating populations are considered. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.14

AFLP MARKERS TO STUDY THE GENETIC RELATIONSHIPS AMONG THE MAIN ITALIAN TABLE CULTIVARS (OLEA EUROPAEA L.)

P. LABOMBARDA, G. FONTANAZZA

Istituto per i Sistemi Agricoli e Forestali del Mediterraneo - Sezione di Olivicoltura di Perugia - CNR, via Madonna Alta 128, 06128 Perugia
p.labombarda@iro.pg.cnr.it

Olea europaea L., AFLP, table cultivars, genetic diversity

AFLP analysis was performed on 19 table and double purpose cultivars: 16 Italian and 3 foreign cultivars (“Gordal”, Picholine” and “Picholine Marocaine”) which showed morphological affinities with some of the Italian cultivars analysed. 

The plant material analysed comes from the Institute germplasm collection where they are in the same environmental conditions.

A total of 185 polymorphic fragments were identified and used to calculate pairwise genetic distances and to subsequently construct a dendrogram which clearly shows the polymorphism characteristic of the cultivars studied. There are not homonyms and synonyms. Particularly interesting is the case of “Giarraffa”, “Gioconda” and “Gordal”. “Giarraffa” is a Sicilian cultivar of ancient origin wide spread in the island and with an economic importance. In Spain according to a diffusion order “Gordal” is the second after the cultivar “Manzanilla” . “Gordal”, as reported by some authors (Mulè et al. 2001), seems to be introduced in Sicily as “Gioconda”. On the contrary our molecular inquiry shows that “Gioconda” and “Gordal” are genetically distinct and that the similarity degree (SC=0.98) between “Giarraffa” and “Gioconda” is higher than the one observed between “Gordal” and “Gioconda”. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.15

USE OF AFLP AND MICROSATELLITE MARKERS FOR GENETIC DIVERSITY ASSESSMENT AND CULTIVAR IDENTIFICATION OF OLEA EUROPAEA L. MEDITERRANEAN GERMPLASM

C. MONTEMURRO*, R. SIMEONE*, A. PASQUALONE**, A. BLANCO*

*) Dipartimento di Biologia e Chimica Agro-forestale ed ambientale (Di.B.C.A.), Università di Bari
**) Dipartimento di Progettazione e Gestione dei Sistemi Agro-zootecnici e forestali (Pro.Ge.S.A.), Università di Bari

Olea europaea L., AFLP, microsatellites, genetic diversity

Olive (Olea europaea L.) is a diploid specie (2n=46) which comprises the cultivated type, var. sativa and the wild type, var. oleaster. The cultivated olive is of Asian origin and derives from the domestication process of O. chrysophilla Laxx. Olive cultivation and diffusion over the Mediterranean basin has a very long history which started from the IV Millenium B.C. 

The ancient origin and ancestral hybridizations within different species of the genus Olea originated a great number of varieties, about 2000, and the existence of cultivar groups with similar morphological traits make difficult the varietal identification only on the basis of the phenotype. Olive, with some important Italian tree crops, like: grapevine, peach, cherry, apple, pear and citrus, has certification proceedings for plant material marketing. For the marketing of tree crops and to satisfy the requirements of the Community Directives (EC Reg. 92/34, 93/48, 93/64, 93/79) an accurate cultivar identification and a guaranty about the plant sanitary state are necessary.

The development of molecular biology allowed to examine olive genetic variation in much grater detail through the use of the large array of DNA (Angiolillo et al., 1999; Sefc et al., 2000). Molecular markers such as: RAPDs, AFLPs, ISSRs, and SSRs), provide an opportunity for olive cultivar discrimination, independent of any influence by environment and developmental stages.

The aim of this research was to use AFLP and microsatellite markers to assess the genetic diversity within a representative sample of Mediterranean germplasm, also considering olive cultivars used for production of Denomination of Protected Origin (DPO) oils.

A total of 60 cultivars native of Italy, Spain, France and Greece was analyzed with three AFLP primer combinations and sixty eight primers pairs of microsatellite markers (Sefc et al., 2000, Carriero et al., 2002, Cipriani et al., 2002). A total of seventy polymorphic AFLP bands and eighty polymorphic SSR bands was obtained. SSR amplified fragments ranged from 110 to 300 bp, while AFLP fragments ranged from 80 to 700 bp. The data obtained were elaborated with the Jaccard genetic similarity coefficient, applying the SAHN clustering method. A dendrogram of genetic distances was elaborated and almost all the cultivars resulted to be distinguishable with a few cases of probable homonymy.

References:
Angiolillo A., Mencuccini M., Baldoni L., 1999. Theor. Appl. Genet.,98: 411-421.
Carriero F. Fontanazza G. Cellini F. Giorio G., 2002. Theor. Appl. Genet. 104: 301-307.
Cipriani G. Marrazzo M.T. Marconi R. Cimato A. Testolin R, 2002. Theor. Appl. Genet. 104: 223-228.
Sefc K.M., Lopes M..S., Mendoca D., Rodrigues Dos Santos M.., Laimer Da Camara Machado M.., Da Camara Machado A., 2000. Molecular Ecology 9: 1171-1173.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.16

MICROSATELLITE DNA SEQUENCES  AND VARIETAL IDENTIFICATION IN OLIVE

V. SARRI*, A. CONTENTO*, M. FREDIANI**, L. BALDONI*** , P.G. CIONINI*

*) Dipartimento di Biologia Cellulare e Molecolare della Università di Perugia, Sezione di Citologia e Genetica, Via A. Pascoli, 06123 Perugia, Italy
**) Dipartimento di Agrobiologia e Agrochimica, Università della Tuscia, Via S. Camillo de Lellis, 01100 Viterbo, Italy
***) CNR, Istituto di Genetica Agraria, Divisione Ricerca di Perugia, Via Madonna Alta, 130 06128 Perugia, Italy

microsatellite DNA sequences, olive, varietal identification

Olea europaea L., the olive, is one of the oldest tree crops, and its agricultural interest is very remarkable in Italy and in the countries of the Mediterranean Basin. In spite of this, knowledge and methodologies allowing us a sure discrimination between more than 2,600 olive cultivars described are to date lacking, in spite of their importance in certifying cultivars and assessing the commercial identity of oils and their origin. Very recently, research work has been carried out in order to identify and characterize olive cultivars by the use of parameters other than the morphological and quantitative traits used up to today, which may be affected by the diversity of enviromental conditions. However, the search for reliable, cultivar-specific markers to be exploited also in  identifying the cultivar/s from which an oil is originated is far from being brought to an end. As a consequence, reliable and feasible protocols for the identification of olive cultivars and then for the traceability and certification of oils are not available.

We carried out research work in an attempt to use simple sequences repeats (SSRs) in the nuclear DNA for the identification of olive cultivars, taking into account that a cultivar would represent a clone, due to vegetative propagation. SSRs isolated by us from a DNA library or found in the literature were  tested for their capability to give polymorphic products when amplified by polymerase chain reaction in different olive genotypes. SSRs able to produce high polymorphism were selected and amplified in 78 olive cultivars from Italy and the other Mediterranean countries. The amplification products were run in 3% sucrose gels and the electrophoretograms were registered. Sucrose gels were chosen, in view of the possible applied exploitation of the method, since they are safe, easy and economical.

We found that the band patterns obtained by summing up the band patterns produced by each of three microsatellite sequences (ssrOeUA-DCA9, ssrOeUA-DCA16, and ssrOeUA-DCA17; Sefc et al., Molecular Ecology (2000) 9, 1171-1193) were unique for 67 out of 78 genotypes studied. Other genotypes could be distinguished by adding the band pattern obtained by using a fourth microsatellite sequence (ssrOeUA-DCA11; Sefc et al., l.c.). Within each of five groups, the cultivars showed the same band patterns. These cultivars are known to represent synonyms, as was confirmed by analyzing their genotypes by AFLP. Plant grown in different environments but belonging to same cultivars showed the same band patterns. Therefore, by using four selected SSRs, ‘bar codes’ were obtained which identify with certainty olive cultivars and can recognize homonymies and synonymies. 

Work is in progress to try the assessment of the origin and commercial identity of olive oils by characterizing  extracted DNAs by the above method.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.17

ANALYSIS OF GYPSY- AND COPIA-LIKE RETROTRANSPOSONS IN THE OLIVE GENOME AND THEIR USE FOR STUDYING GENETIC DIVERSITY 

T. GIORDANI, P. MAESTRINI, A. CAVALLINI, L. NATALI

Dipartimento di Biologia delle Piante Agrarie, Sezione di Genetica, Università di Pisa, Pisa
lnatali@agr.unipi.it 

genome evolution, Olea europaea, olive, retrotransposons

Retrotransposons, or their remnants, usually represent a major fraction of interspersed repetitive DNA in eukaryotes, especially in plant species. It is generally accepted that retrotransposons have played an important role in plant genome evolution. Their amplification and dispersion contributed to genome plasticity perhaps allowing plants the adaptive responses to environmental stresses. 

Analysis of the genomic repetitive component, especially retrotransposable elements, can be useful for studying important features of olive culture, such as the determination of phylogenetic relationships in the genus Olea and its close genera and the accurate identification of olive (Olea europaea L.) cultivars. 

Since retrotransposons (particularly retrotransposons with long terminal repeats, LTR) are usually interspersed in different genomic regions, they may be used to generate molecular markers based on the amplification of retrotransposon flanking sequences, using as primers oligonucleotides designed on LTRs in conjunction with primers containing 3’-anchored microsatellites, i.e. short simple sequences (dinucleotides). The use of retrotransposon sequences to generate molecular markers depends on the availability of conserved LTR regions. In species in which complete retroelements are not available, it is necessary to extend retrotransposon fragments towards 5’- or 3’-ends to isolate putative LTRs. Then, since retroelement sequences are subject to high mutation rates during evolution, the conservation and the redundancy of putative LTR within the genome is to be tested before applying molecular marker techniques.

We have isolated DNA fragments belonging to Ty3-gypsy and Ty1-copia retrotransposons by PCR, using degenerated primers deduced from sequence databases. These fragments include portions of retrotranscriptase and RNAse H of a copia-like element, and portions of retrotranscriptase, RNAse H and integrase of a gypsy-like element. Such fragments have been extended using chromosome walking techniques, to retrotransposon 3’-extremities, i.e. the long terminal repeats (LTRs). Copia- and gypsy-like putative LTR sequences were quantitatively analysed in slot blot hybridisations to evaluate copy number and resulted medium-highly repeated. Southern blot experiments evidenced different hybridisation patterns, i.e., with typical smears and/or heavy hybridisation bands, indicating redundancy of the sequence and its genomic interspersion or with a few light hybridisation bands, indicating the degeneration of the retroelement. Using this approach and sequence comparison, retroelement subfamilies were characterised.

The LTR sequences belonging to different subfamilies can be used to design primers and generate molecular markers. Preliminary experiments showed polymorphism among different cultivars of Olea europaea.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.18

RESEARCH OF SYNONYMS AND HOMONYMS OF OLIVE CULTIVAR SPREAD IN DPO AREAS THROUGH AFLP

O. AMBROSINO*, M. MANZO**, G. PUGLIANO***, L. MONTI*, R. RAO*

*) Università di Napoli “Federico II”, Facoltà di Agraria, Dipartimento di Scienze del Suolo della Pianta e dell’Ambiente sez. Genetica Vegetale
**) Regione Campania - Se.S.I.R.C.A.
***) Università di Napoli “Federico II”, Facoltà di Agraria, Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, sez. Arboricoltura

AFLP marker synonymies homonymies

The number of the olive varieties, in Campania region, can’t be determined with precision. Most of them have been designated by farmers with local and dialectal names. It has been frequently assigned the same name to different cultivars or the same cultivar has been designated with different names. In arboreous species, this confusion is enphasized by insufficient morphological classification of cultivars due to complexity and long lasting of evaluation and requiring great experience. Moreover the environmental factors may strongly influence the phenotype. Therefore there is a great need for the development of new technologies that contribute to correct variety classification and provide rapid methods of early identification of plants diffused in Denomination Protected of Origin (DPO) areas or multiplied in nurseries. 

Most of the varieties of the present study, belong to the list of cultivars included in the disciplinary of production of DPO “extra virgin olive oil” named "Colline Beneventane”, "Colline Casertane" and "Sannio Caudino-Telesino". They were certified for absence of viral infections and described by morphological descriptors. However several plants labelled as DPO varieties but requiring a more detailed classification are present in these areas.  Here we report the AFLP analyses of these plants carried aut in an attempt to clarify the not sufficient morphological descriptions.  

Four combinations of primers were used. The cluster analysis evidenced cases of synonymies and homonymies clarifing that Rotondella di Sanza do differ from other plants labelled as Rotondella. The varieties known as Ravece and Ortice (5 plants analysed) are all synonyms (mean similarity value:  0,99), therefore these varieties should all be enclosed in one group that should be given a new name but in respect to historical traditions here we indicated as “Ravece-Ortice”. Our data confirmed that AFLP markers are effective for variety identification and are able to distinguish synonymies and homonymies in olive. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.19

CROSS-HYBRIDISATION WITHIN THE GENUS OLEA: FIRST ATTEMPTS TO OBTAIN INTERSPECIFIC HYBRIDS

M. E. CACERES, P. LABOMBARDA, P. POLLACCI, M. MENCUCCINI, G. FONTANAZZA

Istituto per i Sistemi Agricoli e Forestali del Mediterraneo, Sezione di Olivicoltura – CNR - Perugia, Via Madonna Alta 128, 06128 Perugia
m.e.caceres@iro.pg.cnr.it

Olea europaea, inter-specific cross, AFLP

A series of interspecific crosses has been undertaken between different species of the genus Olea in the perspective to incorporate in the hybrids, traits related to biotic resistance. Three parental lines  were included in this study: Olea europea (Var. Leccino), Olea cuspidata (origin: Pakistan) and Olea africana (origin South Africa). Viable fruits were obtained in the cross O.africana X O.cuspidata only. Embryos were dissected from mature fruits, sterilised with 10% sodium hypochlorite, rinsed with abundant sterile water and allowed to germinate in vitro. The embryo culture was performed in test tubes containing agar-solidified EZ medium and incubated in a growth chamber with fluorescent light. Embryos derived from open pollination of O.africana were also cultured in the same conditions as control yielding 11 independent genotypes. Out of the 4 viable hybrid seeds, 2 yielded dead embryos, one did not germinate and the other one developed into a viable seedling. To verify the hybrid nature of this seedling, AFLP markers were used to investigate the transmission of paternal alleles into the genome of the putative hybrids. Using a total of 24 primer combinations it was possible to detect 7 AFLPs specific of the pollinating parent that were present in the seedling’s genome thus confirming its hybrid nature. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.20

GENETIC ANALYSIS OF CITRUS RETICULATA BLANCO REGENERANTS OBTAINED FROM IN VITRO ANTHER CULTURE 

B. Chiancone*, M. A. Germaná*, O. Olivares**, L. Navarro**

*) Dipartimento di Colture Arboree, Facoltá di Agraria, Universitá degli Studi di Palermo, Viale delle Scienze 11, 90128 Palermo, Italia
**) Departamento de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias, Apartado Oficial 46113, Moncada, Valencia 46113

mandarins, anther culture, embryogenesis, molecular markers

Mandarins (Citrus reticulata Blanco) and their hybrids are one of the most popular Citrus species worldwide. They are grown in more than 80 countries, representing the 20% of the world Citrus production.

The Mandarino Tardivo di Ciaculli (MTC) and the mandarin Avana are the most important mandarin cultivars in Italy; MTC was selected as a mutation of the Common Mandarin and it is widely cultivated because of its delay in the ripening. In fact the fruits can be collected from the first days of February until middle of April, and although the fruit size is pretty small and with seeds, the flesh quality is really good. Thus, availability of a MTC cultivar with no seeds would have a big impact on the Italian citrus industry.

Since citrus conventional breeding faces different problems related to particular biology of these species, biotechnology appears as an alternative to improve them.

Anther culture is considered a routinely method to obtain haploids. Microspores under some culture conditions may change their gametic pathway that would lead to pollen formation, to develop embryoids or embryogenic callus via androgenesis. Haploid plants may have many applications for citrus breeding: easier mutations detection; double-haploids formation by diploidization of their chromosomes, allowing recovery in just one generation of homozygous plants, what is very difficult in tree plants. Moreover haploid lines can be used for genetic trasformation and as parents in protoplast fusion with diploid plants in order to obtain triploids, which are particularly important since they are seedless.

Actually by anther culture it is possible to recover diploid embryoids too. They can originate from the microspores and then duplicate their chromosomal number, so they are homozygous, or they may come from the somatic tissue of the anther, so they are heterozygous as the mother plant. Those somatic calli and plants have high value in citrus breeding, since they can be used for protoplast fusion, genetic trasformation or in vitro germplasm storage.

This research reports the molecular characterization of several embryoids and plants obtained from anther culture of MTC. The regenerants have been analysed with a flow cytometer to establish their ploidy level, and characterized by microsatellites, in order to determine if they are doubled-haploid or heterozygous somatic plants and to determine possible gametoclonal or somaclonal variation. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.21

SSR AND CAPILLARY ELECTROPHORESIS AS A TOOL FOR DISTINGUISHING ZYGOTIC PLANTLETS IN LEMON INTRASPECIFIC CROSSES

M.-T. SCARANO*, N. TUSA*, L. ABBATE*, D. GRAZIANO*, S. REALE**, S. FERRANTE*

*) C.N.R. IGV-sez di Palermo, Corso Calatafimi 414, 90129 Palermo, Italy
**) Dipartimento S.A.V.A, Università degli Studi del Molise, Via De Sanctis, 86100 Campobasso, Italy

Citrus, protoplast fusion, backcrosses, microsatellite

One of the most important goals in genetic improvement of lemon in the Mediterranean area is certainly the obtainment of new genotypes tolerant or resistant to mal secco disease. To improve tolerance and fruit quality, a diploid lemon cybrid with an intermediate degree of resistance to mal secco disease, spontaneously obtained by symmetric protoplast fusion between ‘Valencia’ (V) orange and ‘Femminello’ (F) lemon, was used as mother plant in backcrosses with a diploid clone of ‘Femminello’ Lemon Tolerant to the Mal Secco disease (LTMS). Backcrosses have been planned because subsequent cycles of sexual hybridization for continued advancement to eliminate negative traits will be possible. ‘Femminello’ lemon reproduces apomictically by nucellar embryony; nucellar embryos are genetically identical to the mother plant, and develop within the same embryo sac together with the zygotic embryo (polyembryony).  Usually we perform flow cytometry (FCM) to distinguish zygotic embryos, but in this case, where both nucellar and zygotic plantlets are diploids, FCM is not able to distinguish zygotics from nucellars, therefore suitable methods are needed for this purpose in order to eliminate useless nucellar plants. Moreover, since the diploid lemon cybrid presents the nucleus of ‘Femminello’ lemon and the mitochondria of ‘Valencia’ orange (as previously detected) this cross is a sort of selfing, because the two parents present almost the same nucleus.  We have regenerated 40 plantlets, which were subjected to microsatellite analysis with the use of capillary electrophoresis fluorescence-based technology for the analysis of the data. Genomic DNA was extracted from young leaves using the CTAB method and therefore seven microsatellite loci were analyzed (http://www.plantbiology.ucr.edu/people/faculty/rooselink.html): TAA1, TAA41, cAGG9, TAA3, CAT01, AG14, CAC39. For all the SSRs, forward primers were labeled with either 6-FAM (blue), HEX (yellow) and TET (green) fluorescent dyes and TAMRA (red)-labeled  used as internal size standard (GeneScan-350). Capillary electrophoresis was carried out on an ABI PRISM 310 (Applied Biosystems) and raw data analyzed with GeneScan software (version 3.1) to estimate the variants size; each DNA band (allele) is then represented by a peak. All the seven primer pairs used in this study showed that the parental plants were heterozygous with two alleles; therefore the zygotic heterozygous plants cannot be distinguished from the nucellars, meanwhile homozygous are certainly zygotics. Among the 40 tested diploids, 3 showed locus segregation, displaying one band, confirming their zygotic origin. Combining SSR method and semi-automated analysis system like GeneScan resulted to be ideal for the molecular analysis of many genotypes and successfully utilized for a large-scale analysis. SSR coupled with fluorescence-band semi-automated allele sizing technology provided doubtless for high genetic resolution and can develop further application in Citrus genetic studies.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.22

HiDRAS: AN INNOVATIVE MULTIDISCIPLINARY EU-FUNDED RESEARCH PROJECT TO BREED HIGH-QUALITY DISEASE RESISTANT APPLES

L. GIANFRANCESCHI, V. SOGLIO

Department of Biomolecular Sciences and Biotechnology, Università degli Studi di Milano, via Celoria 26, 20133 Milano, Italy

apple, fruit quality, molecular markers, QTL mapping

HiDRAS (High-quality Disease Resistant Apples for a Sustainable Agriculture) is a collaborative effort between 11 European groups (University of Milano and University of Bologna, Italy; Plant Research International, The Netherlands; INRA Centre d’Angers, France; Swiss Federal Institute of Technology and Swiss Federal Research Station, Switzerland; Horticultural Research International, U.K.; Federal Centre for Breeding Research on Cultivated Plants, Germany; Centre de Recherches Agronomiques de Gembloux, Belgium; Warsaw Agricultural University and Research Institute of Pomology and Floriculture, Poland) aimed at the identification of genetic factors controlling fruit quality. 

An innovative approach based on the phenotypic and molecular characterization of a large number of related genotypes will be adopted. One of the aims of this project is to provide breeders with new tools, such as molecular markers linked to fruit quality and pathogen resistance, improving “marker assisted selection”. New software will be developed to fully exploit genotypic, phenotypic and pedigree data, consequently fruit quality QTLs will be identified and the transmission of their alleles will be followed along the pedigree. A large number of highly informative markers, including markers from fruit-quality-related genes will be generated and employed to fill in the gaps existing in the apple linkage map already constructed and to increase the efficiency of the QTL analysis. The study of the quality parameters that determine the success of a new apple variety will be carry out testing European consumer needs. In those occasions our scientific research will be presented increasing the trust of European citizens towards disease resistant apples, therefore favoring their diffusion. An apple data repository will be established to collect and share data among participants. Initially database access will be limited but later public access could be partially granted.

Currently a HiDRAS website (http://users.unimi.it/hidras) has been created to allow a world-wide diffusion of the objectives of the project, the work progress and the participants involved.

The original and well concerted multidisciplinary approach, including apple breeding, genetics, molecular biology, statistics and bioinformatics, will ensure the achievement of the project objectives.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.23

ISOLATION OF NEW APPLE CULTIVARS WITH MARKER ASSITED SELECTION (MAS)

S. PASSEROTTI, M. KOMJANC, P. MAGNAGO, E. ZINI, C. TOLLER, P. BALDI, L. ZULINI, I. PERTOT

Istituto Agrario di San Michele all’Adige, via E. Mach 1, 8010 San Michele all’Adige (TN), Italy

apple, Marker Assisted Selection, apple scab

Since 1990 the Agricultural Institute of San Michele all’Adige (TN)-Italy has been involved in genetic researches on apple. In that year programs of classical genetic breeding and molecular studies started in order to obtain cultivars resistant to apple scab and to study genes involved in the interaction with the fungal pathogen Venturia inaequalis (apple scab). In 2000 a new project, “Advanced  Biology Applied to Grape, Apple and Salmons” financed by the CARITRO foundation., started. 

Apple breeding at Agricultural Institute of San Michele all’Adige is aimed at combining high fruit quality with good orchard and conservability performance and pyramiding durable disease resistance for a sustainable production. To select new cultivars with a major defence capacity against the pathogen population is necessary pyramiding different resistance gene in one single genotype. In particular polygenic resistance and major resistance genes should be combined. 

Marker Assisted Selection (MAS) is supposed to be extremely useful in the process of pyramiding resistance genes against the same or different pathogens. It is also possible to pyramide major genes and polygenes, called quantitative trait loci (QTL). Actually there are different molecular markers associated to resistance genes which are produced from different European research groups.

At present, at Agricultural Institute of San Michele all’Adige, 49 different crossings have been considered and about 2800 samples collected. In this crosses it was introgressed: Vf, Vm, Vbj, Va (monogenic), Vb for scab resistance and Pl1, Pl2 for powdery–mildew resistance. Of this 49 crosses: 25 were for Vf, 11 for Vf+Pl1, 7 for Vf+Pl2, 1 for Vf+Va, 1 for Vf+Vbj, 1 for Vf+Vb, 1 for Vf+Vm, 1 for Vm and 1 for Vbj. The molecular markers used in Marker Assisted Selection (MAS) were: AL07 and M18 for Vf, CH05e03 for Vbj, Vm for Vm, AT20 for Pl1 and N18 for Pl2. 

The MAS aim was not only to pyramide genes but also to single out homozygote plants for Vf and plants with recombination effects in the Vf region. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.24

INHERITANCE OF PARENTAL GENOMES IN PROGENIES OF MALUS SP. FROM SEXUAL AND APOMICTIC GENOTYPES AS ASSESSED BY SSR MARKERS

C. BISOGNIN*, W. JARAUSCH**, E.  SEEMÜLLER***, M.S. GRANDO*

*) Laboratorio di genetica molecolare, Istituto Agrario San Michele all’Adige, Trento
**) Centrum Grüne Gentechnik - Neustadt an der Weinstrasse, Germany
***) Biologische Bundesanstalt für Land und Forstwirtschaft – Dossenheim, Germany

apomixis, Malus, apple genome, SSR markers

The ability of flowering plants to propagate by seed is not necessarily linked to sexuality. Some angiosperms belonging to the families of Poaceae, Asteraceae, Rosaceae and Rutaceae commonly reproduce asexually through seed by a process called apomixis. Progenies derived from apomictic parents are genetic copies of the maternal genotype. As apomixis is not obliged into the genus Malus, we planned to carry out controlled crossings between completely no-parthenogenic and highly apomictic apple accessions. Three sexual (M9, P22 and Prima) and five apomictic selection of rootstocks were crossed in different combinations, including some reverse crossings.

More than 1600 seedlings were DNA typed using codominat SSR markers in order to establish their pedigrees and evaluate the possibility to exploit new genetic resources for the breeding of apple rootstocks.

Results showed two main classes of allele combinations in the progenies: one identical to the mother’s genotype and the second having a third allele at each locus segregating from the male parent. Very few hybrids were observed when flowers from apomictic rootstocks were pollinated with M9 variety. The reverse crossings generated a higher percentage of recombinant seedlings together with an unexpected number of self pollination events. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.25

TRANSFERABILITY AND CHARACTERISATION OF 112 APPLE SSRs IN TWO EUROPEAN PEAR PROGENIES 

L. PIERANTONI*, K-H CHO**, I.-S. SHIN**, R. CHIODINI*, S. TARTARINI*, L. DONDINI*, S. SANSAVINI*

*) Dipartimento di Colture Arboree, Università di Bologna, Bologna Italy
**) National Horticultural Research Institute (RDA), Suwon, Republic of Korea

European pear, SSR, linkage map, synteny

European pear is one of the most important fruit species but to date very limited genetic studies have been done. Many reports suggest that simple sequence repeats (SSR) are very useful as markers because they are codominant, highly polymorphic, abundant and reproducible. In the present work it is reported the transferability data of more than one hundred SSR markers in two progenies of European pear (‘Passe Crassane’ x ‘Harrow Sweet’ and 'Abbè Fétel' x 'Max Red Bartlett'). A total of 41 SSR markers have been positioned on a genetic linkage map of former cross population and 20 in the latter one (in this case the mapping work is still in progress). The degree of synteny among the chromosomes that carry two or more SSR markers have been analysed. The alignment of the pear and apple maps both in respect to identification as well as to orientation of the linkage groups confirmed the co-linearity between the two genomes. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.26

CHARACTERIZATION OF ITALIAN CULTIVARS OF HAZELNUT AND PHYLOGENETIC ANALYSIS USING RAPDS MARKERS 

R. DI BONITO, L. BACCHETTA, G. DI STEFANO, C. BERNARDINI, O. PELLICCIA, G. CAVICCHIONI

ENEA, Biotec-Gen, via Anguillarese 301, 00100 Roma

Hazelnut, RAPDs, intraspecific variability, biodiversity 

In this work a molecular analysis of genotypes of hazelnut (Corylus avellana) has been conducted in order to obtain a fingerprinting for the Italian typical cultivars. The results have been compared to the analysis of the nut morphological traits. We have evaluated a collection of 24 genotypes including the main cultivars from the Italian regions of cultivation (Piemonte, Lazio, Campania, and Sicilia) as well as cultivars from Spain, Great Britain, France and Turkey. DNA has been extracted by the method of Doyle & Doyle (1990) and the analysis conducted by the method of Random Amplified Polymorphic DNAs (RAPDs) using arbitrarily chosen oligonucleotide primers. A preliminary analysis of 80 Operon primers has shown 30 primers producing polymorphic and consistent bands on a limited number of samples. Eleven primers have been selected for the analysis of all the genotypes. The results have been used to produce a similarity matrix with the coefficient of Nei and Li (1979) and a phylogenetic analysis. A characteristic electrophoretic pattern has been obtained for each of the cultivars Tonda delle Langhe (Piemonte), Tonda Gentile Romana (Lazio), Tonda Rossa, Tonda Bianca, Tonda di Giffoni (Campania) and  Napoletanedda (Sicilia). The phylogenetic analysis with the UPGMA method has shown the presence of a cluster grouping the cultivars Tonda Gentile Romana, Tonda Rossa, Tonda Giffoni, Tonda delle Langhe. The cultivar Nocchione from Lazio resulted related to some genotypes from Sicily and Campania and Tonda Bianca resulted in a distinct cluster, with cultivars from Great Britain. The morphological analysis has shown a significant variability in the nut characters. The Cv Mansa from Sicily has shown the higher values of nut and seed weight, while valuable characters for nut processing (pellicle removal) were observed in Napuletanedda, Camponica, Tonda delle Langhe, Riccia di Talanico, Tonda Rossa. The evaluation of the nut shape index has shown a spherical form for all the cultivars except Mortarella, S. Giovanni, Mansa, Riccia di Talanico exhibiting long fruits similar to Sivri from Turkey.

Doyle & Doyle 1990. Focus 12:13-15
Nei and Li 1979. PNAS 76:5269-5273

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.27

THE ASCERTATION OF GENETIC CORRESPONDENCE OF ONE GENOTYPE IN ALMOND (AMYGDALUS COMMUNIS L.) FROM APULIA

R. CHAABANE*, F. LAMAJ*, N. LAMASCESE**, D. DE GIORGIO**, P. RESTA*

*) Dipartimento di Biologia e Chimica Agro-forestale e ambientale (DiBCA), Università degli Studi di Bari, Via Amendola 165/A, Bari, Italy
**) Istituto Sperimentale Agronomico, Ministero delle Politiche Agricole e Forestali, Via C. Ulpiani 5, Bari, Italy

cultivar identification, molecular descriptors, multivariate analysis, probability of coincidence, AFLP

Cultivar collections are fundamental for fruit tree breeding, and their maintenance drains significant resources. The study of homonimous and synonimous denominations has a role in the optimization of collections by eliminating duplications and clarifying uncertainties over cultivar classification. The employment of DNA comparisons together with the traditional morpho-physiological descriptors is a powerful approach for the optimization of collections. Fingerprint analyses are particularly well suited as a rapid and precise tool in the field of genetic correspondence in vegetatively propagated crops. However, their application is not immune to mistakes and subjectivity, thus it is necessary to use internal parameters to check the results. One significant test is first to identify an unknown sample within a collection based on the fingerprint alone, and to then establish a conservative estimate of the probability in association with the resulting identity. To this purpose, in a more general research in progress, DNA was isolated from the leaves of an almond cultivar, whose identity remained unknown throughout the steps of the analysis. The AFLP fingerprints obtained were compared with those corresponding to 43 almond cultivars. Based on the presence/absence matrix, both a descriptive analysis by the UPGMA-SM dendrogram and a statistical analysis by the estimation of the probability of coincidence of the DNA profiles were made. Both the unknown sample and the cultivars analysed originated from the collection gathered and maintained by the MiPAF Experimental Station of Bari. This collection includes numerous denominations with diverse diffusion in the Apulian region and, therefore, the group of 43 cultivars analysed is representative of the almond population in Apulia. 

The unknown sample was correctly attributed as being the cultivar “Rossa”. This result demonstrates the possibility of identifying a corresponding cultivar on the basis of leaf DNA analysis, without prior information, and in a relatively short time with a few AFLP primer combinations. In addition, an estimate was determined of the probability that the unknown sample and the corresponding cultivar had the same genotype with respect to the alternative hypothesis that casuality led to the obtained result. In view of these results the possibile applications of DNA analyses in the field of cultivar identification and genetic correspondence are discussed.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.28

MOLECULAR CHARATERIZATION OF OLD SWEET CHERRY GENOTYPES USING SSR MARKERS

E. VARASANO, C. RICCIOLINI, A. PORCEDDU

Institute of Plant Genetics, CNR, Via Madonna Alta 130, 06126 Perugia

sweet cherry, microsatellite, heterozigosity, incompatibility

Sweet cherry, (Prunus avium) is cultivated in temperate regions of the world for its edible fruit. Originated in the Caspian and Black seas, cherry plants spread trhough Europe at the times of Roman Empire. Because the first evidence of cherry cultivation was reported not later than the 16th century it was proposed that cherry domestication occurred indipendently in several areas starting from wild well adapted material. According to this hypothesis the old varieties represent the direct descents of this wild material and it would be ideal for studies aimed at the identification of domestication and adaptation genes.

Microsatellite have emerged as important molecular markers for population studies due to their high polymorphism. In this work we have assesed the variability of 37 old sweet cherry varieties sampled in Tuscany and Umbria at 28 SSR loci. The mean number of alleles was 4,60, the maximum number of alleles per locus was 11 (Bcpt0038) and the minimum 2. The mean heterozigosity was 0,412, the maximum. 0,9714 (Bcpt0038) and the minimum 0,10 (Bcpt-002). The average discrimination power was 0,56, the highest 0,776 (pchgms5) and the lowest 0,151 (Bcpt-004). 

To further characterize these old varieties for breeding purpose we determined the genotype at the S locus by S allele specific PCR. Nine different S alleles were detected which identified 12 different incompatibility groups.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.29

AFLP ANALYSIS OF RELATIONSHIPS AMONG FIG GENOTYPES (FICUS CARICA L.) IN THE APULIA REGION

P. RESTA*, E. FERRARA**, M. ROSELLI*, R. CHAABANE*, F. LAMAJ*, G. FANIZZA*

*) Dipartimento di Biologia e Chimica Agro-forestale e ambientale (DiBCA), Università degli Studi di Bari, Via Amendola 165/A
**) Dipartimento di Scienze delle Produzioni Vegetali, Università degli Studi di Bari, Via Amendola 165/A

germplasm, collection, synonimous, genetic diversity, multivariate analysis

Cultivar selection is needed in order to enhance fig cultivation in Apulia. This task is hampered, among other factors, by the existence of a large number of local denominations. At the beginning of the last century, 94 cultivar names were re-counted in the peninsula of Salento alone. The disformity of fig cultivation also causes an unavoidable depreciation of the product. Thus, it is necessary to shed light on the confusion in the varietal array. Together with morphological characteristics, DNA-based descriptors can be used to study genetic diversity. In addition to ascertaining hypothesised synonimous cultivars, DNA analyses can also be used to estimate the degree of similarity between cultivars, which can be useful information for breeding purposes. This study began by analyzing the collection gathered and maintained  by the Department of Crop Production Science, University of Bari. This collection includes a sizeble number of the denominations known in the Apulian region. The objectives of the analyses were: 1) a preliminary estimate of the genetic diversity present in the collection; 2) the assessment of hypothesised and/or novel cases of synonimous cultivars. To this purpose a sample of the cultivars was analysed with more than 10 primer combinations ( 100 variables) by the AFLP technique.

Firstly, the result of this study shows that enough polymorphism can be detected at the DNA level among Apulian figs and that the different cultivars appear to be genetically distinct. The diversification also appears to be well above that detected by the traditional fruit and physiological trait analyses. Secondly, some denominations, previously hypothesised as being synonimous to the same cultivar, were confirmed by distinct clustering of the genotypes. This was the case for Petrelli, S. Giovanni, Bianco d’Oria, S. Antonio, whose DNA profiles appear virtually identical. The predictive value of DNA analyses for the purpose of cultivar identification and genetic correspondence is further discussed, as well as, their application in germplasm analysis and maintenance.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.30

IDENTIFICATION OF AGRONOMICALLY VALUABLE QTL-ALLELES IN WILD BARLEY (H. SPONTANEUM) THROUGH ADVANCED BACKCROSS QTL ANALYSIS 

V. TALAMÉ*, M.C. SANGUINETI*, E. CHIAPPARINO*, H.  BAHRI**, M. BEN SALEM***, R. ELLIS****, B.P. FORSTER****, S. RHOUMA***, W. ZOUMAROU***, R. TUBEROSA*

*) Department of Agroenviromental Sciences and Technology, University of Bologna, Via Fanin 44, 40137 Bologna, Italy
**) Ecole National D’Agricolture de Meknes, PBS40 Meknès, Morocco
***) Institut National de la Recherche Agronomique de Tunisie, 2049 rue Hedi Karray, Ariana, Tunis, Tunisia
****) Scottish Crop Research Institute, Invergowrie, DD2 5DA, UK

Hordeum vulgare, Hordeum spontaneum, ABQA, drought stress

Advanced backcross QTL analysis (ABQA) allows to quickly identify and exploit beneficial QTL alleles by integrating QTL discovery and variety improvement in a single process (Tanksley and Nelson 1996; T.A.G. 92: 191-203). The strategy relies on the evaluation of backcross (BC) families between an elite variety used as recurrent parent and a donor accession (often a wild species sexually compatible with the cultivated species). ABQA has already proven its validity for the exploitation of exotic germplasm in tomato (Bernacchi et al. 1998; T.A.G. 97: 381-397), rice (Xiao et al. 1998; Genetics 150: 899-909) and recently also in barley (Pillen et al., 2003; TAG. Published online: 2 April 2003).

In this study, ABQA was used on double haploid (DH) lines derived from an H. vulgare x H. spontaneum backcross to identify allelic variation in wild barley valuable for improving grain yield and other agronomically important traits in cultivated barley grown under conditions of water deficit in Mediterranean countries. DH lines were obtained by means of anther culture from BC1F2 plants derived from the cross between Barke (a European two-row cultivar) and HOR11508 (a wild barley accession). The DH lines characterized by negative agronomic traits such as ear shattering, excessive earliness and lateness, low kernel weight and/or dormancy were not considered. In total, 123 double haploid (DH) lines were genotyped with 58 AFLP and 59 SSR markers. The DH lines and the two parental lines were tested under conditions of varying water availability in three locations (Italy, Morocco and Tunisia) in replicated field trials. Data were collected for seven agronomic traits: growth habitus, heading date, plant height, ear length, ear extrusion, grain yield and 1000-grain weight. QTLs were detected by means of ANOVA based on simple regression for each marker. Genome regions were identified as containing a putative QTL when a significant effect was observed at one or more locations with P  0.001 or at two or more locations with P  0.05. Significant QTL effects were identified for all traits. Among the 81 putative QTLs identified, 43 (53%) agronomically favorable effects were contributed by the wild parental line. Interestingly. Although the majority (65%) of QTL alleles increasing grain yield was contributed by H. vulgare, at six chromosome regions (localized on chromosomes 2H, 3H, 5H and 7H) the alleles increasing grain yield were contributed by H. spontaneum. Our results revealed the effectiveness of the ABQA approach for  identifying, in all three locations, QTL regions of H. spontaneum contributing agronomically valuable alleles, thus indicating that ABQA may represent a useful germplasm enhancement strategy for the release of cultivars better adapted to conditions of limited water availability.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.31

SYNTENY RELATIONSHIPS WITH RICE AND TIGHT ASSOCIATION WITH RESISTANCE GENE ANALOGS WAS REVEALED BY THE FINE MAPPING OF THE BARLEY LEAF STRIPE RESISTANCE GENE Rdg2a

D. BULGARELLI*, N. COLLINS**, G. TACCONI*, E. DELLAGLIO*, M. BARAVELLI*, G. TUMINO*, A.M. STANCA*, G. VALÈ*

*) Istituto sperimentale per la Cerealicoltura, Sezione di Fiorenzuola d’Arda, Via S. Protaso 302, 29017 Fiorenzuola d’Arda, Italy
**) Sainsbury Laboratory, John Innes Centre, Norwich, NR4 7UH, UK

barley, leaf stripe, resistance gene analogs, synteny

The single, dominant gene Rdg2a of barley, which confers resistance to the seed-borne pathogen Pyrenophora graminea, is located in the distal region of chromosome 7H. As the first step towards the isolation of the gene, an high resolution genetic map in the region of the Rdg2a locus was constructed by saturation mapping of a large segregating F2 population of 1400 plants (ThibautRdg2a x Mirco). CAPS markers closely associated to the gene were identified. As additional source of markers (and as putative Rdg2a-candidates), also six resistance gene analogs (RGAs), known to map in this chromosomal region, were mapped. The resistance gene Rdg2a was delimited in a genetic interval of 0.18 cM between the RFLP marker ssCH4 and the CAPS markers MWG851. ssCH4 was separated from the resistance gene by two recombination events (0.07 cM), while MWG851 by three recombination events (0.1 cM); both these markers are RGAs. Other RGAs were found to be in linkage with the gene, supporting that this chromosomal region is rich in NBS-LRR sequences. Rice expressed sequences (ESTs) mapped to the distal region of the chromosome 6 were exploited to identify homologs in barley or wheat ESTs and PCR-based markers were developed and mapped; a syntenic relationship with rice was subsequently identified. Markers flanking Rdg2a, derived from rice ESTs information, genetically delimited a contiguous set of a 115 kb rice PAC clones within which any homologous sequences to characterized resistance gene were identified. Thus, either the Rdg2a-homologs gene is not present in rice or the gene is not in a syntenic location.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.32

PHYSIOLOGICAL TRAITS AND ISOTOPIC DISCRIMINATION IN BARLEY GENOTYPES CONTRASTING FOR YIELD UNDER DROUGHT

F. RIZZA*, J. GHASHGHAIE**, C. LELARGE***, L. CATTIVELLI*, N. DI FONZO****, A.M. STANCA*

*) Ist. Sperim. Cerealic., Via S. Protaso 302, 29017 Fiorenzuola d’Arda (PC)
**) Laboratoire d’ Ecophysiologie Végétale, Bât 362, Univ. Paris Sud F-91405 Cedex, France
***) Laboratoire de Métabolisme et Structure des Plantes, IBP Univ. Paris Sud F-91405 Cedex, France
****) Ist. Sperim.Cerealic., SS 16 Km 675, 71100 Foggia

barley, drought, yield potential, yield stability, delta13C

The physiological, morphological and developmental traits contributing to drought resistance are either constitutive or induced or in some cases a combination of the two.

In this work 12 barley genotypes (6 six-rowed type and 6 two-rowed type) contrasting for yield potential and yield stability were selected on the basis of field data recorded during three years (from 1999 to 2001) at Foggia (South Italy), where about 100 genotypes of different origin, year of release, rows type and growth habit were evaluated in rainfed (R) and irrigated trials (I). The mean rainfall was 440 mm, 220 mm of which were received during the barley growing season.

The response of a given genotype in R and I trials was compared for yield and associated traits. Positive, highly significant correlations were found for all traits analysed in field (plant height, heading date, grain yield, kernel number m-2, kernel weight. High yield in both R and I trials was achieved by the most productive genotypes throughout different stategies, probably associated with genotypic traits (higher kernel weight or higher kernel number, reduced plant height, early heading) and conserved under the range of water availability that occurred in the trials.

The 12 selected genotypes were further analysed for carbon isotopic composition (delta13C) of the grains harvested in the last season. The 13C discrimination, as a measure of the integrated WUE during the kernel development (Farquahar and Richards, 1984), was higher in the I trials for all tested genotypes. Higher discrimination is sustained by a higher ratio of intercellular to external partial pressure of CO2 during photosynthesis, due to a higher stomatal conductance. Differences among genotypes for delta13C were significant withinboth R and I trials. The correlation between delta13C and yield was significant when analysed across treatments, but not within treatments. Apparently the stomatal conductance is not a trait that individually controls yield. Indeed, the two genotypes with the lowest and the highest delta13C belong both to the group of the low yielding genotypes. As shown for the other traits, also for the delta13C of the grains the response of the genotypes was conserved in R and I trials.

The overall data are consistent with the hypothesis that selection under favourable conditions allows to identify genotypes adpted to environments subjected to mild drought because the same mechanisms which operate under favourable conditions seem to work even at the levels of water limitation that occurred in our trials

References
Farquahar G.D. and R.A. Richards, 1984.  Aust. J. Plant Physiol. 11: 539-552.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.33

GENOTYPING SINGLE NUCLEOTIDE POLYMORPHISMS (SNPs) FOR SCREENING OF BETA-AMYLASE ALLELES IN BARLEY CULTIVARS

E. Chiapparino*, P. Donini*, R. Tuberosa**, D. O’Sullivan*

*) NIAB, Huntingdon Road, CB3 0LE, Cambridge - UK
**) DiSTA, University of Bologna, Viale Fanin 44, 40127 Bologna ,Italy

beta-amylase, SNPs, functional polymorphisms, barley

Beta-amylase (1, 4-alpha-glucan maltohydrolase; EC 3.2.1.2) is a key enzyme in the degradation of starch in germinating barley, and its activity, in combination with the activities of limit dextrinase, alpha amylase and alpha-glucosidase, is a measure of the total starch degrading activity of barley malt, which is an important characteristic for estimating the quality of malt for beer production.

However, in most commercial malts, beta-amylase activity is insufficient, and the enzyme appears to be rate-limiting for starch hydrolysis during mashing. The lack of activity is due to its thermolability during the mashing proccess which is perfomed at elevated temperature.

Investigation of natural variation in beta-amylase thermostability within cultivated barley led to the identification of 3 alleles, termed Bmy1-Sd1, Bmy1-Sd2L, Bmy1-Sd2H at the Bmy1 locus on chromosome 4H, and the corresponding enzymes (Sd1, Sd2L and Sd2H) possess low, intermediate and high thermostability respectively. 

The functional differences between these three allelic forms reside mainly in three amino acid substitutions (R115C, V233A and L347S) therefore, by genotyping for the three single nucleotide polymorphisms (SNPs) which cause the respective amino acid substitutions, it is possible to identify which allele is present in the variety under study. 

More than 500 cultivated barley varieties, representing a large proportion of the acreage sown to barley in the EU over the last 60 years, have been surveyed for the three known beta-amylase alleles by genotyping the varieties the three SNPs described above. In addition to the previously described Sd1, Sd2L and Sd2H alleles, novel haplotypes (previously undescribed combinations of the three functional mutations) were observed, and these were confirmed by resequencing the relevant fragments of the coding sequence. The predicted functional significance of the observed new haplotypic diversity will be discussed. We conclude that genotyping known functional polymorphisms shows some promise for the early detection of barley lines which carry superior beta-amylase alleles, and may represent a powerful tool for selection strategies for breeders.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.34

Development of an introgression MAIZE library of the early-flowering variety Gaspé Flint into B73

S. Salvi, C. Buonfiglioli, M. Bellotti, S. Vecchi, E. Govoni, R. Tuberosa 

Department of Agroenviromental Sciences and Technology, University of Bologna, Via Fanin 44, 40137 Bologna, Italy

maize, flowering time, QTL, Nearly Isogenic Lines

An introgression library (IL) is a collection of lines each carrying a molecular marker-defined chromosome segment introgressed from a (usually) agriculturally unadapted accession into the genetic background of an elite line (Zamir, 2001, Nat. Rev. Genet. 2: 983-989). The collection has the unadapted accession genome entirely represented among the set of IL lines. Because of their genetic make-up, ILs are ideal tools for mapping and analysing complex traits. Within ILs, multiple QTLs are segregated in single homozygous lines, where can be analysed in an adapted genetic background with epistatic effect reduced to a minimum. IL lines have also the potentiality to directly serve as pre-breeding or breeding material.

Adapting the duration of the cultivation period of a crop to a specific environment is a major target of plant breeding. This is particularly true for maize, a crop of tropical origin now cultivated over a wide range of growing environments. In the cultivated maize germplasm, flowering time is quantitatively inherited and thus amenable to QTL analysis. The aim of this research is to produce a collection of maize IL lines with the genome of the extremely early-flowering variety Gaspé Flint introgressed into the elite genetic background of the medium-late line B73. Among the ILs collection in the B73 background, the Gaspé Flint genome will be fully represented and, therefore, a dissection of the genetic components controlling the large difference in flowering time will be possible. Based on trait differences between the donor and the recipient genotypes, the IL collection will also serve as a permanent source of nearly isogenic material for QTL analysis and cloning for many other morpho-physiological traits, such as plant height, number of nodes and tiller, leaf area, root architecture, yield and yield components (i.e. number and size of kernel, number of ears), heterosis, etc. Specific marker-delimitated chromosome segments could also be utilized in marker-assisted selection programmes. The IL collection is being produced by a molecular marker-assisted backcross based on publicly available SSR markers. The average Gaspé Flint introgressed segment length is 20-30 cM; this will allow for the production of ca. 70-80 IL lines covering the maize genome. A genetic map based on the first generation of assisted backcross will be presented. Interestingly, preliminary QTL analysis showed that a major QTL for flowering time and number of nodes maps on chromosome 8 bin 8.05, where QTLs for flowering time and related traits have also been identified in a large number of mapping populations (Salvi et al., 2002, Plant Mol. Biol. 48: 601-613).

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.35

STUDY OVER THE TIME OF BIODIVERSITY OF ITALIAN RICE CULTIVARS 

R. MANTEGAZZA*, S. GALLIANO*, A. PURICELLA*, M. BILONI**, A. SPADA*

*) Università degli Studi di Milano, Dip. Biol. Sez. Bot. Gen., Via Celoria 26, 20133 Milano, Italy
**) Sa.Pi.Se Soc. Coop., Via G. Mameli 7, 13100 Vercelli, Italy

biodiversity; SSR; rice; Italian cultivars

Rice (Oryza sativa L.) has a great importance for European economy and Italy is the main rice producing country in Europe with 218.675 ha of rice fields and 1.400.000 t of paddy production (data referred to 2002).

Rice was introduced in Italy in the XV century probably by Spanish or Arabs (Piacco 1959). However, little information is available on rice cultivation up to the beginning of the XIX century. At that time new varieties, like Originario Chinese, were introduced mainly from Asian. Moreover mass selection was the main breeding method till 1925 when the first cross was attempted to improve existing cultivars.

Thereafter breeding programs among ancient Italian varieties or with newly imported germoplasm were focused on three main objectives: resistance to fungal pathogens, productivity and quality. This led to a large number of varieties: 126 are listed in 2002 national register.

In this work microsatellite (SSR) markers were used to assess genetic diversity changes in Italian rice varieties over the time. One-hundred-twenty-three Italian varieties were chosen to represent the germoplasm in the years ranging from 1800 to 2001. A total of 11 microsatellite loci were analyzed, showing 68 polymorphic alleles: 24 alleles were found in the 1800 varieties, 64 in the 2001 ones. 

The allelic frequencies and the analysis of polymorphism indicated an increased level of genetic diversity over the time. This is particularly due to the introduction of new varieties from abroad and to the intensive breeding programs of the last 80 years.

By means of microsatellite markers we were able to show how biodiversity is changed in the Italian rice history: although rice was selected for positive agronomic traits, the genetic pool is still large, and new alleles were introduced during breeding programs.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.36

GENETIC DIVERSITY ANALYSIS OF ITALIAN “FARRO” (T. DICOCCUM) POPULATIONS USING AFLP MARKERS

V. TALAMÉ, M. BALLARDINI, E. NOLI, F. D’ANTUONO, R. TUBEROSA

Department of Agroenvironmental Science and Technology, University of Bologna, Via Fanin 44, 40127 Bologna, Italy 

Triticum dicoccum, genetic diversity, AFLP

Triticum dicoccum, also indicated with the term “farro” or “medium farro”, is one of the three species of  hulled-wheat (Triticum monococcum L., Triticum dicoccum Schubler and Triticum spelta L.) which is still traditionally cultivated in limited areas of the central regions of Italy also in poor fertility soils and in presence of environmental constrains.

This species has recently been object of genetics and agronomical studies due to the increasing interest towards “low input” cultivars  in Italy, and in consideration that the T. dicoccum cultivation is disappearing because progressively replaced by modern varieties of bread and durum wheat.

In this study, ten Italian populations of farro, collected from their traditional cultivation areas, were analysed by amplified fragment length polymorphisms (AFLPs) markers in order to better characterize their level of genetic variability. Eight combinations of AFLP primers were used for selective amplification yielding 146 polymorphic AFLPs. On average, each AFLP assay revealed a percentage of 19.1% polymorphic bands; the index of polymorphic content (PIC) and the marker index (MI)  indicated for each AFLP assay  a good discrimination among genotypes.

Based on presence/absence of fragments, the genetic similarity (Dice coefficient) values among genotypes revealed an average value of 0.64 with higher values  within winter habitus populations (GS = 0.79)  and spring habitus ones (GS = 0.83). Cluster and principal component analysis (PcA) clearly showed a good grouping of the genotypes in each single population. The correlation values between similarity estimates computed for each AFLP assay and for assays allowed for the identification of a few representative AFLP assays that may be useful for further characterization of T. dicoccum populations.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.37

GRAIN YIELD, NITROGEN-USE EFFICIENCY AND BAKING QUALITY OF OLD AND MODERN ITALIAN BREAD-WHEAT CULTIVARS GROWN AT DIFFERENT NITROGEN LEVELS

G. GUARDA*, S. PADOVAN*, G. DELOGU**

*) Istituto di Genetica e Sperimentazione Agraria N. Strampelli- Provincia di Vicenza
**) Istituto Sperimentale per la Cerealicoltura Sezione di Fiorenzuola d’Arda (PC)

bread wheat varieties, grain yield, nitrogen use, grain quality

The high yields of today’s modern wheat cultivars imply the use of high input which lead to both higher production costs and a greater risk of environmental pollution. Increasing public awareness of the latter issue, along with growing consumer demand for more healthful products, has led on one hand to greater criticism being levelled at this type of production model and on the other to heightened emphasis on crops grown under integrated-management and organic systems. This applies also to wheat. By contrast, the yield increments registered by the new wheat cultivars over this time have been bolstered by the progressively higher N-inputs. All that has led to the idea that the modern cultivars selected under conditions of high N-input are little suited to low-input conditions with respect to the old wheat populations and cultivars. The present study investigated the responses of grain yield and quality and N-use efficiency at three input rates (N0, N80 , N160 kg ha-1) in a set of 16 of the most representative bread-wheat cultivars from 1900 to 1994. 

The average yield rise throughout the time was 33.5 kg ha-1 year1, due to an increase of 124 kernel number m-2 year-1 and of 0.22 % year-1 for harvest index. In the same time grain N-accumulation increase from 0.21 kg ha-1 year-1 at N0 rate to 0.67 and 0.82 kg ha-1 year-1 for N80 and N160 kg ha-1, respectively, This increase is matched by average yield increments of 44, 50 and 47 kg per kg of N-accumulated. The cultivars exhibited a progressive rise in demand for N-supply over time of release so as to maximize yields accompanied by the upgraded capacity of N-use and an enhanced quality traits: W and P/L rise from values between 65-170 and 0.25-0.39 per cultivar from 1900 to 1970 to 174-241 and 0.48-0.52 for those released after 1970. All the data show that over the last century the goal of upgrading both yield amounts and grain quality for bread-making was successfully achieved. This success also indirectly led to an improved plant nitrogen uptake and use, clear indicators that even under conditions of limited inputs or under organic-farming practices the best results are to be attained by employing not old populations or varieties but modern cultivars, the latter being the only ones with the intrinsic traits capable of ensuring yield and quality at low N supply even though they maximise their traits at high nitrogen inputs.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.38

MARKER-ASSISTED SELECTION WITH SSRs AND AFLPs TARGETED TO LEAF RUST AND POWDERY MILDEW IN WHEAT

M. MACCAFERRI*, M.C. SANGUINETI*, F. ROTONDO*, R. TUBEROSA*, A. DE MONTIS**, E. DE AMBROGIO**

*) Department of Agroenvironmental Science and Technology (DiSTA), Via Fanin 44, 40127 Bologna, Italy
**) Società Produttori Sementi Bologna, Divisione Ricerca, Via Macero 1, 40050 Argelato, Italy

wheat, leaf rust, powdery mildew, marker-assisted selection

In wheat, marker-assisted selection (MAS) for pyramiding disease resistance genes is now feasible thanks to the recent development of a range of molecular tools; in particular, the development of wheat varieties characterised by durable resistance to leaf rust (Puccinia recondita f.sp. tritici) and powdery mildew (Blumeria graminis f. sp. tritici) is a major objective of wheat breeding. In Europe and in Italy, Lr9 and Lr24 for leaf rust and Pm4b and Pm13 for powdery mildew proved to be effective resistance genes. Dominant STSs (Sequence Tagged Sites) are already available for Lr9, Lr24 and Pm13 but are not fully informative as to the genetic constitution of segregating materials, thus limiting MAS efficiency. Our objectives were to screen mapped SSRs and to generate new AFLPs tightly linked to each one of the four selected resistance genes. Four experimental F3 populations, each segregating for one of the four resistance genes, are being used to develop new diagnostic molecular markers. Preference was given to co-dominant AFLP markers or to AFLP band linked to susceptibility.

As to Lr9, microsatellite markers mapped on the distal regions of 6BL were not informative, thus, new AFLP markers were generated in a bulk segregant analysis experiment. About 15,000 AFLP loci were screened for polymorphism on the two contrasting bulks (made by 15 and 11 susceptible and resistant F3 families. The AFLP screening yielded 26 bands putatively associated to Lr9: 5 of them appeared associated with susceptibility, 20 with resistance and 1 locus showed codominance. The relatively high frequency of loci associated to resistance could be ascribed to the different structural features of the wheat and Ae. umbellulata chromosome segments involved in the translocation (Zhang et al. 1998, Theor Appl Genet 96: 69-75). AFLP markers SseGA/MseCAG-200, SseGA/MseTCT-185, SseTC/MseTAG-210 (associated to susceptibility) showed no recombination events with Lr9 over 26 F3 families. The codominant SseAT/MseCTA-410/405 was also validated for tight linkage.

As to Lr24, four informative microsatellite probes were identified on the distal region of chr. 3DL: WMS38, WMS314, WMS664 and WMS645. The probe WMS645 showed a codominant polymorphism, while each of the WMS38, WMS314, and WMS664 loci showed a null allele associated in coupling with the resistance gene, most probably because of the nucleotide divergence between the distal region of wheat 3DL and the introgressed one.

As to Pm4b (distal region of chr. 2L), probes WMS382, WMS311 and WMS846 produced a dominant polymorphism associated to Pm4b resistance allele. Association was detected also for the probe WMS991(codominant marker), but three families, over the 30 tested, showed a recombination event.

As to Pm13, about 13,500 AFLP fragments screened in BSA yielded 36 differential AFLP markers, with 16 of them associated to susceptibility and 12 further validated for tight association to Pm13.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.39

PROGRESS IN DURUM WHEAT BREEDING FOR YIELD AND GRAIN QUALITY DURING THE 20TH CENTURY IN ITALY

P. DE VITA*, O. LI DESTRI NICOSIA**, F. NIGRO**, C. PLATANI**, A. MAGGIO*, N. DI FONZO**

*) Enea C.R. Trisaia, 75026 Rotondella, Matera, Italy
**) Experimental Institute For Cereal Research, Section Of Foggia, 71100 Foggia, Italy

durum wheat breeding, genetic improvement, yield, grain quality 

A group of 14 durum wheat (Triticum durum Desf.) varieties cultivated in Italy during the years 1900-2000 was studied. Four 25-year time periods were considered. For each period, the 2-4 most cultivated varieties were selected. Field trials were conducted at Foggia (Southern Italy) during 2000-2001 and 2001-2002 seasons. Three nitrogen levels were compared in a completely randomised block design with three replications. To understand the morphological and physiological basis for yield improvement during 100 years of selection, and to possibly assist future breeding for yield and grain quality, several traits were considered: grain yield, days to heading, plant height, physiological maturity, harvest index, test weight, weight of 1000 kernels, grain protein content, pigment content and ash content. At harvest, for each experimental plot, two sub-samples of 0.5 m each were collected to assess total dry matter and yield components (kernels m-2, spikes m-2, spikes length, peduncle length, spikelets/spike, kernels/spike). During the growth season, and in coincidence with the heading date, several physiological parameters were measured for each cultivar, including transpiration rate (E), photosynthetic rate (A), stomatal conductance (g) and internal CO2 concentration (Ci). Over the years, a significant genetic improvement was observed for grain yield. A yield increase of 1.40 t ha-1 was observed in modern vs. older cultivars, with an annual genetic gain of about 19 kg ha-1. Based on these results we conclude that the overall potential yield of durum wheat cultivars released in Italy in the 20th century has increased of approximately 44%. Specifically, the improved potential yield was associated to higher kernels number m-2. Yield improvement was mostly associated to larger grain/spike ratios, partially due to the introgression into modern genotypes of dwarf genes from Norin 10. In addition, a larger number of spikes m-2 in modern vs. older genotypes has been found to be correlated both to a reduced number of total tillers and improved tiller fertility. Early heading genotypes, with reduce plant height, have been selected over the last century with the final goal of improving both the adaptability to Mediterranean environments and the harvest index. Regarding grain quality traits, weight of thousand kernels, test weight, and pigment content did not present over time any distinctive trend. In contrast, grain protein contents showed a significant reduction in modern cultivars, which confirmed the negative relationship between protein content and grain yield (R2=-0.60 at p<0.05). This trend was remarkable for all the N levels considered. The linear regression between grain yield and maximum photosynthetic rate (Amax) was significant (R2= 0.40 at p<0.05), indicating that  Amax may be considered as physiological marker to assist future selection for grain yield.  

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.40

FINE MAPPING OF HIGH PROTEIN CONTENT QTLs IN DURUM WHEAT  

A. GADALETA, A. SIGNORILE, A. BLANCO

Department of Environmental and Agro-Forestry Biology and Chemistry, University of Bari, Via Amendola 165/A, 70126 Bari, Italy

protein content, QTLs, durum wheat, microsatellite, AFLP

The lack of sufficient genetic variation for useful traits within the cultivated wheats has limited the ability of plant  breeders to improve grain yield and grain quality. A good source of genetic variation has been found in wild wheats and related species. The wild tetraploid wheat Triticum turgidum ssp. dicoccoides shows particular promise as a donor of useful genetic variation for several traits, including grain protein quality and quantity. 

The development of molecular markers and maps, and the recent strategies for molecular breeding, referred to as advanced backcross QTL (AB-QTL) analysis, have shown that the backcrossing method has considerable potential for the genetic analyses of quantitative traits and as a method of breeding higher yielding and quality crop varieties. 

The objective of the present study was the fine mapping of three high protein content QTLs introgressed from var. dicoccoides into durum wheat cultivar Latino by the backcross inbred line (BIL) method. 
Three markers (Xgwm275-2A, Xgwm577-7B, P39M37(250)-6A), located on chromosome 2A, 6A, 7B, were previously found to be associated to loci for high protein content. Microsatellites and AFLP   markers and the “bulked segregant analysis” were used for the saturation of these 3 regions. Such markers were considered potentially linked to QTL for protein content and then screened on the complete population of 144 F3 progenies. The putative linkage of these markers with protein content QTLs was confirmed by regression  analysis of each marker locus on the F2, F3 and F4 populations (3BIL-85, with  high protein content, backcrossed to Latino with low protein content).

Mapping loci by backcross inbred lines should enable the obtaining of near-isogenic lines (NILs) in which the individual effects of each QTL can be examined in detail without confounding variation due to other putative QTLs. These near-isogenic lines could also be useful  to conduct physiological studies aimed at investigating the potential mechanisms leading to high protein content, to fine-map the position of each QTL, and eventually to map-based cloning.

The relatively low heritability of quantitative traits makes it difficult to select for useful alleles from wild germplasm based on phenotypic evaluation of single plants and/or in single environments so the detection of molecular markers tightly linked to quantitative trait loci is useful for the marker assisted selection in wheat breeding programs. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.41

A DURUM WHEAT LINE PRODUCED BY CHROMOSOME ENGINEERING WITH NOVEL ALIEN TRAITS AND GREAT POTENTIAL FOR VARIETAL DEVELOPMENT

A. GENNARO*, P. FORTE*, G. M. BORRELLI**, M. G. D’EGIDIO***, P. DE VITA**,****, S. RAVAGLIA*****, C. CEOLONI*

*) Dept. of Agrobiology and Agrochemistry, University of Tuscia, 01100 Viterbo, Italy
**) Experimental Institute for Cereal Research, Section of Foggia, 71100 Foggia, Italy
***) Experimental Institute for Cereal Research, 00191 Rome, Italy
****) Present address: ENEA - C.R. Trisaia, 75026 Rotondella, Matera, Italy
*****) S.I.S. – Società Italiana Sementi, 40068 S. Lazzaro di Savena, Bologna, Italy

Triticum durum, Thinopyrum ponticum, leaf-rust resistance, yellow pigment, alien gene transfer

Two genes of breeding value, i.e. Lr19 (leaf-rust resistance) and Yp (yellow pigmentation), closely linked on the 7AgL chromosome arm of the perennial wheatgrass species Thinopyrum ponticum, have been transferred into durum wheat by chromosome engineering. Several 7AL-7AgL recombinant lines were obtained which, as shown by physical and genetic mapping, incorporated alien segments of different size and gene content. Preliminary assessments of relevant agronomic and quality traits have been carried out on those possessing minimal alien chromatin amounts still including the target genes. Advanced selections from the cross of line R5-2-10, harbouring Lr19+Yp in a distal segment spanning 23% of the total arm length, with the well-adapted but leaf-rust susceptible variety Simeto, were tested during the 2001-2002 seasons. Compared to Simeto, the significantly enhancing effect of Yp gene on yellow pigmentation, already evident in early tests of whole-meal carotenoid pigments of such materials, was confirmed by evaluations of semolina colour parameters. No detrimental effects specifically ascribable to the presence of the alien segment were detected for other relevant grain and semolina traits. As to yield, in spite of non significant differences observed as a whole in both years, yet considerable increments with respect to the control were exerted by most R5-2-10 derivatives in the 2001 season, characterized by a heavy leaf-rust epidemic. Comparable or somewhat reduced productions, depending on the lines, resulted from virtually absent leaf-rust pressure (2002 season). The overall characteristics of some R5-2-10 advanced selections make them highly promising candidates for development of novel varietal germplasm, with added quality and nutritional value, able to respond to traditional agricultural systems, but also to meet more recent concerns on environmental sustainability.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.42

MOLECULAR ANALYSIS OF ANCESTRY IN ELITE DURUM WHEAT GERMPLASM BY SSR MARKERS

M. MACCAFERRI*, C. XIE**, M.C. Sanguineti*, R. TUBEROSA*, S. SMITH**

*) Department of Agroenvironmental Science and Technology, Via Fanin 44, 40127 Bologna, Italy 
**) Pioneer-Dupont, Johnston, IA, USA

SSRs, genetic similarity, ancestry, haplotypes, durum wheat

Information on the genetic diversity structure of elite materials is useful for many issues, in particular for the planning and management of breeding programs, for monitoring the whole level of genetic diversity present in a crop gene pool and for germplasm conservation. Also, the molecular (fingerprinting) profiles are useful to elucidate the genetic relationships occurring among cultivars, which ultimately have to be considered for the protection of breeder’s rights (e.g. for essential derivation issues). Recently, molecular markers have also been considered by UPOV as particularly useful to investigate the relatedness between varieties. Among molecular markers, SSRs (Simple Sequence Repeats), in particular dinucleotide genomic SSRs, represent useful molecular markers for such purposes. Durum wheat is an important crop world-wide and is currently the object of a renewed interest: in Italy, more than 100 varieties are registered and commercialised.

A collection of 134 durum wheat accessions was assembled, mainly comprising modern varieties, representative of the modern durum wheat gene pools currently cultivated in some of the most important durum-producer regions world-wide (Italy, France, Canada and USA); additionally, a number of landmark genotypes from the CIMMYT/ICARDA breeding program and cvs. from Austria and Australia were included, as well as cvs or accessions recognised as important foundation genotypes from different breeding pools. Accessions were profiled with 70 dinucleotide markers, which produced a molecular data set with an average of 6.9 different allelic variants per SSR locus. 

Genetic relationships (co-ancestries) among cvs. were investigated by calculating a matrix consisting of all the pair-wise genetic similarities and compared and integrated with the probabilities of ancestry for all 134 cvs. (the latter calculated on the basis of a Bayesian algorithm). Cluster analysis pointed out that the genetic diversity of the elite germplasm herein analysed is highly structured and a number of groups and subgroups of different breeding lineages, mostly consisting of highly related cvs. (in accordance with pedigree information) were identified. Ranking of genetic similarities and probabilities of ancestry were used to predict the most probable parents and the closest relatives of each cv., among those profiled. Case-studies consisting of 15 triplets (i.e., one cv. and both its putative parents) were present in the collection and profiled with molecular markers, as long as 35 cases where a cv. (obtained by single or multiple cross) and one of its putative parents. In most cases, results agreed to what expected on the basis of the corresponding registered pedigree. However,  incorrect pedigrees were also identified; for such cases, robust molecular evidences indicated the involvement of genotypes different from those listed in the pedigree. The analysis of graphical genotypes (map-based haplotypes) allowed for the identification and the evaluation of the extent of the single chromosome regions possibly directly passed through breeding cycles to modern cvs.. Rare alleles at highly variable SSR loci were also considered as signatures of identity by descent.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.43

CONSTRUCTION OF AN SSR BASED LINKAGE MAP OF DURUM WHEAT

I. JURMAN*, M.D. CASTELLUCCIO*, M. WOLF**, M.A. OLIVIERI*, E. DEAMBROGIO***, M. MORGANTE*

*) Dipartimento Produzione Vegetale e Tecnologie Agrarie, Università degli Studi di Udine, via delle Scienze 208, 33100 Udine, Tel. +39.0432.558652, fax +39.0432.558603
jurman@dpvta.uniud.it
**) TraitGenetics GmbH, Am Schwabeplan 1b, D-06466 Gatersleben Germany
***) Divisione Ricerca, Società Produttori Sementi SpA, via Macero, 1, 40050 Argelato, Italy

tetraploid wheat, genetic linkage map, SSRs

We have initiated the construction of a genetic linkage map of tetraploid wheat Triticum turgidum L.  subsp. durum (Desf.) Husnot using segregation data from a population of 249 F5 recombinant inbred lines (RILs) derived from an intraspecific cross between two commercial varieties of durum wheat. The main durum wheat end-use, in Italy, is pasta, and high quality pasta is produced employing durum wheat only. 

Both the parents show high pasta-making quality, but one has a very high protein content and the other has a very high gluten quality.

Microsatellite markers offer a number of advantages being single locus, co-dominant, highly polimorphic markers which can be easily mapped. Thus far a total of 129 microsatellite markers derived from hexaploid bread wheat have been analysed. 

Linkage analysis was performed using the MAPMAKER program with the Kosambi mapping function. A total of 114 loci were mapped at a LOD >  4 on all 14 chromosomes of the A and B genomes. In general , there is a good agreement between the durum map obtained and the Triticum aestivum map (Röder et al, 1998). 

This map will provide a useful tool for detecting economically important quantitative traits and for marker-assisted selection. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.44

MUTATION HOTSPOTS IN GENES INVOLVED IN RESISTANCE IN NICOTIANA TABACUM

M.C. INTRIERI*,**, A. SABATINI**, M. GORI**, R. MULEO*,**, M. BUIATTI**

*) Università della Tuscia, Dipartimento di Produzione Vegetale, via San C. deLellis, 01100 Viterbo
**) Università di Firenze, Dipartimento di Biologia animale e Genetica, via Romana 17-19, 50125 FI

Nicotina, gene resistance, polymorphism, SSR 

Many biodiversity studies within species to date have focused on “neutral” molecular markers, such as RAPD, ISSR and microsatellites. The breeding structure of populations, population bottlenecks and the biogeographical history of a species are expected to affect all markers in similar ways. By contrast, variation in functional regions (expressed or regulatory sequences) might reflect the past influences of selection, which can be different for each gene. Traits of interest include disease resistance, stress tolerance, growth rate, morphology and life-history, which are often quantitative traits that are likely to be affected by many genes. New marker systems for functional genes are now being developed, and previous work from our laboratory, demonstrated the utility of such markers in phylogenetic studies and for the analysis of agronomical traits. Existing sequence information is being used to develop markers that tag variation within the gene or in a flanking region. In this work microsatellites located in functional regions have been developed and studied in Nicotiana tabacum.

Among all the sequences of N. tabacum present in GeneBank, those including complete genes have been analysed for the presence of simple repeats of two and three nucleotides of at least 5 units. From this analysis, SSRs are located preferentially in promoters, introns and UTR of the analysed genes. The classes of genes in which the SSRs are preferencially found are those involved in resistance, and in particular resistance against biotic stress, even if SSRs have been found in other classes of genes, e.g. in phyA third intron or genes involved in metabolic traits. Of 11 primers tested 8 were able to produce simple and reproducible bands, and among those 8 correspond to polymorphic loci.

Genes involved in stress response are the most variable, among the markers analised. These results could suggest the selective advantage of hypervariable sequences in genes involved in defense against pathogens. Further studies on the phenotipic expression of the genes analysed will be necessary for a definitive conclusion. The association between a marker and the phenotypic expression tagged by that marker will depend on the frequency of recombination and presence of recombinational hotspots, the breeding and population structure and the genealogical history of the genomic region. Probably, evolution favoured the presence of mutation hotspots in genes involved in resistance.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.45

JOINT RESEARCH AGREEMENT BETWEEN “ISTITUTO SPERIMENTALE PER L’ORTICOLTURA” AND “BLUMEN” TO IMPROVE SOME TOMATO ITALIAN ECOTYPES

N. ACCIARRI*, G. VITELLI*, G. CAMPANELLI*, E. SABATINI* P. SBARBATI**

*) Istituto Sperimentale per l’Orticoltura ezione di Monsampolo del Tronto (MiPAF)
**) Blumen

Licopersycon esculentum L., Cuor di Bue Ligure, Pera d’Abruzzo, hybrid

There is an increasing interest in some Italian ecotypes of tomato (Licopersycon esculentum L.). In particular, two local types, “Cuor di Bue Ligure” originating from Liguria and Piemonte regions and “Pera d’Abruzzo” from Marche and Abruzzo show good characteristics of the fruit such as shape, taste and absence of aqueous sacs.

It possible to find these ecotypes in little local markets but now an interest by the large-scale retail trade exists.

The main defects of these cultivars are the absence of genetic resistance to diseases, the sensibility to environmental stresses, the short shelf life of the fruits after harvest and the low fruit firmness.

A joint program for the genetic improvement was started by “Istituto Sperimentale per l’Orticoltura” and “Blumen-Ortoricerca” to obtain new lines and hybrids with the typical of the “Cuor di Bue Ligure” fruit and superior firmness and genetic resistance to main diseases.

Blumen-Ortoricerca, using the lines obtained in collaboration with Istituto Sperimentale per l’Orticoltura”, is producing seeds of some  hybrids showing good performances to carried out trials in open field and greenhouse before the registration in  the Ialian “National Register of Varieties”.

The new lines obtained during the breeding program were selected within the original ecotypes to uniform the variability found; lines with different dimension, shape, and color of fruit (with or without green shoulder), and adaptability to open field or greenhouse condition were selected. The improvement for disease resistance was carried out using as starting materials the segregating progenis derived from the cross between the ecotypes with traditional types provided with dominant genes of resistance.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.46

MORPHOLOGICAL AND MOLECULAR CHARACTERISATION OF TOMATO (LYCOPERSICON ESCULENTUM MILL.) LANDRACES FROM CENTRAL ITALY

A. Mazzucato*, E. Bitocchi**, P. Mosconi*, L. Nanni**, V. Negri***, R. Papa**, M.E. Picarella*, G.P. Soressi*, B. Tiranti***, F. Veronesi***

*) Dip. di Agrobiologia e Agrochimica, Univ. degli Studi della Tuscia, Viterbo, Italy
**) Dip di Biotecnologie Agrarie ed Ambientali, Univ. Politecnica delle Marche, Ancona, Italy
***) Dip. di Biologia vegetale e Biotecn. Agro-ambientali, Univ. degli Studi di Perugia, Perugia, Italy

genetic diversity, germplasm, landraces, tomato

In tomato, as in several vegetable crops, Italian farmers have kept a rich heritage of autochthonous landraces that are still widely adopted in small-scale cultivations and consumed in local markets. However, the widening use of F1 hybrids, the decreasing practice of home consumption cultivations and the lack of commercial interest for seed production of these local types are threatening their survivor and the maintenance of their genetic integrity. In this scenario, the study of phenotypic and genetic diversity is important for conserving, evaluating and utilizing genetic resources as well as for determining the uniqueness and distinctness of landrace genotypes in the perspective of their conservation.

In this research, a collection of tomato landraces mainly gathered in Central Italy in local projects by the Universities of Ancona, Perugia and Viterbo, has been grown and characterised at the morphological, physiological and molecular level. The collection included 57 accession of landraces collected from the farmers (43 from Central Italy), 5 vintage cultivars, 8 accessions of commercially available landraces and two wild outgroups. The landraces were highly diversified as for fruit size, shape and destination. 

From 10 plants per accession, raised with standard cultural practices, more than 20 morpho-physiological descriptors have been evaluated together with overall susceptibility to diseases and physiological disorders. One plant per accession, controlled a posteriori for being conform to the expected typology, were analysed with 10 Simple Sequence Repeats (SSR) markers selected from those described in literature and located in both coding and non coding genomic regions.

Descriptive, univariate as well as multivariate statistics were independently worked out for the morpho-physiological and molecular dataset that, nonetheless, deserve both to be integrated in the future. The first observations on morphological traits detected heterogeneity in some landraces and showed the presence of clear off-types in commercial seed lots of local types, indicating that often genetic purity is not a property of both the material conserved by the farmers and that produced by seed companies.

At the molecular level, 10 SSR loci produced in total 48 alleles, whit the number of alleles per locus ranging from 2 to 12. Among SSR loci, the level of genetic diversity measured as expected eterozygosity (He) varied between He=0.07 and He=0.82 with an average value of He=0.40. Observed heterozygosity (Ho) was Ho=0 for four loci while for the other six it ranged between Ho=0.02 and Ho=0.17 with an average value of Ho=0.03. These data indicate a large among locus variation for the level of genetic diversity. This result can be related to the different genomic regions considered. In particular the different loci may be differentially influenced by selection or present a different mutation rate.

Based on SSR data, all the individual analysed were found to be different genotypes with the exception of the “Scatolone di Bolsena” accessions and of two “A pera” accessions. Multivariate analyses generally grouped the landraces into clusters based on fruit characters giving interesting insights as, for instance, the relationship between the “Scatolone di Bolsena”, the “Pantano romanesco” and the “Costoluto fiorentino” types and the cv. Marmande or that between the “Ciliegino” types and the wild taxon L. esculentum var. cerasiforme.

“This research is supported by MIUR in the framework of the project SCRIGNO (Sviluppo e caratterizzazione delle risorse genetiche nativein ortofrutticoltura)”

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.47

MOLECULAR, BIOCHEMICAL AND AGRONOMICAL CHARACTERISATION OF TWENTY POPULATIONS OF ‘PEPERONE DI SENISE’ LANDRACE (CAPSICUM ANNUUM L.)

N. BASER*, G. SONNANTE **, G. LAGHETTI**, G. SARLI**, S. MOLINARI***, G. FANIZZA*

*) Dipartimento di Biologia e Chimica Agroforestale ed Ambientale; Sez. di Genetica e Miglioramento Genetico, Facoltà di Agraria di Bari
**) Istituto di Genetica Vegetale, C.N.R., Bari
***) Istituto per la Protezione delle Piante Sez. di Bari, C.N.R., Bari

characterization, ‘Peperone di Senise’, Capsicum annuum L., molecular markers, agronomical traits

In recent years, conventional agriculture and mechanisation, with the consequent aggression to the environment due to human activity, have caused a dangerous genetic erosion, that is a restriction of the variation of plant species and varieties. Such variation and the development of new local landraces are important resources of the agriculture in the world. As the increasing genetic erosion leads to the uniformity of the genetic background of cultivated crops, the availability of favourable wild genes is becoming crucial for plant breeding. Therefore, characterisation and evaluation of plant population collections stored at gene banks is useful to search for those genes which have adapted in natural conditions to resist pathogens or produce characters of high quality standards. 

Because of international regulations for restriction of the use of artificial food stuffs, the interest in pepper as a condiment and natural colorant has steadily increased. Peppers are rich in carotenoids, which determine the colour of fruits and in the alkaloid capsaicin, giving the characteristic pungency. These two parameters of quality are genetically determined and influenced by environment. The “Peperone di Senise” pepper is the name of a landrace which has been cropped for many years and adapted very well in the Italian region of Basilicata. This landrace has been appointed as IGP by the European Union. In this paper, the agronomic traits of twenty populations of this landrace are reported along with the quality characters detected by a spectroscopic analysis of their content in colour and capsaicin; moreover, the genetic variability of this material has been tested by using RAPD markers. Agronomic traits were characterised using conventional morphological parameters from the whole plants, flowers, fruits and seeds. Qualitative analysis focussed on colour and pungency of fruits; the total extractable colour was assayed and expressed as ASTA international units per gram of paprika, while capsaicin as µg/g of paprika. DNA was extracted from a pool of leaves for each population and RAPD analysis was performed using 20 arbitrary decamer primers. 

The twenty populations were characterized morphologically in detail in three locations of Basilicata region using 35 descriptors (from ‘Descriptors for Capsicum’ – IPGRI 1995). In general the results show a wide phenotypic homogeneity for the whole material even if some populations revealed distinguishing traits (e.g. pungency, fruit shape, corolla colour, growth habit, etc.). Interaction among different temperature and methods of drying with the capsaicin content (almost absent) and the ASTA value (positively correlated) were tested. In some cases (e.g. ‘Conte’, ‘Gazzaneo’, ‘Forte’ and ‘Amendolara’ populations) a different trend was recorded. These tests were carried out on fruits with and without seeds and peduncles. Concerning the yield, the best populations were ‘Forte’ and ‘MG12904’ with 23.9 and 22 t/ha of marketable fruits, respectively. The higher soluble solids content was reached by the ‘CVM’ population (8.8 %) while the lowest by the ‘CV1’ (5.8%).

In conclusion, thanks to the data from three locations and different years (to be completed in the next two years) these twenty populations will be sorted for yield, yield stability and homogeneity, and fruit quality. A considerable degree of variation was observed with RAPD markers. A UPGMA dendrogram based on Jaccard's similarity index allowed to cluster the populations according to their genetic affinities. Other molecular markers such as AFLP are being tested in this germplasm.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.48

SINGLE NUCLEOTIDE POLYMORPHISM ISOLATION IN CAPSICUM SPP.

A. ACQUADRO*, D. LEE**, E. CHIAPPARINO**, C. COMINO*, E. PORTIS*, P. DONINI**, S. LANTERI*

*) Di.Va.P.R.A. settore Genetica Agraria, Università di Torino
**) NIAB - Molecular Research Group, Cambridge, UK 

SNPs, REF-SSCP, pepper,  polymorphism

Single nucleotide polymorphisms (SNPs) are point mutations in which one nucleotide is substituted for another at a particular locus. SNPs are the most common type of sequence differences between alleles and their polymorphism may be used as genetic marker. SNP markers offer great potential for high genome coverage and high throughput analysis in both MAS and basic studies. The identification of SNPs for targeting quality traits, QTLs and disease resistance genes is already advanced on a number of major crops.

We report on a preliminary study aimed at the detection of SNPs in pepper, by REF-SSCP, in genes coding for well-studied enzymes. Fifteen genes from Capsicum annum were selected from the NCBI database for SNP mining. We analysed, for each gene, one fragment of about 600 bp originating from a region overlapping the intron-exon junctions. Fifteen pairs of PCR-primers were designed and employed for the amplification of such sequences in 27 C. annum accessions and 7 pepper species (C. annum L., C. chinense L., C. frutescens L., C. pubescens L., C. chacoense L., C. baccatum L., C. tovari L.). PCR products were analysed by REF-SSCP for the presence of point mutations in different samples, i.e.: the amplification products were digested with MseI or AluI, then denatured to yield single-stranded DNA fragments and rapidly cooled down to 0oC; the ssDNA fragments were separated on polyacrilamide gels and silver stained.

The polymorphic fragments were purified and directly sequenced in an ABI 3100 sequencer using BigDye™ terminator chemistry. The sequences were aligned with Megalign (DNASTAR) using Clustal algorithm. Putative SNPs were identified and validated by Tetra-primer ARMS-PCR. Primers were designed using the software made available on line http://cedar.genetics.soton.ac.uk/public_html/primer1.html.). 

Fifty-seven SNPs were found, of which 34 were in coding region (cSNPs), however only 18 of them caused amino acid changes. Most of the mutations were at the third position of the codon (47%) while 38% and 18% were at the second and the first position respectively. 

The frequency of base substitutions between 5 of the 7 Capsicum species in study was 1 SNP per 48 bp while 1 SNP per 1360 bp between C. annuum accessions.
The strategy applied proved to be a reliable and a cost effective method for SNP characterisation. The use of REF-SSCP offers some economic advantages as it is possible to identify and sequence only the polymorphic amplicons. Nevertheless a complete study of nucleotide variability in this species would require a large genome coverage.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.49

“AMELINA” F1: A NEW HYBRID OF CLUSTER EGGPLANT WITH SMALL FRUITS

G.L. ROTINO*, P. ALBERTI*, N. ACCIARRI**, E. SABATINI**, S. DEGNO***, S. CAMPOCCIA***, O. DI CARA***

*) Istituto Sperimentale per l’Orticoltura Sezione di Montanaso Lombardo (MiPAF)
**) Istituto Sperimentale per l’Orticoltura Sezione di Monsampolo del Tronto (MiPAF)
***) Hybrid Ka D2  

Solanum melongena L., cluster, hybrid

Eggplant (Solanum melongena L.) is a vegetable crop widely spread in Italy, both in open field and greenhouse. In the year 2002, eggplant was grown on 12365 ha, with a total yield of 358000 MT.

Although different fruit types of eggplant are known by the consumers, up to now only two types of eggplant fruit -oval or long shaped and with high weight- are mainly grown in Italy, despite a high variability in fruit color, shape and weight present within the Solanum melongena gene pool.

The Research Institute for Vegetable Crops have started a research program in order to improve and introduce new typologies of eggplant in Italy.

Inside the whole germplasm collected by the Institute, a private seed company –Hybrid Ka D2-showed a notable interest toward the types with cluster fructification and small fruits . A consequent  joint research agreement has been set up in order to obtain new hybrids of this fruit types, that can be usefully grown in greenhouse.

The first hybrid submitted to registration at the National Register of Varieties is “Amelina F1”. This new cultivar, suitable to both open field and greenhouse conditions, is characterized by 4-7 fruits per cluster with contemporary ripening. Single fruits at harvest are 13-18 cm long, the skin is violet and the calyx is green with violet stripes; exposition to the light increase the intensity of the violet color. Fruit weight ranges from 25 to 40 grams. Plant production depends on cycle length. The commercialization can be made in cluster or packaging single fruits.

“Amelina” shows high performances in winter period of cultivation under greenhouse and good adaptation to treatment with phytohormones or bumblebees to improve the fruit set and growth. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.50

AFLP FINGERPRINTING OF DIPLOID MEXICAN SOLANUM SPECIES AND TETRAPLOID DERIVATIVES 

R. AVERSANO, M. CARTOLANO, M.R. ERCOLANO, L. MONTI, L. FRUSCIANTE, D. CARPUTO

Dept. Soil, Plant and Environmental Sciences, University of Naples "Federico II", Portici, Italy

potato, Phytophthora infestans, somaclonal variation, cluster analysis

To introgress resistance to Phytophthora infestans into the cultivated gene pool, sources of resistance were identified in clones of diploid Mexican species S. bulbocastanum (blb), S. cardiophyllum (cph), and S. brachistotrichum (bst). These species are sexually isolated, thus their chromosome complement was doubled in order to successfully perform crosses with S. tuberosum haploids. Several tetraploid derivatives displayed good level of resistance to Phytophthora infestans. Interestingly, one of them derived from susceptible diploids. By contrast, three susceptible regenerants were yielded by resistant diploid clones. To study the genetic variability we were working with, AFLP analysis was performed on 20 tetraploids (11 from blb, 4 from cph, and 5 from bst), and on the diploid genotypes they derived from. Nine primer-enzyme combinations allowed the detection of polymorphisms of interest between species and within tetraploid genotypes within each species. Some AFLP bands present in diploid clones were not found in their regenerated tetraploid derivatives. By contrast, AFLP bands that were lacking in diploid genotypes were ex-novo detected in their derivatives. These events were probably due to somaclonal variation events due to the in vitro chromosome doubling. The genetic material evaluated was clustered based on the matrix of genetic similarities and a dendrogram was constructed to separated four distinct genetic groups. The implications due to this finding are discussed from a genetic and breeding standpoint.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.51

MOLECULAR GENOTYPING OF SOLANACEAE WILD SPECIES FOR RESISTANCE GENES

M.R. ERCOLANO, S. MELITO, R. LANGELLA, A. BARONE, L. FRUSCIANTE

Dept. Soil, Plant and Environmental Sciences, Via Università 100, 80055 Portici, Italy

PCR-marker, Solanaceae, resistance genes, wild species, genetic diversity

The wild Solanaceae species represent an important source of resistance genes to biotic stresses. Numerous genes for resistance to important pests and diseases have been already identified and characterized in such germoplasm. Moreover Recent advances in plant biology and plant-pathogens interactions have resulted in development of new approaches for gene identification. Molecular analysis of plant resistance gene based on sequence homologies showed that they are organized in large multigene family. The characterization of plant resistance allelic diversity could be an important step for genetic improvement of Solanaceae cultivated species. The aim of the present work was to genotype various accessions of Lycopersicon and Solanum genetic resources for the allelic diversity in four resistance genes. In particular, 13 accession belonging to  8 Solanum species and  15 Lycopersicon hirsutum  were tested accessions  a primer-set related to resistance genes. Specific PCR primers were designed using sequences of 4 resistance genes: Pto (a serine-threonine protein kinase) conferring resistance to isolates of Pseudomonas syringae, I2 (a leucine rich repeat protein) conferring resistance to plant vascular disease caused by Fusarium oxysporum f sp. lycoprersici, Sw-5 conferring resistance against the tomato spotted wilt virus, R1 (nucleotide binding domain, leucine-rich repeat) conferring resistance to Phytophthora infestans.

Amplified fragments obtained with R1specific primers differed for number and size. A total of 15 bands ranging from 200 to 1500 bp were generated. Six tomato accessions amplified a fragment corresponding to the expected size of 1400 bp. For Pto, I2 and Sw-5 the presence of polymorphic site potentially differentiating homologues genes  were recognized thought restriction enzyme analysis of PCR products. A good level of allelic polymorphism was detected. In particular the Pto specific primers in  tomato gave a amplified fragment of 1600 bp that after digestion produced the expected restriction pattern in three accession whereas in potato a fragment of 600 bp was generated. Sequencing and analysis of the PCR fragments is in progress.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.52

SELECTION FOR FROST TOLERANCE AND ACCLIMATION CAPACITY OF POTATO GENOTYPES DERIVED FROM SOLANUM COMMERSONII (+) S.TUBEROSUM SOMATIC HYBRIDS

T. CARDI, F. CONSIGLIO, F. FIORENTINO, P. PALMIERI

CNR-IGV, Institute of Plant Genetics, Res. Div. Portici
cardi@unina.it

potato, somatic hybrids, frost tolerance, Solanum commersonii

Frost tolerance is a desirable trait in potato, especially for off-season growth in autumn and winter. Genetic variability for both frost tolerance and acclimation capacity, however, is limited within the cultivated gene-pool. S. commersonii (cmm), is a useful source of genes for both traits, but is sexually incongruent with S tuberosum (tbr). Self and backcross progenies derived from a  somatic hybrid between the two species were analyzed for agronomic and stress tolerance-related traits. The frost tolerance with and without hardening (LT50AA and LT50NA, respectively) of selected progenies is reported in this study.

The LT50NA of 38 BC1 and one self clones varied from –1.8 to –3.3 °C. In similar experiments, that of cmm was equal to –4.6 °C, whereas that of a pool of tbr varieties ranged from –1.9 to –2.5 °C. About 60% of tested progenies were significantly better than the control variety Spunta that showed an LT50 equal to –2.2 °C. The LT50AA of self and BC1 progenies ranged from –2.0 to –5.3 °C, whereas that of cmm and tbr was –8.8 and –2.1 / -3.6 °C, respectively. About 90% of progenies were significantly more tolerant than Spunta. Best clones showed an LT50AA around –5.0 °C, very close to that shown by somatic hybrids.

BC2 progenies were produced by crossing the best BC1 clones with two tbr varieties. Seedlings  of 34 BC2 families were tested in a cold chamber at –4 / -4.5 °C (without hardening) and at –5 / -5.5 °C (after hardening at 4 °C for two weeks). On the whole, 116 out of about 2000 seedlings (6%) survived the freezing test w/out hardening, whereas 13 out 527 (2.5%) the test with hardening. The frost tolerance of a sample of about sixty plants survived in the cold chamber was checked again by means of the electrolyte leakage test. Eleven BC2 clones with LT50NA < -2.5 °C and 13 with LT50AA < -3.5 °C were selected.

Best BC2 clones will be tested for agronomic performance in off-season growth in autumn-winter.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.53

NEGATIVE ASSISTED SELECTION OF HYBRIDS BETWEEN SOLANUM TUBEROSUM AND ITS INCONGRUENT WILD RELATIVE S. COMMERSONII

M. IOVENE, A. BARONE, S. ALBERINO, A. NUNZIATA, L. FRUSCIANTE, D. CARPUTO 

Dept. of Soil, Plant and Environmental Sciences - University of Naples “Federico II”, Via Università 100, 80055 Portici, Italy

AFLP, aneuploidy, resistance to low temperature, resistance to Erwinia carotovora  

A breeding scheme based on odd ploidy production was developed to introduce useful genes from the wild Solanum commersonii (cmm) into S. tuberosum (tbr) genome. Hybrids from 5x x 4x crosses were characterized for traits of interest and selection was assisted by AFLP analysis. As expected, most of the hybrids were aneuploids, with a trend towards a low aneuploid grade. More than 75% of the hybrids resembled tbr phenotype for all the morphological traits considered (plant habit, corolla shape, eye depth) but for stolon length. Screening for resistant traits deriving from cmm was also carried out. As for freezing resistance in non-acclimated condition, hybrids were similar to the cultivated susceptible parent. By contrast, killing temperatures of cold acclimated genotypes were distributed between the wild and cultivated parental values, and ranged from –6.1°C to –2.6°C. Interestingly, some hybrids displayed an acclimation capacity higher than 3°C. A wide variability was also found for tuber soft rot resistance, and hybrids with high levels of resistance were identified. In spite of the fact that aneuploidy has often been associated to reduction of male and female fertility, most of hybrids were fertile after crosses with tbr, making it possible to produce a viable offspring. No significant correlation were found between aneuplody and fertility/resistance parameters.

Negative selection was assisted by AFLP cmm-specific markers, in order to identify hybrids combining noteworthy traits with a low wild genome content. Previously selected cmm-specific AFLPs were used to monitor the degree of wild genome content still present in each hybrid. The percentage of cmm-specific markers ranged from 65% to 95%, with an average value of 78%. To select hybrids with a desirable combination of traits, an evaluation index (E.I) was estimated for each genotype considering the following traits: stolon length, eye depth, specific gravity of tubers, resistance to tuber soft rot, resistance to frost (in non-acclimated and acclimated condition). Hybrids with a higher E.I. and a lower wild genome content than average for both traits were selected for further breeding efforts.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.54

MOLECULALAR CHARACTERIZATION OF LOCAL POPULATIONS OF RADICCHIO (CICHORIUM INTYBUS L.) GROWN IN VENETO

M. BRAKE, G. BARCACCIA, M. LUCCHIN, P. PARRINI

Dipartimento di Agronomia Ambientale e Produzioni Vegetali, Università di Padova – Agripolis, via Romea 16, 35020 Legnaro, Padova, Italy
paolo.parrini@unipd.it

chicory, molecular markers, genetic similarity, genetic diversity

Red or variegated chicory (Cichorium intybus L.) native to, and very extensively cultivated in North-Eastern Italy, locally called “radicchio”, includes different types which represent valuable high-quality crops. The five major types of radicchio cultivated in the Veneto region were investigated by PCR-derived markers to evaluate the amount and partition of genetic variability in local populations and to set up a molecular reference system for the precise genetic identification of different types. Althought a clear-cut morphological differentiation among the five types does exist, their genetic identification is becoming increasingly important. Materials grown are usually represented by local populations known to possess a high variation and adaptation and maintained by farmers through phenotypical selection according to their own criteria.

The experimental material was repredented by 23 local populations, seven of “Rosso di Chioggia” (CH), eight of late “Rosso di Treviso” (TVT), three of early “Rosso di Treviso” (TVP), two “Rosso di Verona” (VR) and five of “Castelfranco” (CF). A total of 797 individual plant DNAs were assayed using random amplified polymorphic DNA markers (RAPD). Five selected operon decamer primers generated a total of 35 markers, 25 of which were polymorphic (71.4%).  The genetic variation was shown to be much higher within than between populations. As a matter of fact, the total Nei’s genetic diversity (HT) was 0.324, while that calculated for single populations (HS) was, on average, 0.217. The high value of the fixation index (GST = 0.331) was consistent with a DNA polymorphism rate more pronounced within populations: the 67% of the total genetic variability observed can be attributed to within populations differences and around 33% is due to differences among the populations. The low value of gene flow (Nm = 1.013) confirms a little exchange of marker alleles among populations. Some type-specific markers that allow type discrimination were found. Further experiments and statistical analyses are in progress. The set up of a molecular reference system seems to be feasible for the precise identification of the single types of Veneto radicchio and suitable for the evaluation of the extent of natural hybridization there can occur between different types.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.55

MOLECULAR AND PHYSIOLOGICAL CHARACTERISATION OF LACTUCA SATIVA EXPRESSING THE ASPARAGINE SYNTHETASE TYPE A OF E. COLI.

D. GIANNINO, C. NICOLODI, G. TESTONE, E. TRASARTI, L. SANTINI, D. MARIOTTI 

Istituto di Biologia e Biotecnologie Agrarie del Consiglio Nazionale delle Ricerche, Sezione Territoriale di Roma, Via Salaria Km. 29,300, 00016 Monterotondo Scalo (Roma)

nitrogen metabolism, genetic transformation, asparagine synthetase A, lettuce

Plant uptake of nitrogen implies the biochemical reduction of nitrates to ammonium, rapidly incorporated into glutamate (Glu) and glutamine (Gln) by the glutamate and glutamine synthases, respectively. The latter also represents the major scavenger of ammonia deriving from nitrate reduction, amino acid degradation and photorespiration. In plants Gln is the nitrogen donor to aspartic acid (Asp) for the asparagine (Asn) synthesis, catalysed by the asparagine synthetase (AS). The latter is inhibited by light, has a high specificity for glutamine and rarely uses ammonium as substrate. Contrary to plants, in prokaryotes the asparagine synthetase type A (asnA) transfers ammonium directly to aspartic acid to produce apsaragine and it is not regulated by light. The genetic transfer of the asnA gene into lettuce was attempted to enhance nitrogen metabolism and to diminish nitrate content, a serious limit for the product quality of this crop. Three cultivars (Cortina, Clinton and Luxor) were transformed with asnA driven by a 35S-derived promoter (pMAC) by A. tumefaciens Transformation efficiency was 70% and the events of single insertion of transgene were 30%. One out of several Cortina primary transformed lines exhibited altered phenotypes with respect to controls, thus it was selected and selfed to reach stabilised transgene expression and homozygosity. Plants harboring pMAC:asnA exhibited a higher growth speed, anticipation of bolting and flowering with respect to wild type Cortina. In greenhouse winter and summer cycles, transformed genotypes featured 1.3-1.4 fold higher fresh and dry weights than controls, due to higher leaf number and average area. The content of Asn, Asp and Gln of pMAC:asnA plants was 2.6 to 4.6 fold higher than controls, whereas Glu variation was not significant. In vitro experiments showed that pMAC:asnA genotypes were more tolerant to increasing ammonium concentrations than controls. Nitrate content of transformed plants is currently being measured and data will be presented and discussed.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.56

GENETIC DIVERSITY AND POPULATION STRUCTURE OF A PHASEOLUS VULGARIS L. LANDRACE MAINTAINED ON-FARM IN CENTRAL ITALY (LAZIO)

B. TIRANTI, V. NEGRI

Department of Plant Biology and Agro-environmental Biotechnology, Section Genetics and Breeding, University of Perugia, Italy, Borgo XX Giugno 74, 06021 Perugia  

Landrace, Phaseolus vulgaris, SSR, genetic diversity, on-farm conservation 

Landraces are balanced, variable populations, susceptible to change in response to environmental and human selective pressures (Harlan, 1975). They present two different levels of genetic diversity: among populations and within population. Population structure of landraces is mostly unknown (Frankel et al., 1995). Several authors showed that a high degree of diversity is maintained in landraces of autogamus species from comparable ecogeographic sites. 

Landraces are still maintained in central Italy. Some landraces of Phaseolus vulgaris, which have been maintained on farm due to adaptation to particular situations, are nowadays cultivated and sold at very high prices due to their organoleptic characteristics. Among them our attention is focused on the ‘A pisello’ landrace from Colle di Tora (Rieti), which, due to particular social reasons, is running  the risk of extinction.

In order to define managing strategies to preserve this landrace for the future, we studied the genetic structure of ‘A pisello’ populations grown by different farmers and levels of variability among and within populations by using SSR molecular markers. 

The high differentiation found in the metapopulation, can be explained by the high sensibility of SSR, isolation of sub-populations during their cultivation, their small size and limited exchange of seeds between farmers. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.57

NEW CLADES OF R-GENES CANDIDATE SEQUENCES HOMOLOGOUS TO NUCLEOTIDE BINDING SITE DOMAIN IN PHASEOLUS VULGARIS L.

F. DEL BIANCO, B. PARISI, P. RANALLI, A. CARBONI*

Istituto Sperimentale per le Colture Industriali, Bologna (Italy)

common bean, root knot nematodes, plant-pathogen interaction, evolution, NBS-LRR resistance genes

The aim of this work, carried out together an extensive breeding program to introduce nematode resistance in common bean, was to exploit the potentiality of Resistance Gene Analogs studies to understand the evolution of plant-pathogen interaction and to elucidate the distribution of these RGAs in Phaseolus vulgaris L. germplasm.

A large number of sequences (more than 1000) homologous to the Nucleotide Binding Site (NBS) domain of NBS-leucine-rich repeat (LRR) resistance genes have been isolated through PCR amplification with degenerate primers, cloned and subsequently sequenced in 7 different genotypes of Phaseolus vulgaris L.: two different cultivars (BAT 93 and Jalo EEP558), respectively representative of the two geographically distinct gene pools (Mesoamerica and the southern Andes), and five accessions from post-domestication centres of diversity (Messico, Peru, Colombia, etc.), showing different rates of resistance to Meloydogine spp. (root knot nematodes). 

Phylogenetic analysis classified these new sequences into TIR (toll and interleukin-1 receptor) and non-TIR subfamilies, by the presence or absence of a region homologous to the TI Receptor domain at the N-terminus and by specific amino acid motifs within the NBS domain itself. Both the TIR and non-TIR groups show long branch lengths and clearly clustered nodes, reflecting a high level of sequence divergence. 

The comparison of these new sequences with those retrieved in different genebanks of Phaseoleae and related tribes, such as Trifoleae, Vicieae, etc., confirmed that major clades within the NBS-LRR family pre-date evolution of these legumes but introduced also the hypothesis of a secondary development and differentiation of specific Resistance Gene Analogs (RGAs) in Phaseolus vulgaris L.; two previously never identified cluster of RGAs within each subgroup (TIR and non-TIR) were retrieved and here described: a huge cluster near the M non-TIR region and another cluster with “intermediate” characteristics within the TIR subfamily. We proposed to call these cluster respectively M6 and N, according to Zhu (Zhu et al., 2002, MPMI 15(6):529-539). 

The presence of new clusters of highly diverged NBS sequences suggests that multiple ways of sequence dispersion are occurring in common bean, but further studies are necessary to confirm the divergence of the RGA family due to plant-nematode evolution. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.58

GENETIC IDENTITY AND SEED PRODUCTION IN HETEROGENEOUS COMMON BEAN LANDRACES (PHASEOLUS VULGARIS L.) OF “NEW” COMMERCIAL INTEREST

P. MASI, G. LOGOZZO, M.A. DILUCA, G. RAIMONDI, P.L. SPAGNOLETTI ZEULI

Phaseolus vulgaris, landrace, certification, genetic markers

Basilicata region (Southern Italy) is rich of highly variable common bean (Phaseolus vulgaris L.) landraces that are the subject of increasing economic interests as typical food productions. The request of larger scale seed production poses a serious risk of genetic erosion and criteria to monitor and certify the  genetic “identity” in heterogeneous landraces is needed. Two common bean landraces Marrozzo (indeterminate growth habit) and Verdolino (determinate growth habit), mixtures of 39 and 22 accessions, respectively, collected in a large area of Basilicata were grown in two environmentally distinct locations to observe the effect of growing environment on their genetic structure. 19 morpho-agronomical traits (9 qualitative and 10 quantitative) and 16 microsatellites loci were scored. 

On the basis of morpho-agronomical traits, “Marrozzo”, widely distributed, did not show significantly different response to different growing environments while Verdolino, for most traits, did show significant differences.

The 16 pairs of SSRs primers detected 75 alleles in Marrozzo and 58 alleles in Verdolino with a polymorphic degree of 100% in Marrozzo and 81.2% in Verdolino. The heterozigosity average observed value (Ho) is 0.011 in Marrozzo and 0.005 in Verdolino. 7 “unique” alleles were detected in Marrozzo and 4 in Verdolino with frequency higher of 40%. Genetic identity of each common bean landrace can be easily assessed on the basis of the presence and the frequency of unique alleles. To avoid genetic erosion the genetic structure of landraces during seed production needs to be monitored. 

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.59

A NEW HYBRID OF CAULIFLOWER “VERDE DI MACERATA” USING CYTOPLASMIC MALE STERILE: “MAGNIFICO F1”

E. SABATINI*, N. ACCIARRI*, G. VITELLI*, G. CAMPANELLI*, M. GALLERANI**, G.P. CRESCENZI**, R. LEVIEIL** 

*) Istituto Sperimentale per l’Orticoltura Sezione di Monsampolo del Tronto  (MiPAF)
**) Clause-Tezier

Brassica oleracea var, Botrytis L., Verde di Macerata, hybrid, male sterile

Italy is the most important center of cauliflower (Brassica oleracea var, botrytis L. ) diversity. In particular, the curd show different shapes and colors. “Verde di Macerata” ecotype has green curds and two genes control the curd color. The cauliflowers with green pigmentation could be an attractive type as the most common white types. The main breeding objectives of this work were to improve plant and curd uniformity and overall quality, and to extend the harvest period. To accomplish these goals a joint research agreement was set up between Istituto Sperimentale per l’Orticoltura and Clause-Tezier France. In particular, we aimed to reduce bractiness, riciness (i.e: early development of flower buds) and looseness which spoil the curds surface. New lines of ‘Verde di Macerata’ type were obtained by means of recurrent selection of segregant population derived from crosses and back-crosses between ‘Verde di Macerata’ and ‘Tardivo di Fano’, a white type with late maturity. Androgenetic lines, obtained through anther culture, were also used. Selected lines with different and enlarged period of maturity were crossed with cytoplasmic male sterile lines to obtain new hybrids F1.

“Magnifico F1” is the first hybrid obtained in the framework of this research agreement is. It is commercialized by Clause-Tezier. “Magnifico F1” is the first hybrid with a period of commercial maturity more late than the original ecotype and the cultivars available until now. In center Italy, with a transplant on 20th-25th August, the harvest begins at half of January. The maturation is concentrated in 10-15 days. The head of “Magnifico” has high specific weight because of the compactness and the semispherical shape. The plant has a good vigor useful for winter production and long vegetative period. The head is not covered by leaf during increasing, this characteristic is important to have a more intense green color at harvest. “Magnifico” shows a good resistance to over maturity and serve the good characteristics of inflorescence from 400 to 2000 grams. 

From the Joint Research Agreement new hybrids of “Romanesco” and “Tardivo di Fano” types are going to be released.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.60

AFLP and ISSR markers utilized in the assessment of genetic variation in Italian Artichoke

N.A. Rey Muñoz**, M.A. Pagnotta*, M.T. Cardarelli**, M. Tucci***, F. Saccardo**

*) Department of Agrobiology and Agrochemistry, Tuscia University, Via S.C. de Lellis, 01100 Viterbo, Italy
pagnotta@unitus.it
**) Department of Crop Production, Tuscia University, Via S.C. de Lellis, 01100 Viterbo, Italy
***) CNR -Institute of Plant Genetics - section Portici, Via Università 133, 80055 Portici (Na), Italy

AFLP, ISSR, landraces, Cynara cardunculus, germplasm

Thirty genotypes of Artichoke landraces of “Romanesco” type have been collected from each of three farms located in Latina province (south of Rome), and thirty genotype from three farmers located in Salerno province in order to assess the genetic variation existing between and within each population. Two different kind of molecular markers, AFLPs and ISSRs, have been used. Fifty bands have been scored for primer combination MseAC-PstCT, 93 bands for primer combination MseTT-PstCA, and 48 for primer combination MseGC-PstAC, while 44 bands have been scored for primer 841 and 7 bands for primer 856. A total of 191 bands have been considered for AFLPs and 51 for ISSRs markers.

The results underlying the presence of a large amount of variation within population (in this case was considered a population constituted by the plants collected from the same farm), ranging from 73% to 85% of the total variation, in relation with the kind of marker used; the differences between plants from different farm, as a consequence, account for only 27 to 15% of the total variation. A similar distribution of the total variation within and between populations has been found by other authors utilizing RAPDs (Lanteri et al. 2001). The polymorphism, utilizing a polymorphism criteria of 95%, was about 77%. This is on averaged higher than the polymorphism found, utilizing RAPDs markers, by Lanteri et al. (2001) on artichoke from Sardinia and by Sonnante et al. (2002) on 32 artichoke cultivars. It is interesting to note that the polymorphism is the different populations is not consistent if AFLPs markers or ISSRs markers were used. Probably, the two kinds of markers used explore different genome regions with different similarity and/or differences between genotypes. The expected heterozygosity (He) could range from 0 to 0.5 and gives an indication if a polymorphic locus has an about equal presence of both alleles (i.e. He close to 0.5) or has an allele very common and the other very rare (i.e. He close to 0). In present study the He indicates that generally the high polymorphism reviled is determined by locus with a good presence of both alleles, hence the variability present within the farmers is definitively high.

The Nei genetic distances between the studied populations are not very high, since there is a high variation existing within population. But, again, the results are lightly different if different markers are considered in the analysis computation.

The present study demonstrates the existence of great variation within farms which still utilized their own landraces. Hence there is material to be conserved; but, the difficulties to found farm that utilize landraces underlay the necessity to conserve the artichoke germplasm and the risk caused by the utilization of a single clone on a huge number of farmers.

The work has been sponsored by CNR Scrigno project.
The authors would like to thank Linda Mondini for her help during some phases of the analysis.

Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress
Verona, Italy - 24/27 September, 2003
ISBN 88-900622-4-X

Poster Abstract - 2.61

EVALUATION OF THE GENETIC VARIABILITY IN 28 ITALIAN EMMER WHEAT (TRITICUM DICOCCUM SCHUBLER) POPULATIONS BY EST, ISSR AND SSR, MOLECULAR MARKERS 

L. MONDINI, M.A. PAGNOTTA, E. PORCEDDU

Dipartimento di Agrobiologia e Agrochimica, Università della Tuscia, Via S.C. de Lellis, 01100 Viterbo, Italy

Triticum dicoccum, ISSR, SSR, EST, Variability

Twenty-eight accessions of T. dicoccum have been evaluated by 6 ESTs (DuPw23, DuPw38, DuPw4, DuPw124, DuPw167, DuPw254), 6 SSRs (WMS372, WMS328, WMS294, WMS92, WMS95, WMS319) and 6 ISSRs primers (811, 841, 847, 848, 856, 857) to determine Nei (Nei, 1972) genetic distance and polymorphism.

The evaluated accessions shown great differences in terms polymorphism. The genetic distance, performed with molecular data, cluster the accessions in four groups: i) most of the PLV accessions (5, 6, 7, 8, 9, 11) plus Molise; ii) Garfagnana, Molisano and Padre Pio and with a less clear-cut Farvento Italia Centrale, Abruzzo, Potenza, Umbria and Leonessa; iii) a very uniform group including Andreoli, Loretucci, Carissimi, Achilli, Santoni, Testa, Graziosi and Cicchetti; and iv) a far a part group with Mosè, Lucanica PLV10 and PLV12. Among the PLV group the PLV 10 and 12 are the ones with higher quality characters, while the others are very similar except the yield and plant height. The two not hulled accessions (Mosè and PadrePio) derived from crosses with durum wheat are put in different groups having a Nei genetic distance of 0.29. Even if, on average, polymorphism at DNA level is 15%, the analysed accessions are significant different for the evaluated characters.

Work sponsored by MiPA project "Risorse genetiche di organismi utili per il miglioramento di specie di interesse agrario e per una agricoltura sostenibile".