SESSION V - SIGA WORKING GROUP

STRESS 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 - 5.01

GENETIC TRANSFORMATION OF STRAWBERRY BY USING A REPEATED VIRAL GENE AS A MODEL FOR VIRUS RESISTANCE

R. DONNOLI, S. COMES, A. FANIGLIUOLO, A. CRESCENZI, I. gRECO, G. MARTELLI, F. SUNSERI

Dipartimento di Biologia, Difesa e Biotecnologie AgroForestali, Università degli Studi della Basilicata Contrada Macchia Romana, 85100 Potenza 

Fragaria x ananassa, Agrobacterium tumefaciens 

The genetic transformation of the cultivated strawberry (Fragaria x ananassa Duch.) have been yet reported (Orlando et al. 1997, Plant Cell Rep. 16: 272-276; Barcelo et al. 1998, Plant Cell Tiss. Organ Cult. 54: 29-36; de Mesa et al. 2000, Aust. J. Plant Physiol. 27: 1093-1100; Ricardo et al. 2003, HortSci. 38: 277-280). Since different transformation strategies have been developed, the techniques are highly genotype-dependent and the transformation frequencies obtained are frequently below 5%. As recently reported by de Mesa et al. (2000) combined techniques (wounding tissue before Agrobacterium infection, by means of particle gun) could improve the transformation efficiency.

On the other hand there are different viruses infecting Fragaria species. Strawberry mottle virus (SMoV) is the most widespread virus worldwide. In single infections, yield losses of up to a 30% have been reported. In mixed infections with, for example, Strawberry crinkle virus SCV), Strawberry vein banding virus (SVBV) and/or Strawberry mild yellow edge virus (SMYEV), the losses can be even higher. Historically, SMoV was often considered to be a mild strain of SCV, and it was not until the mid-1950s that SMoV symptoms were generally accepted as being caused by an aetiologically distinct virus. All the Fragaria species are susceptible to SMoV, though many are symptomless hosts that eventually have reduced vigour and yield depending on the severity of the strain (Mellor & Krczal, 1987, In: Virus Diseases of Small Fruits, USDA Agricultural Handbook 631, pp. 10-16).

Three strawberry cultivars (Clea, Irvine, Paros) useful for South Italy conditions and four accessions of Fragaria vesca, coming from Southern Italy areas, were utilized for genetic transformation by using Agrobacterium-mediated techniques. Four different MS-based media were tested in order to obtain callus proliferation and shoot regeneration. Leaf disks were also co-cultivated with a LBA 4404 Agrobacterium tumefaciens containing a plasmid with the selectable marker gene NPTII, a vital marker like the Green Fluorescent Protein (GFP, Chalfie et al. 1994, Science 263: 802-805) and a repeated viral gene. The infection was performed in a vacuum chamber for 20 minutes.

High percentage (12%) of organogenesis and plant regeneration after Agrobacterium cocultivation were obtained in strawberry (cv “Paros”) by using a MS medium supplemented with 5 µM IBA and 5 µM BAP and the plasmid harbouring the CP of TSWV. A sufficient number of genetic transformation events  were obtained either in strawberry (6%) or in Fragaria vesca (2%) by using the plasmid harbouring the GFP marker. Rarely, genetic transformation events were obtained also by particle bombardment, than the combined transformation technique (de Mesa et al. 2000) would to be tested.

Nevertheless, the genetic transformation protocol for the strawberry genotypes utilized is now optimized, while several experiments will be carry out for the diploid species. 

In the present study, a highly efficient and reliable protocol for plant regeneration and interesting results of Agrobacterium-mediated transformation in strawberry have been reported. The utilization of a repeated viral gene putative responsible for gene silencing was considered for modelling the analysis of virus infection in strawberry, since the Fragaria species was reported as sympotmless hosts of several viruses.

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 - 5.02

ISOLATION OF SINGLE-CHAIN Fv ANTIBODY FRAGMENTS AGAINST POTATO VIRUS X (PVX) FROM PHAGE DISPLAY LIBRARIES

M. DI CARLI, M.E. VILLANI, C. LICO, E. BENVENUTO

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

PVX, scFv, phage display,molecular repertoires

In phage-display libraries, peptides or proteins are expressed as a fusion with the coat protein of the phage allowing a rapid selection of molecules with desired properties by a series of recursive cycles of selection. One of the most important application of phage-display has been the easy isolation of recombinant antibodies with desired specificity; for many application, one of the most versatile antibody format is the single-chain antibody (scFv) constituted by the VH and VL of the two variable domain fragments (scFv). This antibody format is a valuable tool for immunotherapy in plants (Benvenuto & Tavladoraki 1995) or diagnosis.

We isolated scFv antibodies specific to potato virus X (PVX) using two different phage display libraries. One, composed of intrinsically stable scFvs with a repertoire of approximately 5x 107 different clones (the "F8 library" ) (Desiderio et al. 2001), and the other a human germline derived library composed of 6x108 different clones (the "ETH2 library") (Pini et al. 1998).

After four rounds of selection and enrichment ("biopanning") on the immobilised virus, a panel of different phages clones were obtained from each library and individual clones were selected and tested for binding to the virus by enzyme-linked immunosorbent assay (ELISA). Approximately 65% of the clones from the "F8 library" were positive for the PVX while from the "ETH2 library".only 10% of the clones were positive. The clones that showed the highest binding to the virus were chosen for sequencing and further biochemical characterization. These scFvs fragments could represent a valuable tool for inexpensive immunodiagnosis, and once expressed functionally in transgenic plants can also be used for immunomodulation of PVX infection. 

References
Benvenuto & Tavladoraki (1995) Trends Microbiol.3(7):272-5.
Desiderio et al. (2001) J Mol Biol. 13;310(3):603-15.
Pini et al. (1998) J Biol Chem 273(34):21769-76.

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 - 5.03

MODULATION OF PLASMA MEMBRANE H+-ATPASE ACTIVITY IN TOMATO PLANTS INFECTED WITH PSEUDOMONAS SYRINGAE PATHOVAR TOMATO

L. CAMONI, A. GARUFI, P. ADUCCI

Dipartimento di Biologia, Università di Roma “Tor Vergata”, via della Ricerca Scientifica, 00133 Roma

H+-ATPase, Pseudomonas syringae pv. tomato, 14-3-3 proteins, Lycopersicon esculentum, plant-pathogen interactions.

The plasma membrane H+-ATPase is the major ion pump in the plasma membrane of higher plants (Palmgren, 1998). This enzyme generates an electrochemical gradient which provides the driving force for a number of key physiological processes such as stomata opening, phloem loading and root ion uptake.

The important functions of the plasma membrane H+-ATPase suggest that the activity of the enzyme is tightly controlled. Recently, it has been demonstrated that the proton pump is activated by phosphorylation dependent binding of 14-3-3 proteins to the C-terminal domain of the enzyme (Aducci et al, 2002).

Different clues indicate that the H+-ATPase is also involved in the mechanisms of defence during pathogen infection. In fact,the proton pump activator fusicoccin (FC) induces the expression of pathogenesis related (PR) genes and the accumulation of salicylic acid in tomato plants (Schaller and Oecking, 1999; Roberts and Bowles; 1999); furthermore, it has been demonstrated that proton extrusion is an essential signalling component in the HR of epidermal single cells in the barley-powdery mildew interaction (Zhou et al., 2000).

Given the potential role of the H+-ATPase in plant-pathogen interactions, we tested the H+-ATPase activity in different tomato (Lycopersicon esculentum ) ecotypes (San Marzano, Corbarino, Sorrento, Vesuviano) infected with Pseudomonas syringae pv. tomato.

Interestingly, the H+-ATPase from infected plants is purified in activated form. Western blotting experiments demonstrated that higher levels of 14-3-3 proteins are associated to the plasma membrane fraction of infected plants; furthermore, the H+-ATPase purified from these plants displays an higher affinity for 14-3-3 protein in overlay assay.

The significance of H+-ATPase activation in the interaction tomato-Pseudomonas syringae pv. tomato is currently under investigation.

REFERENCES
Palmgren M.G. (1998). Proton gradients and plant growth: role of the plasma membrane H+-ATPase. Advances in Botanical Research 28, 1-70.
Aducci P., Camoni L., Marra M., Visconti S. (2002) From cytosol to organelles: 14-3-3 proteins as multifunctional regulators of plant cell. IUBMB Life 53, 49-55.
Scaller, A., Oecking, C. (1999) Modulation of plasma membrane H+-ATPase activity differentially activates wound and pathogen defense responses in tomato plants. Plant Cell.11:263-72. 
Roberts MR., Bowles DJ. (1999) Fusicoccin, 14-3-3 proteins, and defense responses in tomato plants. Plant Physiology. 119, 1243-50.
Zhou F, Andersen CH, Burhenne K, Fischer PH, Collinge DB, Thordal-Christensen H. (2000) Proton extrusion is an essential signalling component in the HR of epidermal single cells in the barley-powdery mildew interaction. Plant Journal 23, 245-54.

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

Poster Abstract - 5.04

PROJECT EURICE: ANTIFUNGAL GENES IN RICE AS A STRATEGY FOR CROP PROTECTION – THIRD YEAR ACTIVITIES

E. LUPOTTO - (Coordinator) on behalf of the EURICE (QLK5-CT1999-1484) Consortium

Istituto Sperimentale per la Cerealicoltura, via Stezzano 24, 24126 Bergamo (Italy)

biotechnology, crop protection, fungal pathogens, defence genes

The Project EURICE aims to improve and evaluate the use of genes involved in defence against pathogenic fungi in the control of rice blast (Magnaporthe grisea) and other fungal diseases, as major pests for rice in Europe. This approach aims to create new rice genotypes enhanced for resistance and addresses to the obtention of modified cultivars protected against massive attacks of the pathogens, as well as to an evaluation of the genetically modified genotypes in the environment. Three genes of different origin, shown to be implicated in the process of fungal infection, were chosen as potential source of resistance because of their different characteristics and modality of action: the maize gene b32, the fungal gene AFP, and the insect gene cecA. They were initially cloned under the strong monocot promoter ubi1 of maize and introduced into chosen European rice varieties (Oryza sativa L. ssp. japonica). The rice genotypes chosen are representative of the main rice cultivating regions in Europe: Italy, Spain, Greece and France. First events of transformation were subjected to molecular and genetic analyses; expression of the transgenes in leaf tissues at various stages of plant development was confirmed, and seed amplified for the first field trial, which has been carried out in Italy, summer season 2002, and evaluated. Meantime, the most susceptile old Italian variety Maratelli was transformed with single and combination of genes in order to assess the “gene pyramiding” strategy. To date, in vitro assays and pathogenicity tests on primary regenerants and T1 plants confirmed protection against leaf blast for at least two of the three genes under study, being the gene afp the most promising one. During the second year, a potential inducible promoter was also confirmed to be suitable for the project’s purposes: the maize ZmPR4 promoter. Expression vectors were constructed with the antifungal genes driven by p-ZmPR4 and plants are being raised. The conventional field trials carried out in Greece within the project, established an efficient protocol for the evaluation of leaf and neck blast disease in the field, by means of the Honey Comb design, and pathogenicity assays in greehouse conditions supported by confocal microscopy analyses allowed to define parameters for a detailed study of the plant/pathogen interaction. A simple and reproducible Detached Leaf Test (DLT) was sut up, as a tool for testing blast development on young leaves, and proved useful in screening trangenic plants, showing strong inhibition in the blast disease symptoms of transgenic plants. Objectives and detailed results of the third year activities are reported in the poster. The theme of the project is related to project SAFEMAIZE (ICA4-CT2000-30033) on maize, also funded by European Commission within the 5th Framework Programme.

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

Poster Abstract - 5.05

IS S-NITROSYLATION A SIGNALING MECHANISM IN PLANTS?

M.C. ROMERO-PUERTAS*, M. PERAZZOLLI**,*, M. DE STEFANO*, M. DELLEDONNE*

*) Università degli Studi di Verona, Dipartimento Scientifico e Tecnologico, Strada Le Grazie 15, 37134 Verona
**) Istituto di Botanica e Genetica Vegetale, Università Cattolica S.C., Via Emilia Parmense 84, 29100 Piacenza

S-nitrosylation, nitric oxide, signaling

Recent studies point out the potential role of NO as a signal in plants (1). Moreover, NO acts as a key signal in plant resistance to incompatible pathogens by triggering resistance-associated cell death (2).

During plant-pathogen interactions, NO activates the expression of several defence genes (i.e. pathogenesis-related genes, phenylalanine ammonialyase, chalcone synthase). In this context, gene induction is achieved through a cGMP-dependent signaling cascade activated by NO (3). A direct action of NO was proposed to regulate the accumulation of reactive oxygen species (ROS) during plant-pathogen interactions by modulating antioxidant enzyme activities (4).

Recent evidence indicates that NO in animal tissues regulates this diverse biologic processes by directly modifying proteins. NO and related species can oxidize, nitrate or nitrosylate proteins (5). Nitrosylation refers to the binding of a NO group to a transition metal or cysteine residue, is a reversible modification and plays a central role in NO-mediated signalling (5). Accumulating data suggest that many proteins are nitrosylated by NO (for over 100 representative examples are available on line at http://www.cell.com/cgi/content/full/106/6/675/DC1) and that in fact nitrosylation may be a ubiquitous posttranslational modification regulating protein function. Indeed, nitrosylation shares many features in common with phosphorylation, the prototypic posttranslational modification involved in signal transduction regulation. Both modifications are reversible and specific, allowing cells to flexibly and precisely modify protein function in response to environmental signals.

The existence of protein nitrosylation in plants has been studied. Crude extracts from Arabidopsis leaves were subjected to analysis of their nitrosylation patterns by performing SDS-PAGE and Western blotting using Nitro-Glo kit (Perkin-Elmer). Numerous nitrosylated proteins were found by this method in plant tissue. Further studies will be focused on changes in the nitrosylation pattern under different biotic and abiotic stress situations, in order to see whether or not protein nitrosylation is a signal transduction mechanism during plant-pathogen interaction.

1. Wendehenne et al., 2001
2. Delledonne et al., 1998
3. Durner et al., 1998
4. Clark et al., 2000
5. Stamler et al., 2001

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

Poster Abstract - 5.06

PHENOTYPIC AND MOLECULAR CHARACTERIZATION OF ARABIDOPSIS DOUBLE MUTANT nia1, nia2 IN RESPONSE TO PATHOGEN ATTACK 

F. ZANINOTTO, M.C. ROMERO-PUERTAS, M. DE STEFANO, M. DELLEDONNE 

Università degli Studi di Verona. Dipartimento Scientifico e Tecnologico. Strada Le Grazie 15, 37134 Verona

nitrate reductase, nitric oxide, hypersensitive response

Nitric oxide (NO) is an inorganic free radical that act as a signaling molecule with multiple biological functions in vertebrates and in higher plants. NO acts as a key signal in plants resistant to incompatible pathogens by triggering resistance-associated cell death. Two enzymatic sources of NO in plants has been reported, NO synthase (NOS) and nitrate reductase (NR). NR is a central enzyme of nitrogen assimilation catalyzing the transfer of two electrons from NAD(P)H to nitrate to produce nitrite. It may also catalyze a one electron transfer from NAD(P)H to O2 to produce superoxide anion and from NAD(P)H to nitrite to produce NO. 

It has been shown that NR double mutants nia1,nia2 have greatly diminished NR activity and display very poor growth on media with nitrate as the only source of nitrogen.

Arabidopsis plants, both wild type and double mutants nia1, nia2, were grown in a grown chamber in short day conditions for 6-8 weeks. Ammonium sulfate 1 mM was added to mutants as source of nitrate. The response of double mutants against virulent and avirulent pathogen has been studied and compared to that of wild type. DAB and trypan blue staining have been used to measure oxidative burst and cell death, respectively. No difference were found in terms of hypersensitive response (HR) to avirulent pathogen between mutants and wild type plants. Moreover, defense-related genes induction after infection has been studied (gst, pal, pr1). From the analysis of the data obtained results, NR does not appear to be a necessary component of the HR.

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

Poster Abstract - 5.07

MOLECULAR IDENTIFICATION OF MARKERS LINKED TO POWDERY MILDEW RESISTANCE GENES

A.V. CARLUCCIO, A. CENCI, A. BLANCO

Dipartimento di Biologia e Chimica Agro-Forestale e Ambientale, Università degli Studi di Bari, Via G. Amendola 165/A, 70126 Bari, Italy

wheat, powdery mildew, AFLP, mapping

Wheat is the most widely grown and consumed food crop in many parts of the world. However, powdery mildew, caused by the pathogen Blumeria graminis f.sp. tritici, is a destructive foliar disease in regions with a maritime climate. Triticum turgidum var. dicoccoides accession MG29896 is particularly interesting for high seed protein content and resistance to powdery mildew. This accession was crossed to susceptible durum wheat cultivar Latino and a set of backcross inbred lines (BILs) was produced. These lines were essayed for powdery mildew resistance in field condition and a BC5F5  line (5BIL29) showing complete resistance on field conditions. This line was crossed to Latino and the segregation of 400 F2 plants showed resistance was inherited as single and dominant locus. 120 F3 progenies were essayed in field condition to confirm F2 phenotype and to distinguish homozygous resistant and segregant progenies. Pools were produced using DNA from 8 homozygous resistant and from 8 homozygous susceptible F3 progenies and bulked segregant analysis was performed by using SSR and AFLP.

Two hundred microsatellites were assayed between MG29896 and cultivar Latino and about half of them resulted polymorphic. Unfortunately, no one showed polymorphism between 5BIL29 and Latino.

In order to relieve polymorphism, the AFLP technique was used. Genomic DNA was digested with the restriction endonucleases PstI and MseI, and double-stranded adapters were ligated to the ends of the restriction fragments. Pre-amplification was performed using primers specific for adapters including one selective nucleotide, followed by selective amplification using three selective bases. 62 primer combinations were essayed and more than 6000 bands were amplified. About 130 polymorphism were observed between Latino and 5BIL29 whose 16 between susceptible and resistance bulks. Three AFLP markers resulted linked to the resistance gene and were mapped.

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

Poster Abstract - 5.08

FURTHER EVIDENCES FOR THE LINKAGE ASSOCIATION BETWEEN THE OLl-2 GENE OF RESISTANCE TO POWDERY MILDEW AND PCR-BASED MARKERS IN TOMATO

L. RICCIARDI, C. DE GIOVANNI, C. LOTTI 

Department of Agroforestry, Environmental Biology and Chemistry, Section of Genetics and Plant Breeding, University of Bari, Via Amendola 165/A, 70126 Bari, Italy
*Faculty of Agriculture, University of Foggia, via Napoli 26, 71100 Foggia, Italy

Lycopersicum esculentum powdery mildew, molecular markers, marker-assisted selection

Tomato powdery mildew (Oidium lycopersicum) is a recently reported plant disease which has a world-wide spread. The establishment of resistant cultivars appears to be a sustainable way to control the disease and to decrease the requirement for artificial anti-fungal treatments.

A source of genetic resistance due to a recessive gene (ol-2) was found in an accession of Lycopersicon esculentum var. cerasiforme. Starting from this genetic material, in previous works we reported results both on the use of bulked segregant analysis (BSA) applied to an F2 population segregating for the ol-2 gene, derived from the pair-cross between a powdery mildew resistant line of Lycopersicon esculentum var. cerasiforme and on the susceptible cultivar Super Marmande, and the identification and localization in the tomato map of a single RAPD marker (designated as OPU31500) detected in the susceptible bulk. In this work we show the results on further identification of seven AFLP markers linked to the Ol-2 locus and on the conversion of the OPU31500 and the AFLP in co-dominant CAPS markers. Furthermore, by means of a linkage analysis performed in the F2 population, we report the estimation of the distance between those markers and the ol-2 gene. 

The results obtained appear to be useful to develop marker-assisted selection aimed to set up improved lines of tomato resistant to powdery mildew.

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

Poster Abstract - 5.09

IN VIVO AND IN VITRO BIOASSAYS FOR TESTING RESISTANCE TO FUSARIUM SSP. IN WHEAT ENGINEERED WITH THE MAIZE RIP b-32

C. BALCONI*, C. LANZANOVA*, E. BALDONI*, E. CONTI*, M. CATTANEO**, E. LUPOTTO*

*) Istituto Sperimentale per la Cerealicoltura, Sezione di Bergamo
**) Istituto Sperimentale per la Cerealicoltura, Sezione di S. Angelo Lodigiano

Triticum aestivum, Fusarium, Ribosome Inactivating Protein b-32

Fusarium ssp. are widespread pathogens on cereals causing root, stem, and ear rot diseases in both temperate and semitropical areas. A maize gene, encoding a cytosolic albumin (b-32) with RIP (Ribosome Inactivating Protein) homology and biological activity, showing a potential anti-fungal role, was constitutively expressed under CaMV 35S promoter into Triticum aestivum cv. Veery, and six homozygous transgenic lines were used as experimental material. 

Aim of our research is to develop a rapid and reliable screening method to evaluate the response of the transgenic wheat and other wheat genotypes upon artificial Fusarium ssp. inoculation. For this purpose, in vivo bioassays were developed to follow the progression of F. culmorum infection in inoculated wheat germinating seeds and in the subsequent developing seedlings. 

F. culmorum  was grown on PDA plates at 26°C until the mycelium covered the surface of the plate. To set up the optimal conditions for the bioassay, sterile seeds of the non transgenic cv. Veery, were inoculated with a conidial suspension of the fungus by adding 125, 1250 or 12500 spores/seed. Inoculated and control seeds were allowed to continue germination for 7 days. Evaluations of the response in the various genetic materials were performed according to the following parameters: I) percentage of wheat seeds with visible fungus colonization; II) “infection score” related to the seed surface infection extent; III) fungus colony diameter growing around infected seeds; and IV) mycelium colour intensity score. Progression of the infection on the developing vegetative tissues (radicle, coleoptyle) has also been recorded. A concentration of 125 spores/seed was useful to discriminate the response in the tested genotypes. The transgenic lines, expressing b-32 at various levels, showed differentiated and in some cases increased resistance to the Fusarium infection. 

In vitro bioassays were developed to test the inhibitory activity of the maize endosperm RIP and of a commercially available plant RIP (Saporin, from Saponaria officinalis seeds), in controlling F. culmorum growth. For this purpose, known amounts of the RIP extracts were spread on agar medium and fungus radial growth was measured over time. Results indicated that a Saporin concentration of 50 µg/ml in the medium induced a significant fungus growth inhibition (≈ 40%). Current work is in progress in order to evaluate the effect of other plant commercial RIPs and to compare with purified maize b-32 on Fusarium ssp. growth, and its general effect on fungus development.

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

Poster Abstract - 5.10

In vitro anti-fungal activity of the wheat puroindoline proteins

C. Balconi*,**, N. Berardo*, J.E. Sherwood**, M.Motto*

*) Istituto Sperimentale per la Cerealicoltura, Sezione di Bergamo
**) Dept. Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT, USA

puroindolines, anti-fungal activity, in vitro, wheat

The puroindolines (PIN) are wheat endosperm proteins believed to be involved in wheat grain texture, determining grain softness. These proteins have also been shown to have in vitro anti-microbial properties and in vivo activity in transgenic rice plants that constitutively express the puroindoline genes. Because the PIN proteins normally are in wheat endosperm and thus are routinely ingested by humans and animals alike, their use in transgenic crops may be less objectionable than gene products normally not present in plants or in the edible portion of plants.

 The goal of our research has been to determine mode and range of action for the anti-fungal activity of the puroindolines.  The first step was to set up bioassays to test the activity of seed purified PIN proteins against different fungi. Bioassays were developed comparing the anti-fungal activities from a non-PIN-containing control (Langdon Durum wheat, -PIN) and a PIN-containing variety (Langdon 5D(5B), created by substituting chromosome 5B of Langdon with chromosome 5D from Chinese Spring, which contains pin genes, +PIN). We used different approaches to test the sensitivity of different fungi to PIN proteins. The bioassay for yeast-like fungi like Ustilago hordei, Saccharomyces cerevisiae,Candida albicans and Crytococcus neoformans was based on growth in broth culture and quantified by microscopic cell count. All fungi tested were sensitive to PIN, although U. hordei was the most sensitive. High concentrations of PIN extract were fungicidal against U. hordei and fungistatic against the other fungi. Cell morphology was distorted, indicating the PIN was affecting cell membranes or walls. The effect of PIN extract on filamentous fungi was determined using Magnaporthe grisea and Fusarium culmorum. Known amounts of the +PIN and –PIN extracts were spread  on agar medium and the plates were inoculated with a small plug of the test organism. Radial growth was measured over time. Growth of both fungi was inhibited by the +PIN extract. Research is in progress to amplify the knowledge about the range of fungi sensitive to puroindolines and to determine the mechanism for anti-fungal activity of these proteins.

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

Poster Abstract - 5.11

IN VIVO AND IN VITRO BIOASSAYS FOR TESTING RESISTANCE TO FUSARIUM VERTICILLIOIDES AND FOR CONTROLLING FUNGAL GROWTH WITH THE MAIZE ENDOSPERM RIP b-32

C. BALCONI, E. BALDONI, C. LANZANOVA, E. CONTI, A. MAZZOLENI, M. MOTTO, E. LUPOTTO

Istituto Sperimentale per la Cerealicoltura, Sezione di Bergamo 

Zea mays, Fusarium, Ribosome Inactivating Protein b-32

Numerous Fusarium species are widespread pathogens on cereals; they can infect small grain cereals (soft and durum wheat, barley, oats, rice, triticale, sorghum, millet) and maize (mostly F. verticillioides), causing root, stem, and ear rot diseases in both temperate and semitropical areas, including all European cereal growing areas. Some Fusarium strains are also capable of producing mycotoxins which can be formed in infected plants before harvesting, or in stored grains. The occurrence of mycotoxins in cereal grains is a great concern worldwide, because their presence in feeds and foods is often associated with chronic or acute mycotoxicoses in livestock and also in humans. With this respect it is important the development of improved maize genotypes with increased resistance to F. verticillioides  using traditional breeding as well as biotechnology.  

The aim of our research is to develop a rapid and reliable screening method to evaluate maize genotypes for resistance to the infection of this pathogen. For this purpose in vivo bioassays were developed to follow the progression of F. verticillioides infection in inoculated maize germinating seeds and in the subsequent developing seedlings. 

Maize inbred W64A and its isogenic mutant opaque-2, and inbred lines released by the Maize Station of Bergamo were used as the experimental material in order to verify if the indications obtained by the in vivo bioassay for the different genotypes, could be positively correlated to the evaluations of resistance to F. verticillioides in  field experiments. F. verticillioides was grown on PDA plates at 26°C until the mycelium covered the surface of the plate. To set up the optimal conditions for the bioassay, sterile seeds were inoculated with a conidial suspension of the fungus by adding 125, 1250 or 12500 spores/seed. Inoculated and control seeds were allowed to continue germination for 7 days. For each genotype, records of the percent of maize seeds with visible fungus colonization and an “infection score”, related to the seed surface infection extent, were registered. The progression of the infection on the developing vegetative tissues (radicle, coleoptile) has been also recorded. Results indicated that the concentration of 125 spores/seed, and the second day after inoculation as timing for the infection evaluation, are the optimal conditions for the bioassay. Under these bioassay-conditions, the mutant opaque-2, lacking the endosperm Ribosome Inactivating Protein (RIP) b-32, appeared more susceptible to F. verticillioides infection both at level of germinating seeds and developing seedlings. The association of increased F. verticillioides susceptibility with RIP deficiency in opaque-2 mutant, confirmed previously reported indications about the role of b-32 in defence mechanisms against fungal pathogens. In this context, in vitro bioassays were developed to test the inhibitory activity of the maize endosperm RIP and of a commercially available plant RIP (Saporin, from Saponaria officinalis seeds), in controlling F. verticillioides growth. For this purpose, known amounts of the RIP extracts were spread on agar medium and fungus radial growth was measured over time. Preliminary data indicated that a minimal Saporin concentration of 30-40 µg/ml in the medium was required for significant fungus growth inhibition. Current work is in progress in order to evaluate the effect of purified maize b-32 on Fusarium verticillioides growth, and its general effect on fungus development.

The work is developed within the framework of the Eu-funded project SAFEMAIZE (ICA4-CT2000-30033, Coordinator: Prof. Dave Berger, Univerity of Pretoria, SA) in FP5.

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

Poster Abstract - 5.12

INDIVIDUATION OF CDNA-AFLP MARKERS IN APPLE PROLIFERATION PHYTOPLASMA RESISTANT APPLE ROOTSTOCKS

M. MOSER*, W. JARAUSCH**, M. KOMJANC*, E. VINDIMIAN*, E. SEEMUELLER***, R. VELASCO*

*) Istituto Agrario S.Michele a/A Via E.Mach, 1 38010 S.Michele a/A
**) Centrum Grüne Gentechnik (CGG) Neustadt an der Weinstrasse, Germany
***) Biologische Bundesanstalt für Land- und Forstwirtschaft (BBA) Dossenheim, Germany

apple proliferation, cDNA-AFLP, differential screening

Apple proliferation (AP) disease is associated with phytoplasmas, phloem – restricted bacteria of the class Mollicutes, which are naturally transmitted by leafhopper or psyllid vectors but which can also be spread by man through grafting or vegetatively propagated AP-infected material.

Witches brooms, enlarged stipules and small sized fruits with incomplete coloration are the most typical symptoms of AP. Their gravity depends on the infected varieties. 

Unfortunately, phytoplasmas cannot be cultured on artificial medium and can only be maintained in the infected plant. The introduction of methods based on DNA research could greatly improve the studies on phytoplasma-plant interaction. 

In the past 15 years resistant genotypes of apple rootstocks were selected and re-crossed with commercial genotypes in Germany. These rootstock selections were demonstrated to be field resistant to AP disease and experimentally inoculated plants showed a high rate of recovery. They are to vigourous to be of commercial interest but they are a possible starting material for  new analyses to understand the mechanism of the disease and for the marker assisted selection of new AP phytoplasma resistant rootstocks.

The following genotypes have been used in order to characterize the resistance phenomenon:
M9: Malus domestica rootstock, susceptible genotype
4551(M. domestica cv. Laxton Superb x M. sieboldii): field resistant rootstock selection, the resistance phenotype is inherited by M. sieboldii
H0909 (4551 x M9): backcross of 4551 with susceptible M. domestica genotype, still field resistant.

The objectives of this work are:
•	Selection of cDNA-AFLP markers suitable for a marker assisted selection in the crossing of resistant rootstocks and commercial non-vigourous rootstocks
•	Investigation of the differential expression of the plant genes during the infection by the AP phytoplasma 
•	Identification of possible mechanisms involved in the resistance or tolerance to AP phytoplasma.

The two techniques used are cDNA-AFLP and Differential Screening.

Several bands have been isolated by cDNA-AFLP which are present in both the resistant genotypes but not in the susceptible genotype (M9). Our intention is further to identify a pool of resistance related genes by differential screening using the following subtractive cDNA libraries H0909-D4551; D4551-M9; H0909-M9.

Results obtained from differential screening and cDNA-AFLP will be discussed.

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

Poster Abstract - 5.13

HCRVF2 GENE, CLONED FROM THE VF REGION, IS ABLE TO CONFER RESISTANCE TO SCAB (VENTURIA INAEQUALIS)  IN GALA APPLE TRANSFORMED PLANTS

E. BELFANTI*, E. SILFVERBERG-DILWORTH**, M. BARBIERI*, S. TARTARINI*, A. PATOCCHI**, J. ZHU*, ***, B.A. VINATZER*,****, L. GIANFRANCESCHI**,*****, C. GESSLER**, S. SANSAVINI*

*) Dipartimento di Colture Arboree (DCA), University of Bologna, Italy
**) Plant Pathology, Institute of Plant Science, Swiss Federal Institute of Technology, ETH Zurich, Switzerland
***) Current address: Dept. of Horticulture, Laiyang Agricultural University, Laiyang, Shadong, P. R. China
****) Current address: Dept. of Molecular Genetics and Cell Biology, University of Chicago, USA
*****) Current address: Dept. of Biomolecular Sciences and Biotechnology, University of Milan, Italy

Venturia inaequalis, Malus x domestica, Vf-resistance gene cloning, genetic transformation, GM-apples

Malus floribunda 821 carring the Vf gene has been used in breeding programs for over fifty years as resistance source to apple scab. Cultivars derived from this wild species showed to be resistant to the infection of Venturia inaequalis but at a lower level than M. floribunda 821 and this is probably due to the loss of some minor genes during the gene introgression. The characterization of the Vf gene started at Bologna’s DCA and Zurich’s ETH and led to identify its position inside a region of 350kb on chromosome 1. Three similar expressed sequences, named HcrVf1, HrVf2 and HcrVf4, were then isolated from this region and they were homologous to the Cf resistance gene family of tomato. HcrVf2 gene was introgressed into Gala leaf and internode explants by Agrobacterium tumefaciens mediated transformation. Several transformed lines expressing the transgene were obtained and preliminary in vitro results  indicated an increase of resistance (Barbieri et al., 2003). These plants were than evalutated in greenhouse by infection with a conidia suspension of Venturia inaequalis. The results show that the introgression of HcrVf2 gene is sufficient to transform the susceptible cultivar Gala into a resistant one. However it’s not yet possible to affirm that HcrVf2 is the only gene at Vf region that confer resistance toward scab infection as the effect of the other expressed sequences remains to be investigated. 

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

Poster Abstract - 5.14

STUDY OF THE EXPRESSION OF A CHITINASE FROM AUTOGRAPHA CALIFORNICA MULTIPLE NUCLEAR POLYEDROSIS VIRUS IN TOBACCO PLANTS

S. ARCIELLO, M. LABRIOLA, R. RAO

Dipartimento di Scienze del Suolo, della Pianta e dell’Ambiente, Università degli Studi di Napoli “Federico II”, Via Università 100, Portici

Virus gene, protoplast transformation, chitinolytic activity

Biotic stresses represent the main cause of loss of production in agriculture. Pest control actually is achieved through chemical pesticides causing problems of toxicity and environmental safety.  The genetic engineering of plants, powerful tool to tranfer resistance genes to crops, can overcome limits connected with strategies actually used to protect plants. 

Much attention has been addressed to chitinases for their specific hydrolytic activity towards chitin a structural homopolymer which has been found in the cell wall of fungi and in the peritrophic membrane and the exoscheleton of insects. The chitinase encoding gene has been expressed in E. coli cells and the purified protein proved to inhibit Botrytis cinerea and Alternaria alternata spore germination and to perforate peritrophic matrix of Bombyx mori larvae in in vitro assay. The recombinant enzyme showed both endo and exo chitinase activities as in the virus infected cells. With the aim to find out the best condition to express the ChiA protein in plant, Nicotiana tabacum has been stably transformed with two different contructs: the wild type chiA gene harboring  a carboxy-terminal ER retention motif (KDEL) and a mutated gene missing the KDEL which was replaced with the YGAL sequence. 

ChiA production in transgenic tobacco plants transformed with the two contructs was assessed by immunoblotting which revealed that KDEL substitution caused a reduction in protein accumulation. Moreover both native and mutated ChiA protein exherted enzymatic activity.

Localization studies of the recombinant proteins carryed out on protoplasts and media immunoblotting, demonstrated that wild type ChiA accumulated mostly inside the cells while mutated protein was preferentially secreted into the media. Chitinolytic assay performed on secreted proteins showed that ChiAYGAL stored its enzimatic activity proving that despite protein production was reduced, KDEL substitution did not compromise  protein functionality. We expect that the different localization of the recombinant protein may be useful to protect the plants against pathogenic fungi (secreted protein), and phytophagous insect (retained protein).

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

Poster Abstract - 5.15

MOLECULAR CHARACTERIZATION OF TWO cDNAs CODING FOR ACETYLCHOLINESTARESE FROM THE PEACH POTATO APHID MYZUS PERSICAE

S. CASSANELLI*, B. CERCHIARI*, S. GIANNIN*, D. BIZZARO**, G.C. MANICARDI*

*) Dipartimento Interdisciplinare di Scienze Agrarie, Università di Modena e Reggio Emilia, Reggio Emilia
**) Istituto di Biologia e Genetica, Università Politecnica delle Marche, Ancona

acetylcholinesterase genes, aphid, Myzus persicae, pesticide resistance

Insecticide resistance is the major obstacle to control of agriculturally important pests. This worldwide problem has been documented for over 500 arthropod species, particularly among insects and mites. Resistance results in increased pesticide application frequencies, increased dosages, decreased yields and environmental damage. Given the tremendous difficulty and investment associated with development of new, safe and cost-effective insecticides, there is a grave need to preserve the efficacy of current and future insecticides. For these reasons, it is essentially to understand the mechanisms by which insects and mites acquire resistance so that we can intelligently design strategies to delay its onset.

Among pest crop insects, the peach potato aphid Myzus persicae is without any doubts one of the more representative species and one of the more serious pest on a wide range of agricultural and horticultural crops, in which it can cause substantial direct feeding and cosmetic damage as well as transmitting more than 100 virus diseases (Cravedi and Cervato 1991; Barbagallo et al., 1996; Bianco and De Luca, 1997).

Acetylcholinesterase (AChE) is the target site for two important classes of insecticides, organophosphates (OPs) and carbamates. Widespread use of these compounds has led to the development of resistance in many insect species, often due to an insensitive form of the enzyme. A modified acetylcholinesterase (MACE) that confers specific insensitivity to pirimicarb and triazamate has been previously reported in Myzus persicae (Moores et al., 1994; Foster and Devonshire, 1999). In order to determine the genetic basis of this insensitivity, we have amplified the AChE gene from both sensitive and insensitive italian populations of M. persicae using RT-PCR. However, no mutations were identified which could account for this insensitivity. The identification of a second AChE gene in the aphid species Schizaphis graminum (Gao et al., 2002) has led us to design a second couple of primers which led to the amplification of a sequence that differed from M. persicae AChE1 gene. However, no mutational changes were identified between sensitive and insensitive M. persicae populations also regarding this second AChE gene.  Southern blotting and multiplex-PCR experiments demonstrate that both genes are present in a single copy and they are not differentially expressed between sensitive and insensitive populations.

The above mentioned data clearly demonstrate that the molecular basis of AChE resistance in italian population of M.  persicae remain largely unknown.

References:
Barbagallo S, Cravedi P, Pasqualini E, Patti I, 1996 . Bayer S.p.A. Milano Divisione Agraria. 123 pp.
Bianco M, De Luca M, 1997 . Terra e vita, supplemento al n° 12: 4-6.
Cravedi P, Cervato P, 1991 . Informatore fitopatologico, 64:32-34. 
Foster SP, Devonshire AL, 1999 - Pestic. Sci., 55: 810-814.
Gao JR, Kambhampati S, Zhu Y, 2002. Insect Biochem Mol Biol 32: 765-775.
Moores GD, Devine GJ, Devonshire AL, 1994 - Pesticide Biochem. Physiol., 49: 114-120.

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

Poster Abstract - 5.16

SPECTROPHOTOMETRIC ANALYSIS OF ANTHOCYANIN REACTION IN POTATO SOLANUM TUBEROSUM (+) S. PINNATISECTUM SOMATIC HYBRIDS AFTER PHTHORIMAEA OPERCULELLA ZELLER (LEPIDOPTERA: GELECHIIDAE) LARVAL FEEDING

S. MUSMECI*, A. LAI**, S. ARNONE*

*) ENEA CR  Casaccia, BIOTEC-GEN, Via Anguillarese 301, 00060 Roma
**) Enea C.R. Frascati, FIS – LAS, Via Enrico Fermi 45, 00044 Frascati (Rm)

PTM, anthocyanin, Solanum pinnatisectum, tuber resistance

Potato tuber moth (PTM) (Phthorimaea operculella Zeller) is one of the most important insect pests for this crop. Originated from South American tropical and subtropical areas, it found a suitable habitat in Mediterranean Basin. The larva is mining both leaves and tubers, emphazising its damaging effects especially in Third World Countries, where suitable insect-proof stores for tubers are not available. At the moment, different agronomic and biological control techniques are available in order to reduce the pest impact both in field and in stores, including the use of genetically resistant plants.  Natural leaf resistances, conferred by glandular trichomes (Musmeci et al., 1997; Malakar and Tingey, 2000) or leptines (Westedt et al., 1998) have been combined into a unique genotype genetically modified with the gene Cry I (Westedt et al. 1998) expressing Bacillus thuringensis toxin in tubers too. Concerning natural tuber resistance, the wild species S. pinnatisectum shows complete larval resistance (Arnone et al. 1998)  and the somatic hybrids S. tuberosum (+) S. pinnatisectum (Menke et al., 1996) demonstrate an intermediate level of resistance (Arnone et al., 1999) to PTM. This resistance could be partially due to glycoalkaloids (Sinden, 1986). However, the breakdown of resistance (from 77,2 % to 38,57 % of larval mortality) on pre-treated tubers at 4 °C and at 38 °C for 4 days, suggests the presence of unstable compounds directly involved in the resistance or related to the inducible defence mechanisms. In addition, during bioassays, an anthocyanin reaction at the preferential larval entrance site (tuber eyes) was always observed. The anthocyanin production, visible at 24 hrs after inoculation, very evident at 48 hrs and increasing with larval feeding, could be related with a direct response of the genotype to specific insect compounds playing the role of elicitors. In fact, preliminary observations showed that the spot formation is induced after a real chewing larval behavior vs. a generic artificial wounding. It is possible to hypothesize that the fast anthocyanin reaction is a plant protective mechanism related to the production of active oxygen species and to limit lipid peroxidation (Baker and Orlandi, 1995). Active oxygen species and lipid peroxidation play a central role in production of plant defence signal molecules (such as salicilic acid and jasmonic acid) against insects and pathogens (Walling, 2000). 

In order to better understand the relationship between the anthocyanins, resistance mechanisms and elicitation specificity, bioassays with spectrophotometer  (Mancinelli, 1984 modified) have been undertaken in different experimental conditions (antibiosis tests with newly emerging larvae, treatments with larval regurgitant, or artificial scratching). Anthocyanin amount is periodically recorded at 48 hrs and after 6 days, by taking out a tuber tissue carot (ø 5mm h 2mm). Subsequentely, a second check is necessary to record larval mortality and surviving larval weight. The results of this work are presented.

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

Poster Abstract - 5.17

RESISTANCE OF LIMA BEAN TO BEAN WEEVIL LARVAE MAY BE ACCOUNTED FOR BY THE STABILITY OF ITS SEED PROTEINS IN THE GUT OF THIS BRUCHID SPECIES

M. FILEPPI, F. SPARVOLI, R. BOLLINI

Istituto di Biologia e Biotecnologia Agraria, CNR, Via Bassini 15, 20133 Milan, Italy

Acanthoscelides obtectus, lectin-related proteins, Phaseolus seeds, resistance 

The bean weevil Acanthoscelides obtectus represents a major thread for bean seeds during storage.

On common bean seeds (Phaseolus vulgaris), each female lays several eggs, the larvae then penetrate the seed to emerge as mature adults in about thirty days. This cycle can be repeated a number of times on each seed and may lead to complete loss of  the stored commodity.

A diffuse level of resistance to this bruchid has been found in lima bean (Phaseolus lunatus), where adult emergence occurs in a number of days almost doubled compared to common bean, known to be a susceptible species.

Common bean seeds contain a family of abundant lectin-related proteins that can be regarded as defense proteins. Among them, arcelin has been related to resistance against Mexican bean weevil (Zabrotes subfasciatus) and resistance apparently is due to poor digestibility of this protein.

In lima bean seeds, proteins of this class, namely ARL (arcelin-like) and AIL (alpha-amylase inhibitor-like) are particularly abundant.

As a preliminary attempt to identify resistance factors in lima bean, the digestibility of its proteins has been investigated. The presence of undigested seed proteins has been estimated by western blot analysis of total protein extracts from developing larvae guts. Large amounts of both phaseolin and lectin-rlated proteins apparently survive in the guts. On the contrary, in control common bean seeds only faint amounts of phaseolin survive digestion. In an attempt to correlate one or more major lima bean proteins to resistance, two protein fractions, enriched in phaseolin and in the lectin-related ARL and AIL respectively, have been prepared from dry seeds and their toxicity assayed in artificial seeds. At the concentration investigated, no correlation between protein fractions and resistance has been found. Proteins in the partially purified protein fractions survive a long time in the gut, suggesting their poor digestion is due to intrinsic features rather than to the presence of proteinase inhibitors. Indeed, lima bean seed proteins remained undigested when incubated in vitro with proteolytic enzymes extracted from the gut of third instar larvae raised on common bean seeds whereas they were promptly digested if heat denatured before the incubation.

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

Poster Abstract - 5.18

MOLECULAR RESPONSE TO SALT STRESS OF THREE ITALIAN POPULUS ALBA GENOTYPES FROM DIFFERENT LATITUDES

I. BERITOGNOLO*, M. PIAZZAI*, R. MULEO**, I. PAOLUCCI*, E. KUZMINSKY*, M. SABATTI*, G. SCARASCIA-MUGNOZZA*

*) Dipartimento di Scienze dell’Ambiente Forestale e delle sue Risorse, Università degli Studi della Tuscia, via S. Camillo de Lellis snc, 01100 Viterbo
**) Dipartimento di Produzione Vegetale, Università degli Studi della Tuscia, via S. Camillo de Lellis snc, 01100 Viterbo

Populus alba, salt stress, H+ATPase, gene expression, Real-Time PCR

Soil salinity is one of the most significant abiotic stresses for cultivated as well as for forest plants. Because of their high genetic variability in salt tolerance, forest trees could serve to recover and cultivate lands affected by secondary salinisation. Populus spp is considered a model plant for research on forest trees. It is also promising as biomass producing crop, for CO2 sequestration and bio-remediation of degraded soils. Populus alba is present as natural species in Italian riparian habitats and shows a relatively high salt tolerance when compared to other cultivated and natural species, such P. nigra, P. deltoides, P. x euramericana and P. trichocarpa.  Salinity imposes two stresses on plant tissues: a water deficit and ion specific stresses resulting from altered K+/Na+ ratio and Na+ and Cl- concentration that are toxic to plants. Plants respond to elevated Na+ concentrations by maintaining low cytosolic Na+ concentrations and a high cytosolic K+/Na+ ratio. The strategies to achieve that include Na+ extrusion and/or the intracellular compartmentalization of Na+ in the vacuole. This active transport depends on the proton gradients established by proton pumps. Plasma membrane H+ATPase (proton pumps) plays an important role in the establishment and maintenance of ion homeostasis during salt stress. In experiments on herbaceous plants it was observed that salt stress can induce the differential expression of H+ATPase genes in tolerant genotypes when compared to less tolerant ones. H+ATPase genes are considered as good physiological markers of salt stress. 

The objective of the present work is to investigate the molecular basis of salt tolerance in P. alba by using H+ATPase as candidate genes. In our study we compared three P. alba genotypes selected from natural populations and adapted to a range of ecological conditions: genotype 6K3 from Bormida valley in Piemonte region (Lat 44°30’ N), genotype 2AS11 from Sele valley (Lat 40°38’ N) in Campania region and genotype 14P11 from Sinni valley (Lat 40°09’ N) in Basilicata region. The three genotypes studied were selected on the basis of their origin and for the divergence of morphological and phenological adaptive traits. Salt stress experiments with potted cuttings were set-up in green house conditions. The degree of salt tolerance was assessed as difference in relative growth rate and ecophysiological parameters (transpiration, water potential, photosynthesis) between stressed and control plants. The three genotypes were submitted to irrigation with NaCl solution and showed a different tolerance to salt stress, being 14P11 the more tolerant to high salinity levels. These genotypes could represent valuable genetic resources and an interesting experimental model to study the physiological mechanisms leading to salt tolerance. Leaf samples were collected during the salt stress experiment at different levels of salinity and used for molecular analyses. Some fragments belonging to P. alba H+ATPase genes were cloned by PCR and the obtained H+ATPase sequences were used to design specific primers for RT-PCR. First analyses of gene expression by RT-PCR showed a differential expression of H+ATPase genes in salt stressed plants when compared to control plants. We are carrying out quantitative analyses of gene expression by Real Time RT-PCR (Light Cycler, Roche) in order to quantify the transcript level of H+ATPase genes relatively to housekeeping genes (Elongation Factor, 18S). Results of quantitative RT-PCR will be presented in correlation to plant growth and salinity level and the possible role of H+ATPase genes in salt tolerance will be discussed.

SESSION V - SIGA WORKING GROUP

PLANT PHYSIOLOGY

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

Poster Abstract - 5.19

A GENE-TRAPPING APPROCH TO STUDY THE CORRELATION BETWEEN CALMODULIN AND PCD IN ARABIDOPSIS THALIANA

A. DE FRANCESCO, M. LANDONI, F. FOGLIA, C. TONELLI

Università degli Studi di Milano, Dipartimento di Scienze Biomolecolari e Biotecnologie, Via Celoria 26, 20133 Milano

Gene-trap, Calmodulin, PCD, Arabidopsis

We report the preliminary characterization of a calmodulin 2 (cam2) mutant, isolated in the Exotic (Exon trapping insert consortium) collection of Arabidopsis, obtained using a gene-trapping system based on the Ac/Ds transposons of maize.

In the cam2 mutant the transposon, containing the GUS reporter gene, is inserted in the At2g41110 gene, corresponding to Calmodulin 2 (Cam2).

Calmodulins (CaMs) are members of Ca++-binding proteins family, identified in all the eukaryotes examined. When CaMs bind to Ca++, there is a conformational change and the activated protein is able to interact with its targets.

In Arabidopsis there are six Cam genes, with a highly conserved primary structure, and others Calmodulin-like genes.

The consequence of the loss of function of Cam2 is an altered phenotype: cam2 plants are shorter than wild type and the leaves of the rosette show necrotic lesions very early during development.

Cell death in plants can occur as necrosis or as genetically defined programmed cell death (PCD), an active process involved in various developmental processes and in the interaction with pathogens.

To better understand the nature of these lesions we have used histochemical analysis to reveal the presence of specific markers associated with PCD. 

We have also analyzed in wild type and mutant plants, the expression level of PR (pathogen-related) genes, constitutively expressed in “lesion mimic” mutants and SAG (senescence-associated) genes, known to accumulate during senescence.
Finally, we have grown wild type and cam2 plants in different media to verify if these necrotic lesions could be a consequence of a nutritional deficiency.

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

Poster Abstract - 5.20

Characterisation of the enzymes involved on carotenoid pigment biosynthesis

A. Cenci, S. Somma, A. Blanco

Dipartimento di Biologia e Chimica Agro-Forestale e Ambientale, Università degli Studi di Bari, Via G. Amendola 165/A, 70126 Bari, Italy

durum wheat, carotenoid, sequence analysis, EST, BAC library

Breeding for durum wheat quality mainly concerns protein quantity and quality and carotenoid pigment content to give appreciable end-products. Carotenoid pigments confer to pasta the yellow amber colour requested and have also an antioxidant activity against photo-oxidant effects of light energy and against free radicals. These characteristics make them important components for their commercial, nutritional and healthy value. Semolina pigment content is a quantitative trait with high heritability values. It is controlled by QTL detected on several chromosomic groups. 

Several enzymes are involved in carotenoid biosynthesis: nucleotidic sequence of these enzymes are available in gene databases for many species, but not for wheat. To identify the structure of these genes, comparison was performed by using mRNA and genomic sequences from different species. Rice, maize or Arabidopsis sequences were used to select wheat Expressed Sequence Tags (EST) on which specific primers were designed. A durum wheat Bacterial Artificial Chromosome (BAC) library was screened by Polimerase Chain Reaction (PCR) and BAC clones containing sequences for the carotenoid pathway enzymes were isolated. For each enzyme, BAC clones deriving from different genomes were distinguished by length polymorphism, conformation-sensitive gel electrophoresis or enzymatic restriction pattern. Chromosomal localisation was performed by using Langdon substitution line set. Sequencing and genetic mapping will be performed for the most interesting genes.

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

Poster Abstract - 5.21

EVALUATION OF CAROTENOIDS CONTENT IN MAIZE KERNELS USING NEAR INFRARED REFLECTANCE SPECTROSCOPY (NIRS) 

N. BERARDO*, O. V. BRENNA**, A. AMATO*, P. VALOTI*, V. PISACANE*, M. MOTTO*

*) Experimental Institute of Cereal Research, Maize-Section Bergamo, Italy
**) Department of Food Science and Microbiology, University of Milan, Italy

carotenoid, NIRS, maize, genotypes

Maize is one of the most important agronomic crop in the world. It provides food and feed for human and  resources for many unique industrial and commercial products. Maize is in North Italy irrigated plane one of the major incoming cultivation for its high biomass production and high nutritive value. Two general classes of carotenoid pigment, carotenes and xanthophylls, are primary responsible for yellow color of maize grain. The composition of carotenoids and tocopherols in grain has been studied extensively due to their importance in animal nutrition. The carotenoids are important feed constituents of maize because carotenes are precursors of vitamin A, while xantophylls impart a desiderable yellow color to egg yolks and the skin of poultry. Carotenoids, have a range of diverse biological functions and actions, especially in relation to human health. Recent studies have shown that the antioxidant carotenoids and tocopherols may have the potential to reduce the risk of many chronic diseases such as cancer and cardiovascular disease. In general, zeaxanthin and lutein are the major carotenoids present in maize kernels. One problem to measure level of carotenoid pigments in food plants by genetic approach strategies has been the complex and time-consuming analyses required to quantitate the individual carotenoids. Here we reported the carotenoid contents in maize kernel genotypes obtained by  near infrared reflectance spectroscopy (NIRS). Wide differences among kernel samples were obtained for the major carotenoids examined in this study with the following fractions: violaxanthin, lutein, zeaxanthin, isolutein, cryptoxanthin and carotenes. 

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

Poster Abstract - 5.22

MOLECULAR BASIS OF RESISTANCE TO ALS-INHIBITORS HERBICIDES IN SCIRPUS MUCRONATUS L. (PALLA)

L. SCARABEL*, N. CARRARO**, M. SATTIN*, S. VAROTTO**

*) Istituto di Biologia Agroambientale e Forestale (IBAF)-CNR-sez. Malerbologia, viale dell’Università, 16 Legnaro (PD)
**) Dipartimento di Agronomia Ambientale e Produzioni Vegetali, Università degli studi di Padova,  viale dell’Università, 16 Legnaro (PD)

Acetolactate synthase (ALS), herbicide resistance, point mutation, polyploids, rice fields

Acetolactate synthase (ALS) is the target site of five herbicide families known as ALS-inhibitors: sulfonylureas (SU), imidazolinones (IM), triazolopymiridines (TP), pyrimidinylthiobenzoates (PTB) and sulfonylaminocarbonyltriazolinones. During the ‘80s and ‘90s the use of these herbicides increased rapidly worldwide because of their excellent selective control of the main broadleaf weeds by using low dosages. Anyway, only five years after the commercial introduction of the SU (chlorsulfuron) in 1982, the first case of resistance was observed in Lactuca serriola (Mallory-Smith et al. 1990). Since then there has been a steady increase in the number of species affected by ALS resistance: to date at least 25 monocot and 55 dicot species (Heap, 2003).

In rice crops, repeated use of ALS inhibitor herbicides has led to the development of ALS-resistant biotypes. At least three infesting rice species developed populations resistant to ALS-inhibitors in Italy: Alisma plantago-aquatica, Scirpus mucronatus and Cyperus difformis. In most cases, the field-selected ALS-inhibitors resistance is linked to an altered ALS enzyme that is no longer sensitive to the herbicide (target-site resistance). The resistance is due to the possession of particular point mutations in one out of five specific highly conserved regions (Domains) of the ALS gene sequence, that determine one amino acid substitution in the protein sequence. It has become evident that each amino acid substitution shows a peculiar pattern and level of resistance to different ALS-inhibitors. 

Three populations of S. mucronatus were screened in our labs: two were selected by the bensulfuron-methyl (SU), while the third one was sensitive to ALS-Inhibitors. One population (9711) presented a high resistance to SU and a low resistance to TP, while the second one (9719) showed a high resistance to both  SU and TP. To elucidate the molecular basis of resistance, DNA and RNA were extracted from plants grown in the greenhouse and survived to a treatment with ALS inhibitors at field dose. Degenerated specific primers were constructed and used in  PCR and RT-PCR amplifications in order to amplify genomic DNAs and/or cDNAs encompassing the five conserved Domains of the ALS gene. Then, RACE experiments were performed in order to obtain the full-lenght cDNA of the ALS gene of  S. mucronatus.
In population 9711 the nucleotide sequence of Domain A differed from that of the susceptible  population by a single nucleotide mutation at the variable Pro codon at position 197 (where the amino acid position refers to the Arabidopsis precursor ALS), predicting a Pro to His substitution. Specific primers will be used to amplify the conserved  ALS Domains of resistant byotypes in population 9719, that is characterised by a different pattern of resistance.
S. mucronatus is a polyploid plant. Southern analysis using ALS gene as a probe showed tha the species has at least four ALS sequences in its genome. With the aim to characterize the level of ploidy and to understand how the different ALS genes are expressed and regulated in different tissues and during plant development further experiments are in progress.

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

Poster Abstract - 5.23

EFFECT OF THE REMOVAL OF THE C-TERMINAL REGION OF GF14-6, A MAIZE 14-3-3 PROTEIN, IN THE INTERACTION WITH THE H+-ATPASE

S. VISCONTI, P. ADUCCI

Dipartimento di Biologia, Università di Roma “Tor Vergata”, Roma

14-3-3 proteins, H+-ATPase, protein-protein interaction

14-3-3 are a family of conserved proteins widespread in all eukaryotes. They are involved in the regulation of many cellular processes, such as the cell division and differentiation, apoptosis, gene transcription, targeting of proteins to cellular compartments and in the coordination of multiple signal transduction pathways. 

In plants, 14-3-3 proteins possess also some peculiar features; in fact they are involved in the regulation of metabolic enzymes, among which the nitrate reductase and the sucrose-phosphate synthase, and in the regulation of the H+-ATPase, the most important ion pump present in the plasma membrane of plant cells. Recently, a role of 14-3-3 proteins in germination, senescence and in plant responses to abiotic and biotic stresses has been suggested.

These functions are always ascribable to the ability of 14-3-3 to interact with target proteins. Generally, the interaction with 14-3-3 proteins requires the phosphorylation of specific serine/threonine residues present in the 14-3-3-binding motif, even though some proteins are able to interact with 14-3-3 in a phosphorylation-independent manner. 

An amphipathic groove responsible of binding with target proteins has been identified by crystallization studies and mutants analysis of 14-3-3. Recently it has been suggested that the C-terminal region of 14-3-3 proteins could play a regulatory role in the interaction with target proteins by occupying the intramolecular ligand-binding groove. Interestingly, the selectively cleavage of the C-terminal region of a 14-3-3 isoform has been observed during germination of barley embryos; suggesting that the C terminus removal could represent a post-translational regulation mechanism of the 14-3-3 proteins.

In order to investigate about the role of the C-terminal region of the GF14-6, a maize 14-3-3 isoform, we produced a mutant lacking of the last 22 amino acid residues and expressed it in Escherichia coli as a GST-fusion protein. The deletion mutant was then tested for the ability to interact with the H+-ATPase and to stimulate its activity.

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

Poster Abstract - 5.24

A. THALIANA NONSYMBIOTIC HAEMOGLOBIN 2 (AHB2): EXPRESSION PROFILE ANALYSIS AND CHARACTERIZATION OF TRANSGENIC PLANTS ALTERED IN THE LEVEL OF THE PROTEIN

C. ANZI, D. EDERLE, E. ZAGO, M. DELLEDONNE

Università degli Studi di Verona, Dipartimento Scientifico e Tecnologico, Strada Le Grazie 15, 37134 Verona

nonsymbiotic plant haemoglobin, gene expression

Two different groups of haemoglobins have been found in plants: symbiotic and non-symbiotic. The first group has been well characterized in legumes and other symbiotic plants, and its function during the symbiotic process is now clear. On the other hand, nonsymbiotic plant haemoglobins (nsHb) represent a challenge for researchers as they are widespread in plant kingdom, but we are still far from finding their proper (if any) physiological function. Phylogenetic analysis showed the existence of two class of nsHb in dicots. Arabidopsis thaliana possesses two non-symbiotic haemoglobins,  AHb1 (class 1) and AHb2 (class 2). AHb1 is induced in root and rosette leaves by low oxygen levels. AHb1 has a very high oxygen affinity. Analysis of plants grown on MS0 medium revealed a low constitutive level of AHb2 transcript in rosette leaves of A. thaliana and the induction of AHb2 transcription was observed during cold stress. AHb2 shows a lower affinity for oxygen than AHb1. Here we show that the level of protein is extremely stable. Recombinant AHb2 protein, expressed in E. coli, was purified and used to obtain polyclonal rabbit antisera for western blot analysis. We never observed changes in protein amount, no matter what the level of the corresponding transcript, suggesting a post transcriptional control of AHb2 level. Moreover, to get a deeper insight, we monitored mRNA and protein level in plants grown in soil. This analysis showed a different AHb2 transcription profile between plants grown on MS0 medium or in soil, but once again, in both growth condition protein level didn’t change. To test whether an increased level of GLB2 has effects on plant physiology, we generated transgenic plants containing 35S::AHb2 construct. We selected three lines: one showing a lower protein level than wild type, not detectable with western blot analysis; the other two lines having higher protein content. Their phenotypic and molecular characterization is presented.

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

Poster Abstract - 5.25

DEVELOPING CHICKPEA SEEDS ASSIMILATE SULPHUR, AND INCREASE THEIR ACCUMULATION OF SULPHUR AMINO ACIDS IN RESPONSE TO INCREASED DEMAND FROM AN ADDED TRANSGENE ENCODING A SULPHUR RICH PROTEIN

P. CHIAIESE*, L. MOLVIG*, C. HOCART**, T.J.V. HIGGINS*, L.M. TABE*

*) CSIRO Plant Industry, GPO Box 1600 Canberra ACT 2601, Australia 
**) Research School of Biological Science, GPO Box 475, Canberra, ACt 2601, Australia

chickpea, sulphur amino acids, sunflower seed albumin

Increase evidence supports the hypothesis that reproductive sink organs are active in sulphur assimilation. In this stud, developing embryos of chickpea (Cicer arietinum L) were demonstrated to contain significant reserves of sulphur in an oxidised form. The concentration of oxidised sulphur decreased while the concentration of reduced sulphur and nitrogen increased during embryo maturation, consistent with the premise that oxidised sulphur is reduced and assimilated in the developing embryo. It was shown that developing chickpea cotyledons could incorporate the sulphur atom from sulphate into protein, and that embryos contained significant amounts of two enzymes of the cysteine biosynthetic pathway. Transgenic chickpea containing a seed-expressed gene for a sulphur-rich sunflower albumin, contained more seed methionine and less oxidised sulphur than controls, suggesting that introduction of an additional sulphur demand stimulated sulphur reduction in the transgenic seeds. Seeds of two transgenic lines that accumulated large amounts of the sunflower albumin had undetectable levels of oxidised sulphur, altered total seed protein profile, and diminished activities of proteinase inhibitors, compared to non-transgenic seeds of the parental chickpea cultivar. It appeared that reduced sulphur sequestered into sunflower albumin was supplied by additional sulphur assimilation in the developing embryo, as well as some diversion of sulphur amino acids from endogenous seed protein.

SESSION V - WORKING GROUP

geneticalLY modified organisms

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

Poster Abstract - 5.26

TROPOS METHODOLOGIES APPLIED TO AN ANALYSIS OF ACTORS AND GOALS DEPENDENCIES I N THE AGROBIOTECH DEBATE

F. MARIN*,**, P. BRESCIANI**, L. MARTINELLI*

*) Istituto Agrario San Michele all’Adige, 38010 San Michele all’Adige (TN)
**) ITC-irst, Divisione Sistemi Ragionamento Automatico, 38050 Povo (TN)

actor dependencies, communication of informations, requirement engineering, Science & Society

Even tough interest for science is becoming an integrating part of our culture, the relationship Science-Society is still difficult. In general, public information on research progress is an hard task that needs to be managed with extreme expertise. This is particularly truth in the case of biotechnology, where communication results critical, since emotional, political and cultural aspects merge with the scientific impact of the innovative technology. Recently, we embarked in a project aiming to study the problems related with the communication of agrobiotechnology. The debate on this topic is quite sparkling, involving various levels of interests, roles and actors of the whole society (researchers, students, NGO’s, institutions, families, associations etc.), each group aiming to play an active role in the “dialogue”.

The huge amount of available information on the subject may increase the communication difficulty within the various categories. Besides, information overload and often distorted communication make matters worse. Thus, there is a need to rationally organize the available information. The tool we propose for handling this problem is a specific methodology named Tropos, conceived in the context of the so called requirement engineering, a sub-discipline of the software engineering. This was initially proposed by Prof. E. Yu and Prof. J. Mylopulous of the Toronto University, and currently is also wildly  exploited at the University and at the ITC-irst of Trento. Tropos is an Agent oriented Software Methodology, based on the analysis of the actors, goals and relationships involved in a specific context. In our case, the context is defined within the communication of agrobiotechnology field. Tropos formalizes the results of a previous analysis with diagrams showing the most important relationships and needs to be considered in the final organization of the communication system. Starting from the study of the existing context of agrobiotechnology, we defined some categories of relevance in the debate (i.e. scientist, communicators, citizens, institutions, farmers etc.), and for each we defined specific interests and goals. The crucial aspect of the overall analysis is properly focusing the various actors, with the aim of enlightening behaviors and choices based on their identities, goals, and mutual dependencies. As an example, we pointed out a dependency between the “actor-Citizen” and the “actor-Insititution”, based on the claim for food security regulation of the first and the need of social and political consent of the second. Then, we pointed out some questions such as “Who is who?”, “What does one want to do?”, “Why?”, “How can we balance the different interests?”. Finally, all the relations we characterized, were modelized on the basis of the Tropos diagrams, that can progressively describe the actual state of our study, and help us in analyzing the complex system “agrobiotechnology”. In our diagrams we can distinguish needs and priorities, roles and dependencies of the various actors involved. We can compare these elements with the different possible communication tools (i.e. web sites, publications, etc.) in order to propose the most suitable communication solution which would satisfy the requirements previously defined and marked by the diagrams. This solution would result in a new technical actor to be involved in the overall organization (i.e. a technological system), with the role of supplying a significant contribution to the improvement of the actual communication system. The final result would be a best communication tool fitting the requests of each actor.

This research was supported by the project OSSERVA3 of the Autonomous Province of Trento.

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

Poster Abstract - 5.27

Gene flow in wild population of Lactuca serriola analyzed by SSR markers 

M. VISCHI, I. SCOTTI, E. VALENT, S. DELLACASA, A.M. OLIVIERI

Dipartimento di Produzione Vegetale e Tecnologie Agrarie, Via delle Scienze 208, 33100 Udine, Italy  

lettuce, gene flow, SSR markers, introgression  

This study was carried out in the frame of the EU project ANGEL (Analysis of gene flow from crop to wild forms in letture and chicory and its population-ecological consequences in the context of GM-crop biosafety). Lactuca serriola is closely related to cultivated lettuce and was selected as a model system  to establish the degree of outcrossing under field circumstances taking in account its adaptation to a wide range of environmental conditions and the high degree of invasiveness.

Five wild lettuce populations were identified in the North Est of Italy and in Switzerland around Fribourg. About 50 plants for each population (or, where possible, the whole population) were sampled, and their position recorded. A molecular fingerprint of each plant was obtained using 11 SSR markers developed in L. sativa. Allele frequencies were calculated for each locus and each population. The number of alleles per locus was between 3 and 11. The level of heterozygosity was generally low but was higher in populations growing in more central locations relative to the species range (S. Dorligo, Italy). High level of genetic similarity was detected between population except for one population (Amaro, Italy) growing in conditions of spatial isolation.

No linkage disequilibrium was detected both for single populations and for the 5 populations considered as a  whole, suggesting levels of recombination high enough to prevent disequilibrium to take place. 

Gene flow between population was calculated according to the private allele method and by calculation of Fst value. Both methods revealed an absent or very low gene flow with the only exception of two Italian populations (Mossano, Pozzuolo). Combining the molecular profile of plants with their position on the field, it was possible to analyze genotype distribution and heterozygote interspersion. By analysing spatial autocorrelation of genotypes, seed dispersion range was tentatively estimated to be effective up to 5 m. The limit of seed dispersion was estimated at 20 m, since genotypes were randomly distributed over this distance.

This could be used as a guideline for establishing safety distance in case of transgenic cultures being grown together with natural relatives. Further analysis is nevertheless needed to better define population structure and to directly assess outcrossing level in this species.

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

Poster Abstract - 5.28

ABSENCE OF GENE FLOW IN AN ENVIRONMENTALLY CONTROLLED EXPERIMENTAL GM-RICE FIELD TRIAL

E. LUPOTTO*, J. MESSEGUER**, A. REALI*, E. CONTI*, E. BALDONI*, C. LANZANOVA*, A.  MAZZOLENI*, I. SALVALAIO***, S. RUSSO***
 
*) Istituto Sperimentale per la Cerealicoltura, Sezione di Bergamo (Italy)
**) Institut de Recerca i Tecnologia Agroalimentaries – I.R.T.A. Cabrils, Barcelona  (Spain)
***) Istituto Sperimentale per la Cerealicoltura, Sezione di Vercelli (Italy) 

biotechnology, environmental control, gene flow

Transgene flow from engineered crops to other cultivars or to their wild and weedy relatives is one of the major concerns in relation to the ecological risks associated with the commercial realease of transgenic plants. Rice is a strictly autogamous species and spontaneous outcross is rare, although possible. Previous experiments reported on possible gene flow in open field conditions favoring pollen flow from GM-lines to cultivated rice in proximity (Messeguer et al., 2001). The establishment of strategies aimed at controlling and minimising gene flow and an experimental assessment of the risk associated to experimental conditions adopted for GM-crops segregation, are of major interest for supporting actions of the competent authorities in the matter of deliberate release notifications for scientific studies.

An experimental design has been set up and evaluated with the aim to detect, if any occurred, gene flow related to the trait “herbicide resistance” in rice, obtained with the cassette 35S CaMV-bar, conferring resistance to L-glufosinate, active principle of the herbicide Basta (Liberty) (AgrEvo). A set of three independent events were established as homozygous lines in the spanish rice cultivar Senia. They exhibited clear and stable resistance to the commercial formulation of Liberty, at a level of 1.7g/l. Series of 30 plants per GM-line were sown in an environmentally controlled area, isolated with: I) open space kept weed-free; II) a cultivated rice variety with higher size and later flowering time respect to the GM-lines. Two hemi-circles around the GM-plot, in the direction of dominant breeze were sampled, the closest at two meters and the second at three meters, with a total number of 160 panicles sampled, taking panicles contemporary flowering to the GM-lines. On an indicative number of 20000 seedlings germinated and sprayed with Liberty, no one resulted L-glufosinate resistance, thus confirming the reliability of the isolation measures adopted. Furthermore, weedy rice, also called red rice, spontaneously proliferates in rice fields and represents a real weed for the rice crop. Complete eradication of red rice is almost impossible due to its characters of shattering, dormancy, and persistence. Gene flow to spontaneous red rice is also of major concern when adoption of herbicide resistance is foreseen, since in a short run it can be envisaged that most of the weedy rice might introgress the resistance gene. Strict avoidance of this occurence is therefore required. Thirty-five spontaneous red rice plants growing at various orientations and distances from the GM-plot were sampled with harvest of panicles contemporary flowering with the GM-plants. On an indicative number of 11000 red rice seedlings treated with Liberty, no one resulted resistant to the herbicide, thus confirming the complete avoidance of cross-contamination during the trial. Molecular studies confirmed absence of the bar gene in some seedlings exhibiting a sligth tolerance in the beginning of their development, which was in any case lethal afterwards. The results obtained from this specific environmental monitoring confirmed the efficacy of the measures adopted for the experimental  trial in GM-rice lines isolation.

The research is performed within the framework of the project EURICE (QLK5-1999-1484) co-funded by the European Commission within the 5th Framework Programme.

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

Poster Abstract - 5.29

MITOCHONDRIAL DNA VARIABILITY IN RPL5-RPS14 GENOME REGION OF SOLANUM SPP. INVOLVES MULTIPLE RECOMBINATION EVENTS

N. SCOTTI*, L. MARECHAL-DROUARD**, T. CARDI*

*) CNR-IGV, Institute of Plant Genetics Res. Div. Portici, Via Università 133, 80055 Portici (NA), Italy
**) IBMP, Institut de Biologie Moleculaire des Plantes du CNRS, 12 rue du General Zimmer, 67084 Strasbourg cedex, France

potato, somatic hybrids, mitochondrial DNA, homologous recombination

The mitochondrial genome of higher plants shows much more variation in size and structure than any other genome type investigated. This variability is associated with a high frequency of DNA recombination across homologous repeated sequences of variable size. Somatic hybridisation is a powerful method to produce plants with new combinations of nuclei, plastids and mitochondria. Mitochondrial DNA (mtDNA) in somatic or cytoplasmic hybrids (cybrids) often contains regions contributed by both fusion partners as well as novel fragments not seen in parents. 

Southern analysis of Solanum commersonii, S. tuberosum and their somatic hybrids (SH) with rpl5 and rps14 gene probes detected polymorphisms between parental genotypes, and a novel fragment in eight somatic hybrids. This novel fragment could be generated by inter-parental recombination and/or by a selective amplification of pre-existing substoichiometric sequence arrangements. In order to understand the origin of this new fragment, several molecular analyses (cloning, sequencing and PCR) were carried out on mtDNA of parents and two somatic hybrids SH9A and SH9B, regenerated from the same callus, and male fertile and male sterile, respectively. PCR analysis showed that the S. commersonii rpl5-rps14 arrangement, is present at substoichiometric level on S. tuberosum mtDNA. Furthermore, in addition to the two stoichiometric rpl5-rps14 arrangements, SH9A mtDNA contains the third new substoichiometric rpl5-rps14 region. 

The stoichiometric and substoichiometric rpl5-rps14 arrangements detected in the mitochondrial genome of the SH9A somatic hybrid and a computer-assisted sequence similarity analysis strengthened our hypothesis that the recombinant molecules we observed could be the result of inter-parental recombination. The sequence similarity searches detected upstream to rpl5 gene a short repeat of 7bp (R1) involved in a rare recombination event found in a single wheat regenerant (Hartmann et al. 1994), whereas revealed a break point of homology 161 bp (R2) downstream of the rps14 gene (rps14-cob intergenic region) in S. commersonii. 

From the above findings, we propose two models that explain the mechanism responsible of the generation of the different rpl5-rps14 arrangements showed in SH9A. The models and the experimental results demonstrate how somatic hybridisation and resultant mitochondrial fusion may enhance mitochondrial recombinations between short repeats or activate recombination between non-recombinogenic sequence. In addition, we showed that in our somatic hybrids the rearrangements observed are not random; in fact the breakpoints resulting from fusion, upstream and downstream rpl5 and rps14 genes, respectively, were located near the evolutionary breakpoints of the Angiosperms (Quiñones et al. 1996) suggesting that the rpl5-rps14 can be defined as a “hot spot” of recombination in the mitochondrial genome of higher plants.

References
Hartmann C., Récipon H., Jubier M.F., Valon C., Delcher-Besin, E., Henry Y., De Buyser J., Lejeune  B., Rode A. (1994) Curr Genet, 25, 456-464
Quiñones V., Zanlungo S., Moenne A., Gómez I., Holuigue L., Litvak S. , Jordana X. (1996) Plant Mol Biol, 31, 937-943

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

Poster Abstract - 5.30

STABLE PLASTID TRANSFORMATION OF POTATO CV. DESIREE WITH A GENE INVOLVED IN FATTY ACID DESATURATION

D. Gargano, W. Craig, P. Lenzi, N. Scotti, S. Grillo, T. Cardi

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

plastid transformation, potato, transgenic plants, unsaturated fatty acids

In comparison with nuclear transformation, the transformation of the plastidial genome shows several advantages, such as: transgene containment caused by maternal inheritance of cytoplasmic organelles, precise transgene insertion in a defined plastome region by homologous recombination, as well as high and stable gene expression and protein accumulation. In crops other than tobacco, however, its efficiency and applicability are rather limited, and reports of successful transgene expression are still scanty. Herein we report the integration and expression of a Delta-9 desaturase gene, controlling the insertion of double bonds in fatty acid chains, in the plastidial genome of the widely used potato cv. Desiree. A higher content of unsaturated fatty acids is a desirable trait for stress tolerance of higher plants as well as for their nutritional value.

In the process of devising a plastid transformation protocol in potato we tested the regeneration potential from leaf explants of various genotypes using a number of either single or double-step regeneration protocols. The cv. Desiree showed profuse shoot proliferation on several media and thus was selected for transformation experiments. Optimal conditions for gene delivery using the biolistic approach were set up using different vectors and shooting parameters. In experiments with the nuclear vector pGUS-HYG (courtesy of Dr. T. Kavanagh, Ireland) up to 4.1 hpt+ shoots per bombardament were obtained.

As far as plastid transformation was concerned, the gene encoding a Delta-9 stearoyl-ACP desaturase, isolated from the wild potato species S. commersonii (Grillo et al. 1996 In: Physical Stresses in Plants, 163-169) was cloned into the plastid vector pZS197 (Svab and Maliga 1993 Proc. Natl. Acad. Sci. Usa 90, 913-917) and transferred in the potato plastome by the biolistic approach. Using a single step regeneration protocol and 500 mg l-1 of the antibiotic spectinomycin for selection, we obtained seven explants with green calli from eight bombardaments; so far, two of them regenerated shoots. PCR analysis with primers annealing to the flanking regions of the inserted sequence revealed the right integration of the transgene and the complete homoplasmy of the transformed cells. Further, Northern analysis showed the correct transcription of the transgene. Western analysis is under way to verify the presence and the level of protein production in the plastid.

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

Poster Abstract - 5.31

PHOTOSYSTEM II BASED BIOSENSORS: OVEREXPRESSION OF D1 PROTEIN IN E. COLI

G. TESTONE*, D. GIANNINO*, E. PACE**, D. MARIOTTI*, M. T. GIARDI**

*) Istituto di Biologia e Biotecnologie Agrarie (IBBA), CNR, Area della Ricerca di Roma, Via Salaria Km. 29,300, 00016 Monterotondo Scalo (Roma), IT
**) Istituto di Cristallografia (IC), CNR, Area della Ricerca di Roma, Via Salaria Km. 29,300, 00016 Monterotondo Scalo (Roma), IT

psbA gene, D1 protein, Chlamydomonas reinhardtii, biosensors

The development of biosensors responds to the demand for in-situ, rapid, simple, selective and inexpensive tools for pesticides monitoring. A biosensor is defined as a analytical device, incorporating a biological or biomimetic sensing element that provides selectivity and is closely associated to a transducer system. Several currently used herbicides mostly affect the photosystem II (PSII) complex of plants and impair the light dependent phase of photosynthesis. The PSII is a multi-enzymatic chlorophyll-protein complex located in the thylakoids of algae, cyanobacteria and higher plants and it  may turn useful as a component for herbicide biosensors upon extraction from the organisms. The PSII-D1 and D2 proteins together are responsible for the electron transfer to the plastoquinone in the electron cascade of photosynthesis, though D1 is the major target of herbicides. In order to construct a new class of biosensors, wild type, naturally mutated and PCR-mutagenised D1 proteins from Chlamydomonas reinhardtii were over expressed in E. coli to: a) produce bacterial liquid culture based biosensors and b) purify D1 proteins to be fixed on supports, which are necessary for new biosensor prototypes. Here, data on the efficiency of D1 overexpression in E. coli BL21-SI (gateway cloning technology), protein yield and sub cellular location, and D1/herbicide interaction assays will be presented and discussed. 

SESSION V - WORKING GROUP

BIODIVERSITY AND GENETIC RESOURCES

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

Poster Abstract - 5.32

CHARACTERISATION OF DNA EPISOMES IN TUBER SPECIES

C. COSENTINO, B. LAZZARI, A. VIOTTI

Istituto Biologia e Biotecnologia Agraria, CNR, Milano

tuber species, episomes, T. borchii

Total DNAs from ascomata of different Tuber species were purified by CsCl gradient centrifugation and further treated with RNase and proteinase K. The analysis by agarose gel electrophoresis and ethidium bromide staining of eight different species displaies discrete size bands that migrate faster with respect to bulk DNA. Each species shows a specific band pattern with apparent electrophoretic mobility ranging between 0.7 and 11 kb. Among the various Tuber species, T. borchii was chosen as the elite one since in vitro liquid and solid cultures of hyphae can be obtained and as it is one of the best characterised at molecular level. T. borchii shows two main groups of faster migrating bands or episomes in the 7.0 (1Tb) and 2.5 (2Tb) kb region. Similarly, Tuber magnatum shows one major group of bands around 2.5 kb (1Tm). Episomes were found to be degraded by DNaseI while were resistant to both ExoIII and lambda-exo activities, suggesting a covalently circular closed feature for these episomes. In Southern blot analysis each group of episomes results to be specific within the same species and also among the other species. In particular, 1Tb and 2Tb were recovered in in vitro hyphae cultures. Preliminary data by northern blot analysis also indicate that episome probes hybridise to RNA molecules of different sizes suggesting a possible role in expressing their genetic information.

Currently, episome DNA fragments are under cloning and subsequent DNA sequences will allow a more precise identification of their functional roles.

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

Poster Abstract - 5.33

MOLECULAR CHARACTERIZATION OF NATURAL POPULATIONS OF TUBER MELANOSPORUM THROUGH AFLP AND SSR MARKERS

C. RICCIONI, A. RUBINI, F. TOPINI, F. PAOLOCCI 

CNR Istituto di Genetica Vegetale Sez. di Perugia, Via Madonna Alta  130, 06128 Perugia, Italy

truffle, SSR, AFLP, Tuber melanosporum

T.melanosporum is a symbiotic truffle species for which a strictly selfing reproductive mode and the lack of a genetic structure, at least in a survey of populations from the northernmost range of species distribution (France and North Italy), it has been reported (Bertault et al. 1998). Under this scenario, the organoleptic differences among truffles of different proveniences were accounted to environmental causes only. 

In attempt to verify whether such a thesis sounds also when populations from all the different production sites are considered, we decided to collect samples from natural truffle grounds distributed all over Spain, France and Italy and analysed them by means of AFLP and SSR markers.   

A total of 180 specimens were tested by 4 AFLP primer combinations which revealed a strong genetic homogeneity in T.melanosporum (16% of polymorphic traits). AMOVA analysis showed that, despite a high intrapopulation variability (76%), a significant percentage (24%) of species variability was partitioned among populations. The genetic distance among populations was measured by the Nei’s index (Nei, 1978) and the PCA (Principal Coordinate Analysis) showed that, although most of the populations are grouped in a strict area of the diagram, some of them (for instance Subasio and Cerreto, both from Umbria) are well split off. In sum, no significant correlation emerged between genetic and geographic pairwise distance values among the 19 T.melanosporum populations, however few of them could be typed for the presence of exclusive and/or more frequent than in other groups, AFLP bands.

The T.melanosporum genetic polimorphism was also assayed by STMS analyses. Three polymorphic and single-locus SSR loci (ME2, ME4 and ME7) were selected and screened over 80 ascocarps belonging to 8 populations from Italy, France and Spain. None of the individuals was found to be heterozygous at any of the loci. ME2 and ME4 were quite uniform showing only two allelic forms, but interestingly, one allele of the ME2 locus was present only in the Navarra population. On the other hand, the ME7 locus was more polymorphic and appeared in 11 allelic forms, two of them were exclusively present in two populations already typed by AFLPs (Capodacqua and Cerreto).

Both AFLP and SSR-based germplasm screenings confirmed a strictly selfing reproductive mode in T.melanosporum which makes it possible the presence of independent lineages within single populations or even within single natural truffle grounds. It is of remarkable applicative interest the finding that some populations can be simultaneously typed by two independent molecular markers, such AFLPs and SSRs are. It is therefore conceivable that, at least for few populations, distinctive genetic markers can be used as tools to trace truffles according to their proveniences.   

References

Bertault, G., Raymond, M., Berthomieu, A., Callot, G. and Fernandez, D. (1998) Trifling variation in truffles. Nature   394, 734.
Nei, M. (1978) Estimation of the average heterozygosity and genetic distance from a small number of individuals. Genetics 89: 583-590

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

Poster Abstract - 5.34

SSR MARKERS TO ASSESS GENETIC STRUCTURE AND POPULATION DYNAMICS IN T.MAGNATUM

A. RUBINI, F. PAOLOCCI, F. TOPINI, C. RICCIONI, S. ARCIONI

CNR Istituto di Genetica Vegetale sez. di Perugia, Via Madonna Alta 130, 06128 Perugia, Italy

Truffle, SSR, Tuber magnatum

Among symbiotic Tuber spp., T.magnatum is the truffle species producing the most appreciate and valuable ascomata, which are only harvested in Italy and some Balkan areas. Despite its economic relevance only few studies have been so far carried out to shed light into its genetic structure and populations dynamics. Therefore SSR containing loci have been cloned and tested on 370 T.magnatum samples, grouped into 28 populations, collected over the species range of distribution in a search for patterns of genetic structure at two hierarchical levels: within and among populations. Although the average value of gene diversity and mean number of alleles resulted to be not significantly different among populations according to the Kruskall-Wallis ANOVA, some northernmost populations showed an average variance in repeat number higher than all the others and some population-specific or group-specific alleles were identified for southernmost populations too. Better still, we found a significant, although low, correlation between the genetic and geographic distance matrices (Fst: R = 0,278; P = 0,00423; Ds: R = 0,39889 p = 0,001; (delta micron)2: R = 0,4848, p = 0,0006) and between the pairwise values Fst/(1-Fst) versus the log of the geographical distance (R = 0.278; p = 0,00417). It is therefore more than likely the presence of an isolation by distance effect in T.magnatum and the possibility of differentiating among T.magnatum truffles of different geographical provenience as a tool for origin marks. 

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

Poster Abstract - 5.35

Red and white rice genotypes have different antioxidant accumulation in the grain

A. gianinetti*, F. Finocchiaro*, B. Ferrari*, G. Galaverna**, N. Ferrari**, N. Pellegrini***

*) Istituto Sperimentale per la Cerealicoltura, sezione di Fiorenzuola
**) Dip. di Chimica Organica e Industriale, Università di Parma
***) Dip. Sanità Pubblica sezione di Igiene, Università di Parma

rice, proanthocyanidins, antioxidant activity

Rice is one of the most consumed cereal in the world. It is employed as polished grain, i.e. deprived of hulls embryo and kernel coats (pericarp fused with seed coats, and aleuronic layer). With red grained rice it is meant a rice, either cultivated or weedy, whose kernel is contradistinguished by a brick-red (reddish brown) colour, visible after removing the husk (floral bracts) which covers the caryopsis. As polishing leaves a few portions of kernel coats, if some red grains from weedy rice were originally present in the batch, the marketed product could show a varying percentage of red striped grains.

The chemical identification of pigments, distinguishing them from standard white-grained rice, is still uncertain. It has been observed that the reddish coloured testa is associated with the presence of a class of polimeric polyphenols (proanthocyanidins) and  it has been observed also that red grained rice has a great antioxidant activity and epathoprotective action, due to its high level of antioxidants compounds.

Aim of this research is to establish the antioxidants composition (tocols, polyophenols, oryzanols) and the total antioxidant activity of whole grains in a red variety (S. Eusebio) and in a white rice variety (Loto) cultivated in the same farm. Samples of dehulled rice flour were analysed.

For these purposes, an extraction procedure has been developed, which consists of extraction of the milled rice sample with three different solvents of growing polarity: hexane, acetone/water + 1% HCOOH, methanol/water + 1% HCOOH. The polyphenols characterisation was carried out with RP-HPLC analysis (water/acetonitrile gradient with 0.2% of formic acid or NaCl) equipped with Photo Diode Array detector and mass spectrometer ZMD Micromass with electrospray interface. Polimeric polyphenols were characterised with HPLC-DAD-MS.

The Total Antioxidant Activity (TAA) was measured by the ABTS radical cation  assay in all three fractions obtained with the three solvents. It was also evaluated the content of tocols (tocopherol and tocotrienol) and oryzanol in the hexane fraction. The HPLC-DAD-ESI-MS analysis has demonstrated that the red variety has high levels of polimeric polyphenols that belong to proanthocyanidin class. These polymers are completely absent in the white rice. The TAA was measured with the ABTS-flow injection method. The red rice has a very high TAA, if compared with the white variety (almost 5 times higher), primarily due to the high contribution of the acetonic fraction. This fraction contains the proanthocyanidin polimers, which, then, appeared to be the compounds that primarily characterize the red variety analyzed and differentiate it from the white varieties commonly cultivated.

In view of the high levels of proanthocaynidins in this red grained rice and  the possible use of  this rice variety as a source of  dietary antioxidant, it will be interesting to evaluate the presence and the concentration of these compounds also in other cereals with reddish testa. After using red grained rice line as a model matrix, we are currently screening barley germoplasm for red grained genotypes. 

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

Poster Abstract - 5.36

ABUNDANCE, DISTRIBUTION AND PHYLOGENETIC RELATIONSHIP OF LTR-RETROTRANSPOSONS IN THE RICE (ORYZA SATIVA L.) GENOME

A. ZUCCOLO, M. MORGANTE

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

retrotransposon, Long Terminal Repeats, recombination, expansion, reduction

The analysis of the repetitive component of a genome can serve two different purposes: on one hand it can shed light on the evolutionary history of the genome, on the other it can be of great help for an accurate annotation of a genomic sequence. The rice genome (1C = 420 Mbp) is being completely sequenced but the knowledge of the composition of its repetitive component is still lacking. In order to study LTR-retroelements distribution and phylogeny in Oryza sativa L., 343 PAC and BAC clones sequenced by the Rice Genome Program were initially considered. An overall non redundant sequence space of 43 Mbp was analysed. The identification of complete LTR-retrotransposons was carried out on the basis of the results of dot matrix analysis to reveal the presence of the LTR (Long Terminal Repeats). 314 complete elements were identified and classified as members of the Ty3-gypsy family (230 elements) or Ty1-copia (73 elements) family; 11 elements were not classified because of the lack of the pol domain. Elements were classified into subfamilies on the basis of LTR sequence similarity. An overall estimate of the distribution of partial and complete LTR-retroelements in the rice genome was obtained using similarity searches on a larger set of sequenced BACs spanning more then half of the rice genome (303 Mbp) and on the entire sequence of chromosome 1. Almost 16.4% of the genome fraction considered is represented by LTR-retrotransposons that seem to be evenly dispersed across the genome. Solo LTRs that are the result of different types of recombination events are almost 2 times more frequently found than entire elements, showing that mechanisms are active that can reduce genome size. In order to understand the evolution of the rice genome in comparison to that of the maize genome a phylogenetic analysis of elements using conserved domains from both species was performed that revealed differential amplification of some subfamilies in the two species. Sequence divergence between the LTRs from each element was used to estimate the time of their insertion into the genome. Most elements appear to have inserted less that 5 million years ago.

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

Poster Abstract - 5.37

Sequence variation at the dhn4 locus in wild and cultivated barley

S. GIULIANI*, M. MACCAFERRI*, S. SALVI*, R. TUBEROSA*, D.W. CHOI**, Y.J. CLOSE**

*) Department of Agroenviromental Science and Tecnology, University of Bologna, viale Fanin 44, 40127 Bologna, Italy
**) Department of Botany & Plant Science, University of California, Riverside, CA, 92521-0124, USA

drought tolerance, Hordeum, natural allelic variation

Dehydrins (LEA D11 proteins) are water-soluble lipid-associating proteins that accumulate during low-temperature or water deficit conditions, and play a role in freezing and drought-tolerance in plants. It has been suggested that DHNs act as stabilizers of membranes or proteins. DHNs exist as multi-gene families in plants. The most complete information about Dhn genes is in barley where until now 13 different classes have been identified and mapped on 5 chromosomes (Choi et al, 1999, TAG 98: 1234-1247). Dehydrins are characterized by an abundance of charged and polar amino acids, and contain consensus amino acid sequence domains termed Y-, S-, and K-segments. These conserved domains are pieced together in a consistent manner, interspersed by other lesser conserved and usually repeated domains (phi-segments) (Close 1996, Phys. Plantarum 97: 795-803). 
Allelic differences in the protein-coding regions appear to derive mainly from duplications of entire phi-segments or single amino-acid substitutions, suggesting that polypeptide structural constraints have been a strong force in the evolution of DHN alleles. 

We have attempted to explore natural allelic variation in cultivated barley Hordeum vulgare and its wild progenitor H. spontaneum at the Dhn4 locus, which encodes a major drought-induced and seed protein. In this study, we included accessions of H. spontaneum from a wide range of collection sites and cultivars of H. vulgare as a representative sample of barley germplasm cultivated in Europe and North America. DNA sequencing of Dhn4 showed three major types of allelic variation, two at the protein level and one regarding the intron. At the protein level, we found a variable number of phi-segments between the K1 and K2 domains, ranging from six to nine in total. The majority of the accession analysed shows a phi-7 structure in which two central domains are missing. Moreover, variable amino acids (AAs) or AA clusters have been identified at four different regions within the ORF. In H. spontaneum two very different intron classes have been identified. Interestingly, the genetic variation at Dhn4 appears non-randomly organized in the H. spontaneum germplasm, with the intron and exon variants combined in two haplotype classes (spanning the Dhn4 full lenght sequence); only one of these two haplotype classes has been found in H. vulgare. More rearrangements, with several haplotypes, have been observed in the phi segment array region. In total, seven haplotypes were found in the germplasm accession herein analysed. Excellent opportunities exists for developing high-throughput assays targeted to the relevant SNPs and other sequence polymorphisms of Dhn4.

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

Poster Abstract - 5.38

DEVELOPMENT OF A EUROPEAN COMMON BEAN (PHASEOLUS VULGARIS L.) CORE COLLECTION BASED ON PHASEOLINE ELECTROPHORETIC PATTERNS

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

Dept. Biologia Difesa e Biotecnologie Agroforestali, Università degli Studi della Basilicata Potenza

Phaseolus vulgaris, phaseoline, gene pool, core collection 
 
Archaeo-morphological, biochemical and molecular evidence suggest that common bean (Phaseolus vulgaris L) was separately domesticated in Andean and Mesoamerican centres (Gepts, 1988; Delgado-Salinas et al. 1988; Gepts and Debouck, 1991).

Particularly, electrophoretic analysis of phaseoline, the major seed storage proteins, permitted to discriminate the two gene pools: Andean (“T” and “C” type) and Mesoamerican (“S” type) ones.

At different times both gene pools Meso-american and Andean were introduced in Europe as confirmed by phaseoline patterns. The distribution of genetic diversity on the basis of Meso-american and Andean germoplasm in Europe is poorly known.

In this study a collection of 544 European accessions of common bean (Phaseolus vulgaris L) from several genebanks (IPK, NGB, CUB, ANP, etc.) was analysed using SDS-PAGE phaseoline patterns (Romero et al., 1975).

Over all, the 76.3 % of accessions is from Andean gene pool (45.59% phaseoline type “T” and 30.70% type “C”) and the remaining is from Mesoamerica (type “S”).

As reported by other authors (Gepts, 1988) we found that phaseoline type “C” from Andean gene pool is the most frequent both in Italian and Iberian accessions. Type “T” is the most frequent Andean type among accessions from other origins. the Type “S” (from Mesoamerican gene pool) did show high frequencies (32.5%) in the germplasm from eastern-Europe and about 50% of type “S” from southeast Europe group are from Albania.

These results, in agreement with the previous studies, show a different contribution of the two American gene pools  to the colonization of Europe and the high adaptive ability of this species.

On the basis of phaseoline type frequencies and country of origin  a “core” collection of 300 accessions has been constituted representing the contribute of American gene pools to European germplasm. 

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

Poster Abstract - 5.39

VARIATION IN ITALIAN ACCESSIONS OF PHASEOLUS COCCINEUS

A. ACAMPORA, M. CIAFFI, A.R. PAOLACCI, O.A. TANZARELLA

Dipartimento di Agrobiologia e Agrochimica, Università della Tuscia
Via S. Camillo De Lellis, 01100 Viterbo
tanzarel@unitus.it

Phaseolus coccineus, seed proteins, molecular markers, runner bean

P. coccineus, or runner bean, is one of the five cultivated species of Phaseolus which were introduced from Central America in the 16th century. During the last decades there has been a progressive decrease of the common bean cultivation area, that from more than 400,000 ha in the 40’s has dropped to about 11,000 ha, the situation for P. coccineus is even worst. Moreover, a single variety, Bianco di Spagna, is grown in most of the very limited cultivation area of P. coccineus in Italy, whereas several local types are grown on very limited and marginal areas. This situation has determined a dramatic erosion of the genetic variation in the Italian germplasm  of P. coccineus. A tendency to rediscover traditional and abandoned foods is spreading among consumers and the market is appreciating some previously neglected agricultural products, among them also some local varieties of common bean. Also P. coccineus has the potentiality of being included among valuable traditional foods. A key step for its promotion is represented by the assessment of the genetic variation existing in Italian germplasm, including the stored collections and the cultivated materials. It would be important, in particular, the evaluation of the nutritional characteristics, which are mostly related to the protein composition of the seeds.

The aim of this paper was the assessment of genetic variation in P. coccineus using molecular and biochemical markers. Some of the 53 available accessions, from different Italian regions, were collected directly from farmers, others from different Italian Institutions, most of them were furnished by IPK, Gatersleben, Germany. The total proteins of ten seed for each of 27 accessions of P. coccineus were analysed by SDS-PAGE electrophoresis. The electrophoretic patterns showed a wide variation both within and between the analysed accessions, as expected in an allogamous species. Some genotypes, in particular, showed the lack of the phaseolin fraction, the most important and abundant seed storage protein of Phaseolus, and of the fraction corresponding to the lectins, that are important both negatively, as antinutritional factors, and positively, for conferring aspecific resistance to insects. The variation within and between the Italian accessions of P. coccineus has also been assessed using different molecular markers. Among ten screened RAPD markers, only three were able to detect variation between single genotypes of different Italian accessions and were selected for further analyses, also some combinations of AFLP primers showed a very low level of variation. Only one of seven analysed ISSR markers detected polymorphism among the Italian accessions. Finally, IRAP and REMAP, two markers based on retrotransposons previously applied in barley (Kalendar et al., 1999. Theor. Appl. Genet. 98:704-711), have been developed in P. coccineus on the basis of the conserved LTR region sequence of the Tpv-2 family of retrotransposons, which has been studied in P. vulgaris (Garber et al., 1999. Plant Mol. Biol., 39:797-807). Some of the analysed primer combinations, in particular of IRAP, produced several polymorphic bands. Also some accessions from Central America were comprised in the analyses with the different markers. The polymorphism resulted much higher than between Italian accessions, suggesting a low level of genetic variation in the Italian germplasm of P. coccineus. 

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

Poster Abstract - 5.40

GENETIC DIVERSITY IN PHASEOLUS VULGARIS AND P. COCCINEUS REVEALED BY PLASTIDIAL AND NUCLEAR MICROSATELLITE MARKERS

L. NANNI*, D. SICARD*#, J. ACOSTA**, R. PAPA*

*) Dipartimento di Biotecnologie Agrarie ed Ambientali, Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy
rpapa@univpm.it
**) Instituto Nacional de Investigaciones Forestales y Agropecuarias, Chapingo, México
#Permanent address : UMR de génétique végétale, INRA/UPS/CNRS/INA Gif-sur-Yvette, France

Phaseolus spp., SSR, cpSSR, breeding system, population structure

We investigated on the level and distribution of genetic diversity in the autogamous species P. vulgaris and the allogamous P. coccineus. Two sample of both species representing  different geographical scales and different level of sympatry were studied. The first sample contained 67 wild and domesticated accessions from the domestication centres of the two bean species: 24 accessions of P. coccineus from Meso-america; 19 accessions of P. vulgaris from the Andean gene-pool and 24 accessions of P. vulgaris from the Meso-american gene-pool. The second sample contained 115 accessions collected on single plant basis from 6 wild populations: 3 natural populations of P. vulgaris and 3 natural populations of P. coccineus collected in 5 different sites located in the northern area of the state of Morelos, Mexico. In one collection site the two bean species were sympatric. Two types of SSR markers were used: nuclear SSR markers developed from gene sequences including metabolic enzymes and genes related to plant defense and plastidial SSRs (cpSSR) previously shown to be commonly present on the chloroplast genome in gymnosperm and angiosperm.  The eight SSR markers previously developed in P. vulgaris, were polymorphic both in P. vulgaris and in P. coccineus. Out of eight cpSSRs tested in P. vulgaris and P. coccineus, three cpSSRs gave polymorphism within and among the two bean species. Results indicate that both nuclear and cpSSR represent a good tool in order to study the genetic diversity in the two Phaseolus species. The population structure, the observed and expected heterozygosity revealed by these markers were in agreement with the contrasting breeding system of P. vulgaris and P. coccineus.

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

Poster Abstract - 5.41

COMPARATIVE ANALYSIS OF RAPD AND AFLP POLYMORPHISMS IN A CORE COLLECTION OF GRASSPEA (LATHYRUS SATIVUS L.)

G.B. POLIGNANO*, C. LOTTI**, V. ALBA*, C. DE GIOVANNI***, P. DELL’ORCO***, L. RICCIARDI***

*) IGV- C.N.R., Via Amendola 165/A, 70126 Bari, (Italy)
**) DiSA, Università degli Studi di Foggia, Via Napoli 26, 71100 Foggia, (Italy)
***) DiBCA, Università degli Studi di Bari, Via Amendola 165/A, 70126 Bari, (Italy)

Lathyrus sativus L., genetic diversity, ODAP content, molecular markers

Grasspea (Lathyrus sativus L.) is a leguminous crop belonging to the tribe Viciae. For its rusticity, drought tolerance, adaptability to different soils and adverse climatic conditions, it is grown either for stockfeed or human consumption in many Asian and African countries and in the Mediterranean basin. Although rich in proteins (up to 35%), the utilisation of the grain is limited due to the presence of the neurotoxic compound 3-(-N-oxalyl)-L-2,3 diaminopropionic acid (beta-ODAP) that causes neurolathyrism.

The importance of this legume crop in developing countries has promoted the establishment of several breeding programmes mainly aimed at reduce ODAP content in the seeds. In order to define genetic variability and to select grasspea lines with a low ODAP content, a characterisation of a large germplasm collection of Lathyrus from both Italy and foreign countries was performed at the Plant Genetic Institute of Bari (Italy) by scoring bio-agronomic and biochemical data. On the basis of this information, a core collection of 44 entries was selected taking into consideration ODAP content, 100-seed weight, grain protein content.

PCR-based markers have been successfully used to detect and assess the genetic variation in our selected core collection. A study was performed on the 44 entries using 36 pair-wise RAPD primer combinations (DP-RAPD, double primer RAPD) and 5 AFLP primer combinations. A dendrogram of genetic similarity was obtained from polymorphic bands scored in both molecular analyses, comparing the results to detect the efficiency of the two classes of markers to reveal intraspecific polymorphism in the species.

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

Poster Abstract - 5.42

ANALYSIS OF SEQUENCE POLYMORPHISMS IN CODING AND NON CODING REGIONS OF TAP2 GENE FOR THE STUDY OF INTERSPECIFIC DIFFERENTIATION IN TOMATO

A. SCIALPI, P. BOGANI, M. BUIATTI

Dipartimento di Biologia Animale e Genetica “Leo Pardi”, Università di Firenze

anionic peroxidase, Lycopersicon, sequence analysis, genetic diversity, phylogenesis

Plant peroxidases oxidize polyphenols, flavonoids and alkaloids (Gaspar et al., 1982; Blom et al., 1991) or phytoregulators such as IAA (Gazaryan et al., 1996) catalyzing the reduction of hydrogen peroxide into water, using electrons from diverse donor molecules.

Tap2 gene codes for a higly anionic peroxidase ABA-induced in suberizing tomato fruits. The gene consists of three exons and two introns and the sequence has been deposited in the GenBank (Robert and Kolattukudy, 1989).

In this work we report data obtained from the sequencing of the full-length gene in eleven species of the genus Lycopersicon with the aim to evaluate their genetic diversity and species relationships on the base of sequence level variations both in coding and non coding regions of the gene.

The primers utilized for PCR amplification were located from 329 to 348 nt the forward, and from 2525 to 2505 nt the reverse one, resulting in the synthesis of a fragment 2196 bp long, containing the three exons interrupted by two intron sequences.

Preliminary results obtained show differences in the levels of polymorphism between exons and introns determining different topologies of phylogenetic trees according to the functional units used.

Particularly our results focus on the understanding of the informative value of sequence variations depending on their localization in the gene in order to provide further informations for the study of genetic diversity and, hence, the dynamics of the genome evolution.

References
Blom T.J.M., Sierra M., van Vliet T.B., Franke-van Dijk M.E.I., de Koning P., van Iren F., Verpoorter R., Libbenga K.R. (1991) Planta 183: 170-177
Gaspar T., Penel C., Thorpe T., Greppini H. (1982). In: Peroxidases 1970-80. A survey of their biochemical and physiological roles in higher plants, University of geneva, Switzerland.
Gazaryan I.G., Lagrimini I.M., Ashby G.A., Thorneley R.N.F (1996). Biochem J. 313: 841-847.
Roberts E., Kolattukudy, P.E. (1989). Mol Gen Genet, 217: 223-232.

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

Poster Abstract - 5.43

MOLECULAR CHARACTERISATION AND TRACEABILITY OF PROCESSED COFFEE

S. Doveri*, L. Palmieri**, C. Martellossi***, G. Graziosi***, D. Lee*, P. Donini*

*) NIAB Huntingdon Road CB3 0LE Cambridge - UK
**) Dept of Environmental Science University of Parma, Parco Area delle Scienze 11/a, 43100 Parma, Italy
***) Dept of Biology, University of Trieste, Piazzale Europa 1, 34127 Trieste, Italy

coffee, quantitative PCR, SSR, microsatellite, traceability

Coffee products on the market are usually derived from one of two species: Coffea arabica (Arabica) and Coffea canephora (Robusta), or consist as a blend. 

Robusta derives its name from its disease resistance properties. It delivers a strong, full-bodied coffee with a high caffeine content (4.5% compared to 1.7% for Arabica). Robusta coffee is characterised as  “neutral” and weak-flavoured, while coffee obtained from Arabica is a milder, fruitier and acidulous beverage of higher quality. The market value of traded Arabica beans is higher than that of Robusta, thus there is business potential and commercial need for diagnostic tests that discriminate the two species both in starting raw materials and in final products. 

Several reports exist on the determination of coffee species composition of products based on chemical analyses (caffeine, sterols, terpenic alcohol, minerals and phenolic acids). However, these methods suffer from severe limitations and are hardly suitable for quantifying species composition of blends.  

One solution to the industry’s problems with authenticity is a DNA-based diagnostic test that would enable the buyer to determine the market value of traded commodities and serve as a quality assurance tool for guaranteeing products.

Our study illustrates the DNA-based approach to the determination of genetic identity of the two coffee species in blends, and the development of quantitative-PCR methods with the aim to quantify the presence of each species in a blend. SSR (microsatellite markers) analysis and the Lightcycler PCR platforms have been used in our study on C. arabica and C. canephora.

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

Poster Abstract - 5.44

MOLECULAR MARKER CHARACTERISATION OF OLIVE OIL VARIETY COMPOSITION

L. Palmieri*, S. Doveri**, N. Marmiroli*, P. Donini**

*) Dept of Environmental Science University of Parma, Parco Area delle Scienze 11/a, 43100 Parma, Italy
**) NIAB Huntingdon Road CB3 0LE Cambridge - UK

Olea europaea, olive oil, SSR, SNP, RealTime PCR, traceability

Olive (Olea europaea) is the most important oil-producing crop in the Mediterranean. Its cultivation is expanding due to the increase in oil demand following dietary trends and perceived health benefits. The quality and market value of olive oils are defined by several parameters, ranging from the chemical composition to the nutritional and organoleptic properties, to the area of olive production which is often associated to specific varieties, and to the industrial processing of oil. An expanding market for a high value agricultural product is exerting a pull for novel methods that could be used for quality assurance, for assisting trade of raw and finished products, and for the protection of brands. Research focussed on the authenticity and provenance of agricultural products has recently received the attention of both the EU and National programmes. While the chemical analysis of olive oils allows the assessment of most quality parameters such as fatty acid composition1, triglyceride composition2, sterol composition3 and general chemical composition4, it is largely unsuitable for the determination of variety composition. 

We report on the variety molecular identity characterisations of DNA from both olive tissues and olive oil. Our studies are progressing in the frame of two FPV EU-funded projects (DNA-TRACK and OLIV-TRACK), and with the support of the UK Food Standards Agency.

In order to pursue the molecular determination of oil PDO, DOC and variety authenticity, we have been facing several technical challenges, starting from the development of methods that would allow the isolation of good quality DNA from olive oil.

Our study has progressed on the genetic identity characterisation of olive oil varietal composition using microsatellite markers (SSR), and on the quantification of the relative proportion of different varieties entering the composition of oil blends.

We have determined that amplifiable DNA is recoverable from filtered and unfiltered olive oil and that  SSR markers are suitable to distinguish between different varieties. Moreover we are developing a set of SNP markers from functional genes that will be used to quantify genotypes within oil blends using fluorescent probes in Real Time PCR. In addition the SNP markers will be employed in the study of genetic diversity in functional genes involved in fatty acid biosynthesis pathways. Our research is thus resulting in the production of advanced technical protocols for commercial diagnostic testing, in the production of markers, and in basic scientific knowledge on the genetic diversity of olive and in the study of functional diversity at specific loci.

References:

1) Stefanoudaki E. et al, Am Oil Chem Soc 76:623-626 (1999)
2) Stefanoudaki E. et al, Food Chem 60:425-432 (1997)
3) Leardi R. et al., Riv Ital Sost Grasse 64: 131-136 (1987)
4) Aparicio R. et al., Grasas Aceites 41(1):23-39 (1990)

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

Poster Abstract - 5.45

GENOMIC CHARACTERIZATION OF OLEA EUROPAEA VARIETIES BY 26S RFLP BASED POLYMORPHYSM AND FISH TECHNOLOGIES 

L. BRUNO*, T. PANGARO*, R. COZZA*, D. GIANNINO**, M.B. BITONTI*

*) Università degli Studi della Calabria-Dip.to di Ecologia, Ponte P. Bucci, cubo 6B, 87030 Arcavacata di Rende (Cosenza)
**) Istituto di Biologia e Biotecnologia Agraria (sez.di Roma), Via Salaria km 29300, 00016 Monterotondo Scalo (Roma) 

Olea europea, RFLP, FISH

Olive (Olea europaea L.) is one of the most characteristic species of the Mediterranean area and one of the oldest agricultural tree crops. Breeding of olive-trees has been limited due to the long juvenile phase and the lack of the phylogenetic relationships among Olea species. Within the Olea species, two sub-species are distinguished: sativa and sylvestris. The former includes the cultivated varieties which are self-incompatible and thus vegetatively propagated; the latter harbours varieties which only reproduce by sexual means. Olive feral forms derive from crosses between varieties belonging to sativa and sylvestys subspecies. Molecular markers of polymorphism have turned to be powerful tools to improve classification of Olea groups, define phylogenetic relations among cultivars, distinguish close related varieties and screen for genotypes in genetic breeding programme. In the present study, RFLP markers and FISH techniques were used to analyse a set of Olea europea cultivars representative of Calabria and ancestral forms from Sardinia. An rDNA 26S derived fragment of Olea europaea was used to probe genomic DNA restricted with different enzymes and distinctive polymorphism was revealed between Calabrian cultivars and the Sardinian wild forms. Inter-cultivar polymorphism is currently being analysed and cluster types will be possibly presented. Southern analyses with rare cutting enzymes suggests that rDNA cistrons are organized in at least four main polymorphic clusters in both Calabrian cultivars and Sardinian forms. Moreover, FISH performed on Calabrian cultivars displayed at least four clear signals near the nucleolus region in the interphasic nuclei. 

SESSION V - WORKING GROUP

PLANT REPRODUCTION

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

Poster Abstract - 5.46

CYTOMIXIS IN ALFALFA (MEDICAGO SATIVA L.)

A. MARIANI, M. BELLUCCI

Institute of Plant Genetics - Research Division of Perugia, CNR, via della Madonna Alta 130, 06128 Perugia, Italy

alfalfa, cytomixis, genetic control, pollen viability 

Cytomixis has been defined as the migration of chromatin between adjacent cells through cytoplasmic connection channels. It occurs in a great number of plant species and has mainly been observed in pollen mother cells. In the present study we report the identification of a few cytomictic alfalfa plants, characterized by very little or no seed production. Cytomixis had never before been observed in alfalfa. Those alfalfa plants, the “mother plants”, were selfed and crossed with a normal control plant. Microsporogenesis analysis was performed on the mother plants, on the S1 and F1 plants and on the controls. The S1 and F1 plants, like the mother plants, were found to be cytomictic.  Single or multiple chromatin bridges between two or more meiocytes were observed almost exclusively in prophase I. Some completely empty meiocytes were also observed. In addition to cytomixis, other meiotic abnormalities were found. Control plants showed an almost regular meiosis. The highest values of cytomixis were observed in the mother plants, and the lowest in their F1 progenies. Variability of cytomixis in the F1 plants is probably due to a heterozygotic condition of the parents for this trait. No significant correlation was found between cytomixis and pollen viability, even if the cytomictic plants showed low values of pollen viability. Our results indicate that in  Medicago sativa cytomixis is very likely a natural phenomenon under direct genetic control, whose appearance does not seem to be determined by environmental factors. A possible association of cytomixis with the reproductive system, and the role of cytomixis in plant evolution are discussed.

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

Poster Abstract - 5.47

METHODOLOGIES FOR THE MOLECULAR CHARACTERISATION OF POLLEN MUTANT

M. VIANELLO, E. GATTI, M. VILLA, M. SARI GORLA

Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Via Celoria 26, 20133 Milano

pollen mutants, gaMS-1, maize, development

The purpose of this research is the study of the genetic control of pollen development and function in maize. The knowledge of the genetic and molecular components regulating these processes can allow the development of new strategies of genetic improvement of important crops. Furthermore this is an excellent model for important basic researches on developmental processes, including establishment of cell polarity, cell fate determination, cell differentiation and morphogenesis. 
For these studies pollen mutants represent an important tool. We have isolated many mutants of pollen development, both pre-meiotic and post-meiotic. The latter are characterized by a 1:1 ratio of fertile and sterile pollen grains within a single anther. In particular, gaMS-1 produces mutant grains that at anthesis are still at a stage of maturation corresponding to that of a binucleate microspore.
However, the analysis of this type of mutants presents some drawbacks, and it is important to set up some specific strategies for its optimisation.
First of all, due to the sterility of the mutants grains, the mutation can be maintained only in heterozygous condition and it is not possible to obtain homozygous plants. Consequently, it is not possible to obtain a homogeneous pollen population, exclusively composed by mutant pollen grains. Thus, in order to analyze transcripts or proteins differentially expressed by the two types of grains it is necessary to compare wild type pollen with a mixture of normal and mutant grains derived by segregating plants. This method greatly reduces the power of resolution of the analysis, since only quantitative differences can be detected.
To overcome this problem, a method for the separation of the two pollen types produced by heterozygous plants has been developed to obtain suspensions containing up to 95% of each grain type, by using a gradient of Percoll, from 100% up to 40%. 
A further limitation is the material availability: due to the above-mentioned problems, large amounts of it are normally very difficult to be obtained. Thus it is important to develop systems for the analysis of DNA of a limited number of grains. From the suspension of the pollen previously separated by Percoll, a manual removal of the about 5% of different grains of pollen was made, using a glass capillary. The DNA from the cleaned pollen suspension has been amplified directly, avoiding the extraction protocol. The pollen has been homogenised, and the obtained suspension submitted to a PCR reaction.
Fresh pollen, collected during flowering time, is maintained in 70% ethanol. The amplification of DNA from these fixed pollen grains has been performed following the above mentioned protocol.
A RNA isolation protocol (LiCl-method) has been set up, to be used on limited amount of pollen.

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

Poster Abstract - 5.48

SEEKING FOR PETUNIA HYBRIDA MIP1 GENE

M. VIVIANI, G.B. TORNIELLI, M. PEZZOTTI

Dip. Scientifico e Tecnologico, Università degli Studi di Verona

mei2, mip1, meiosis

Sexual reproduction is a fundamental way to provide genetic variability and meiosis is one of the most important steps of this process. Since meiosis is highly conserved among eukaryotes, the knowledge acquired in simple organisms, like Schizosaccharomyces pombe, can be used to understand this phenomenon in higher organisms. Yeast mei2 and mip1 genes play a very important role in meiosis. Mei2 protein, in the cytoplasm, is responsible for the synthesis of pre-meiotic DNA, while, in the nucleus, promotes meiosis I. Mip1 protein interacts weakly with Mei2 in the cytoplasm, probably helping Mei2 protein folding, but its biological function may be more complex.

Several sequences with homology to the yeast mei2 and mip1 genes were identified in some higher organisms, among which we recently isolated mei2 (Moretti et al. 1999) from Petunia hybrida.

This study reports the isolation and the molecular characterisation of mip1 from P. hybrida. The isolation of mip1 was carried out by screening a cDNA library of P. hybrida ovaries performing PCR with degenerated primers. These primers were designed on the conserved blocks resulted from the alignment of S. pombe Mip1 protein with seven homologous proteins belonging to other eukaryotes (H. sapiens, M. musculus, D. melanogaster, A. thaliana, C. elengans, S. cerevisiae). The entire sequence, 4467 bp, has been isolated using semi-nested PCR. Tissue specificity was performed on various plant organs of P. hybrida by Real Time RT-PCR. mip1 was highly expressed in the ovary, while it was expressed at basal levels in petals, sepals and stems. There were no detectable levels of mip1 transcripts in the leaves.

In order to investigate the role of mip1 in P. hybrida we will construct silenced mutants of this gene using the RNA-antisense method.

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

Poster Abstract - 5.49

Identification of an activation-tagged arabidopsis MUTANT Defective in embryo sac development

G. CREMONA*, F. CONSIGLIO***, A. ERRICO*, R.A. BRESSAN**, C. CONICELLA***

*) DISSPA Dept. Soil, Plant and Environmental Sciences, University of Naples "Federico II", Via Università 100, 80055 Portici, Italy
**) Dept. Horticulture & Landscape Architecture, Purdue University, West Lafayette, Indiana (USA)
***) CNR-IGV, Institute of Plant Genetics, Research Division Portici, Via Università 133, 80055 Portici, Italy

Arabidopsis, activation tagging, mutant, sexual reproduction 

Meiosis is a particulary significant cellular event in plant sexual reproduction because it separates the sporophytic and gametophytic generations. Plants carrying meiotic defects often show low gamete viability and low fertility, and in some cases complete sterility. Meiosis is a complex multistep process and the complexity of events suggests that many genes are involved and are tightly regulated to ensure each meiotic step. Many meiotic genes have been reported in various eukaryotic organisms, but relatively little knowledge is available on the molecular details of meiosis in flowering plants. Recently, Arabidopsis thaliana (Ath) has become a model to study meiosis in plants. The availability of a great number of T-DNA and transposon mutated lines represents a powerful tool to isolate meiotic mutants and to identify the corresponding tagged genes.

More than 300.000 activation tagging T-DNA mutant lines of Ath were generated at Purdue University (USA). A secondary screening of this collection identified several putative meiotic mutants showing reduced fertility. One line (b16) was confirmed as containing the mutation for more than one generation. The low fertility phenotype of this mutant co-segregates with the insertion marker. The genetic lesion responsible for this phenotype was identified by TAIL-PCR of the DNA flanking the insertion site. Genetic analysis of the mutant gene is being tested by crossing the mutant line to wild type to determine the inheritance control. A preliminary self-cross segregation analysis of b16 evidenced that the mutation is sporophytically expressed, monogenic and dominant. Histologycal and cytological analysis are being performed on silique and floral buds, and first evidences show that a low number of seeds occurs in the silique depending on development arrest of megaspore mother cell before embryo sac formation.

Further molecular and functional analisys will be performed to assess the rule of the mutated gene in the meiotic pathway. 

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

Poster Abstract - 5.50

MOLECULAR GENETIC APPROACHES TO STUDY APOMIXIS IN HYPERICUM PERFORATUM L.

F. ARZENTON*, H. BÄUMLEIN**, F.MATZK**, F. BLATTNER**, G. BARCACCIA*, M. LUCCHIN*

*) Dipartimento di Agronomia Ambientale e Produzioni Vegetali, University of Padua, Agripolis, viale dell´Universitá 16, 35020 Legnaro, Padova, Italy
margherita.lucchin@unipd.it
**) Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Corrensstrasse 3, D-06466 Gatersleben, Germany

Apomixis, FCSS, AFLP, cDNA-AFLP, H. perforatum L.

Asexual seed formation (apomixis) bypasses meiosis and fertilization of egg cell. The isolation of the apomixis genes is of great interest because this could enable the introduction of apomixis into sexual crop and thus the fixation of heterosis or of any elite genotype. St. John´s wort (Hypericum perforatum L.) reproduces mainly by facultative aposporous parthenogenesis. We consider H. perforatum a good model plant for apomixis research mainly because of its small genome size (1C=0,650pg), the existence of apomictic and sexual plants that are cross-compatible, the great variability in the mode of reproduction and its use as a medicinal plant. In order to understand the genetic control of apomixis in H. perforatum we carried out molecular analysis at the genomic (1) and at the transcriptional (2) level:

1. Several apomictic and sexual genotypes clearly characterized for the mode of reproduction by means of flow cytometric seed screen (FCSS) (Matzk et al. 2001) were analysed using the AFLP technique. The genotypes originated from different collection sites or botanical garden in Europe. Therefore they represent a very differentiated genetic background and showed a very high amount of polymorphic genomic DNA. Seven markers were selected as apomicts-specific (present in all the apomictic lines and absent in the sexuals). The large genetic variability between the genotypes and the clear discrimination of the contrasting mode of reproduction by the apomict-specific fragments support the hypothesis that this genomic level approach could lead to the isolation of markers linked to the apomixis trait. We detected the segregation patterns of the seven fragments in a triploid F1 population obtained by crossing a completely sexual diploid with an obligate apomictic tetraploid. The mode of reproduction of the entire F1 population is going to be determined by means of FCSS. This analysis will enable us to verify whether the seven markers are correlated with one of the two components of apomixis since the genetic control of the components of apomixis is likely to be independent in H. perforatum (Matzk et al. 2001). The subsequent conversion of the apomixis related AFLP markers into simple single locus PCR markers might provide a useful tool to greatly facilitate apomixis research in H. perforatum. 

2. FCSS and molecular markers have enabled the evaluation of the mode of reproduction of 15 Italian ecotypes and the genetic variability among and within these ecotypes, respectively. The most similar ecotypes (1 and 13) showed differences in the mode of reproduction (high incidence of apomixis and reduced level of parthenogenesis in aposporous embryo sacs, respectively) and were chosen for further analysis. The reproductive organs of both ecotypes have been analysed by means of cDNA AFLP technique in order to assemble a mini library of pistil specific ESTs. All cDNA samples were investigated using cDNA pools from somatic tissues such as leaves and stems as controls. This approach enabled us to select several mRNAs specifically expressed in the pistils of both ecotypes or of just one of the two. Fragments sequencing is in progress in order to select ESTs with interesting homologies (specific of the pistil or of the reproductive phase) that will be used as probes in Southern analysis on the F1 population.

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

Poster Abstract - 5.51

AN AUXIN-REPORTER GENE DETECTS ALTERED IAA CONTENTS IN TOMATO FLORAL MUTANTS

M. ROSSI*, R. CACCIA*, F. SALAMINI**, A. MAZZUCATO*, G.P. SORESSI*

*) Dipartimento di Agrobiologia e Agrochimica, Sezione di Genetica, Università degli Studi della Tuscia, Via S.C. de Lellis snc, 01100 Viterbo, Italy
**) Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné Weg 10, 50829 Köln, Germany

tomato, auxin, floral mutants, reporter genes

Auxin is involved in the control of several growth and developmental processes in plants. Notwithstanding this importance, the molecular mechanisms underlying auxin action are to date poorly understood. The characterization of mutants, whose phenotype is putatively determined by altered hormone levels or perception, may shed light on the role played by hormones in different aspects of plant developmental biology, physiology, biochemistry and molecular biology. In this perspective, the tomato is a favoured experimental species, since more than one thousand monogenic mutants have been described to date that cover all aspects of plant growth and development.

In this research, the auxin accumulation dynamic has been analysed in a number of tomato floral mutations by means of a reporter construct where the auxin-inducible promoter of the Agrobacterium tumefaciens gene 5 was cloned upstream of the uidA gene (GUS). The mutants selected for the analyses were: uniflora (uf, that bears inflorescences reduced to a single flower), lateral suppressor (ls, that, besides the lack of lateral shoots, also shows lack of the corolla segments), stamenless 2 (sl-2, that shows homeotic conversion of stamens into carpels), fasciated (f, that shows flowers with supernumerary stamens and carpels) and parthenocarpic fruit-2 (pat-2, that under cold nights and incresing photoperiod shows autonomous ovary growth and parthenocarpic fruit production). All the mutants where crossed with plants harbouring the reporter construct in homozygosis and F2 families were screened for the presence of the transgene. From kanamicin - resistant wild-type (WT) and mutant plants segregating in the F2 families, plants organs (roots, leaves, petals, stamens, ovaries and fruits) were collected and subjected to the histochemical GUS assay. Non transgenic specimens, used as negative control, were always devoid of reporter signal. In turn, remarkable, organ-specific differences were reported between mutant and WT plant tissues. 

WT roots showed strong auxin accumulation at the root tip and at the adventitious root primordia. The uf, sl-2 and f mutants showed no difference from the WT at the root level. Differently, pat-2 and ls roots showed a lower IAA content. Signal intensity was also overall associated to root vigour. In leaf tissues, for the WT and all mutants the signal was absent or very weak, with the exception of sl-2 that showed higher IAA content. 

The WT ovary showed an increasing auxin content through its development into fruit, up to a maximum that was reached at the mature green stage. While the uf ovary did not show any difference from the WT, pat-2 and f ovaries and their young fruits had a stronger reporter signal, in agreement with the putative role of the hormone in parthenocarpic development and in organ number specification. However, auxin content in the pat-2 ovary was lower than in the WT at later stages of fruit development, as it was in the partially sterile ls mutant; such observation supports the hypothesis that fruit IAA is essentially contributed by the seeds.

The reported results on auxin distribution in tomato WT and floral mutant plants confirm the important role played by this hormone in regulating flower and fruit development and growth.

SESSION V - WORKING GROUP

FRUIT TREES AND WOODY PLANTS

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

Poster Abstract - 5.52

STUDIES ON IN VITRO EMBRYO CULTURE OF OLIVE 

P. POLLACCI, M. MENCUCCINI 

Istituto per i Sistemi Agricoli e Forestali del Mediterraneo (ISAFOM), Sezione di Perugia

Olea europaea, embryo culture, germination, in vitro culture 

The olive tree (Olea europaea L.) has a long-juvenile phase in field condition (some cases more than 10-15 years to reach flowering) and this make very difficult the genetic improvement by the traditional breeding technique.

Some authors found olive embryonal dormancy and germination of seeds are slow and uneven events, and these make the propagation of new genotypes more difficult. 
 
The purpose of this work was to study the effect of time and temperature storage and teguments presence on growth of olive mature embryos. Fruits of cv Leccino, obtained by free-cross-breeding, were randomly sampled on November. After removing the fleshy exocarp and mesocarp, stones (endocarp, seed coat, endosperm and embryo) were washed and dried. Stones were broken and seeds were sterilized by mild shaking for 10 minutes in a 10% commercial preparation of sodium ipoclorite (4,9 % active Cl) and abundantly rinsed in sterile water. The embryos extracted under sterile conditions, were cultured in test-tubes with agar medium (EZ) in growth chamber at 241°C to the light. In vitro cotyledons, radicle and epicotyle development percentage and the presence/absence of callus were evaluated after 8 weeks, whereas in vivo plantlets survival was evaluated after 12 weeks from transplant in greenhouse. Stones, seeds (seed coat, endosperm and embryo) and embryos have been treated as  following:
A)	Stones to 4°C for 2 months, then embryos in EZ in growth chamber;   
B)	Stones to 4°C for 10 days, then embryos in EZ in growth chamber;   
C)	Seeds to 4°C for 2 months, then embryos in EZ in growth chamber;   
D)	Seeds to 4°C for 1 month, then embryos in EZ in growth chamber;   
E)	Seeds to 4°C for 1 month with water, then embryos in EZ in growth chamber;   
F) 	Seeds to 4°C for 10 days, then embryos in EZ in growth chamber;   
G)	Embryos in EZ to 4°C for 2 months, then in growth chamber;
H)	Embryos in EZ to 4°C for 1 month, then in growth chamber;
I)  	Embryos in EZ to 4°C for 3 months, then in growth chamber;
L) 	Embryos in EZ to 4°C for 10 days, then in growth chamber; 
M)	Embryos in EZ to 4°C for 5 days, then in growth chamber;
N) 	Embryos in EZ immediately  in growth chamber (control).

Results shown the developed cotiledons percentage of  "D", "E" and "F" theses was consistently higher (100 %), at contrary of the "N"  thesis (40 %). The root development was observed in 55% of the cases for the thesis "D" and 40-45% in the theses "A", "E" and "F", while only 30% in the control. Similar trend was also estimated in the growth of the epicotyle. The thesis "G" has a strong presence of callus (a double number of cases in comparison to the control). Plantlets survived in vivo after the acclimatation in greenhouse have reached 80 % in the thesis " F " against 30 % of the control.  

In conclusion,  cold storage (4°C) for  10-30 days increases in vitro growth of mature embryos and in vivo percentage plantlets survival in comparison with the embryo culture standard procedure.

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

Poster Abstract - 5.53

CHARACTERIZING THE ACYL CARRIER PROTEIN GENE IN OLIVE

L. BALDONI, C. RICCIOLINI, A. PORCEDDU, E. VARASANO, C. GUERRERO

CNR - Istituto di Genetica Vegetale, Sezione di Perugia

Lipid metabolism in olive has been mainly studied at biochemical level but only few works were addressed to the identification and characterization of genes involved in the process of oil accumulation in the fruits, such as the Stearoyl-ACP Desaturase and the Diacylglicerol Acetyl Transferase (DGAT).

Plants need Acyl Carrier Protein (ACP) for the synthesis of fatty acids in membranes and for lipid accumulation. Numerous isoforms of ACP have been described in different plant species, permitting the fine regulation of fatty acid synthesis. ACP is a small protein (approximately 9,500 Da) which plays an important role as cofactor in all reactions of fatty acid synthesis in the chloroplast transferring new acyl groups to the borning chain up to the production of the first unsaturated fatty acid (oleic acid). ACP is a very asbundant component of the fatty acid synthesis, due to the fact that most of the enzymes require acyl-ACP in their reactions. It has been the first protein of plant lipid metabolism to be purified and the first whose gene was cloned. Numerous ACP genes and cDNA clones have been characterized in plants, like in Brassica, spinach, Cuphea and Arabidopsis. 

The role played by ACP in the process of oil synthesis in olive fruits is under study and, in this context, the identification and characterization of the ACP gene were carried out. A cDNA library from developing fruits has been screened with an heterologous probe. From partial cDNA clones the 5’ end of the messanger was obtained by a 5’RACE experiment. The complete 763 bp cDNA clone, coding for a peptide of 134 residues including the signal peptide followed by the mature protein, has been obtained and its sequence shows high homology with that of other plant species. From the sequencing of the RACE product resulted the presence of four different clones almost identical but differing in three nucleotides and a deletion. These differences caused a change in the protein sequence only in two cases. The expression of the ACP gene during olive fruit ripening has been studied by Northern analysis of RNA from the Hojiblanca cultivar. The level of ACP messanger starts to increase when the oil synthesis starts, as it happens for an other important oil synthesis gene in olive, the Stearoyl-ACP Desaturase. The isolation of various genomic clones of ACP in different plants has shown that the corresponding genes present a mosaic conserved structure with four exons interrupted by three introns and the position of these introns is the same in many species. By PCR amplification of the genomic regions corresponding to the different parts of the cDNA clone it was possible to clarify the position and sequence of introns in the olive gene. A fragment from the cDNA clone has been used for Southern analysis in order to establish the number of ACP gene copies present in olive.

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

Poster Abstract - 5.54

THE TRACEABILITY ALONG THE OLIVE OIL CHAIN: RECOVERY AND CHARACTERISATION OF DNA FROM VIRGIN OLIVE OIL

N. LOMBARDO*, E. MUZZALUPO*, E. PERRI*, R. TESTOLIN**

*) Istituto Sperimentale per l’Olivicoltura, Rende (CS)
eperri@irsaolivicoltura.it
**) Dipartimento di Produzione Vegetale e Tecnologie Agrarie, Udine, Italy

Olive oil, Olea europaea L., RAPD-PCR, traceability

The characterisation of the genetic identity of DNA recoverable from olive oil could facilitate the assessment of its place of origin and conformity to type thanks to the particular regional spread of olive cultivars and to their different contributions to the olive oil mixture as stated by each Protected Designation of Origin regulation. The application of random amplified polymorphic DNA techniques requires that intact DNA should be recovered from virgin olive oil. Very recently, the recovery of intact DNA from virgin olive oil has been accomplished by using proteinase K. In the present work the performances of four new different enzymatic mixtures for oil production were compared. The recorded data demonstrate that the olive oil obtained by using two new different enzymatic mixtures treatment during the malaxation process provide DNA amenable to RAPD-PCR amplification. This is an important result because the new enzymatic mixtures are cheaper than proteinase K and they are usually employed by food industries. come from cheese production.

This research was carried out by a financial contribution of the Project MIUR-MIPAF “Valorizzazione di prodotti tipici: definizione di parametri e tecniche a garanzia dell’origine e qualità (VATIPICI)”. Paper n. 3.

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

Poster Abstract - 5.55

CONSTRUCTION OF A BACTERIAL ARTIFICIAL CHROMOSOME LIBRARY OF A SEEDLESS CLONE OF CITRUS SINENSIS CV “VANIGLIA”

S. FERRANTE, G. GERACI, D. GRAZIANO. M.-T. SCARANO

C.N.R. IGV-sez di Palermo, Corso Calatafimi 414, 90129 Palermo, Italy

BAC, genomics, sweet orange, DNA cloning

Isolation of genes controlling expression of important agronomical traits is at heart of plant genetic improvement. For this purpose, cloning of  large DNA fragments within proper vectors represents an outstanding tool for genome analysis. BAC vectors (Bacterial Artificial Chromosome), due to high cloning efficiency, clone stability and ease of use, represent, currently the system of choice for the construction of large insert genomic DNA libraries. These vectors utilize the E. coli single-copy fertility (F plasmid) and can maintain genomic DNA fragments up to 350 kb. A prerequisite for the constitution of large fragment DNA libraries is the isolation of DNA of high molecular weight (megabase-size) and high quality.  We followed the protocols previously proposed by Zhang et al. (1995) isolating nuclei from fresh leaves and subsequently embedding them in low melting agarose plugs. To prepare high molecular weight restriction fragments we applied the procedure from D.G. Peterson and the vector pIndigoBAC-5 (Epicentre) has been used because purchase dephosphorylated and linearized with HindIII restriction enzyme. Pulsed Field Gel Electrophoresis (PFGE) has been carried out with Gene Navigator System (Amersham Pharmacia Biotech) provided with and hexagonal electrode to generate homogeneous electric field (CHEF). Size-selected DNA fragments have been isolated from agarose via electroelution with Electroelution System Model 422 (BioRad). After ligation of insert DNA with the chosen vector, to form BACs, transformation of competent cells (TransforMax EC100 Electrocompetent E. coli, Epicentre) has been performed with BioRad Gene Pulser II apparatus. The most common form of insertional inactivation (alpha-complementation) has been utilized to select recombinant clones. Because the mean DNA length in the plugs resulted to be greater than 600 kb, the plugs have been evaluated adequate for the library construction. In order to obtain an high number of transformants, in the ligation the optimal molar ratio of size-selected DNA/vector was determined to be 6/1. The high number of colonies observed per plate (430 cfu/plate, 8.6 cfu/microl), the reduced number of blue-colored colonies (non-recombinant clones) and false positives detected, confirms that the tested procedure resulted to be efficient and, therefore, useful for the subsequent phases of clone collection. The described methodology has been conceived to generate clones with inserts of exogenous DNA between 100-350 kb. In this project, the mean insert length, estimated on a random sample of white colonies, was equal to 135 kb. It is possible that screening of a greater number of clones could determine a reduction of the estimated mean insert length, without nevertheless compromize the efficacy of the library under construction.

Peterson D.G., Tomkins J.P., Frisch D.A., Wing R.A., Paterson A.H. 2000. Construction of plant bacterial artificial chromosome (BAC) libraries: An illustrated guide. J. Agric. Genomics 5 (www.ncgr.org/research/jag).
Zhang H.B. , Zhao X., Ding X., Paterson A.H., Wing R.A. 1995. Preparation of megabase-size DNA from plant nuclei. The Plant Journal  7(1): 175-184.

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

Poster Abstract - 5.56

STRUCTURE, ORGANIZATION AND SEQUENCE COMPOSITION OF THE GRAPE (VITIS VINIFERA L.) GENOME 

G. FAES*,**, M. MOROLDO*,**, F. CATTONARO*, A. ZUCCOLO*, P. FONTANA**, R. VELASCO**, M. MORGANTE*

*) Dipartimento di Produzione Vegetale e Tecnologie Agrarie, Università degli Studi di Udine, via delle Scienze 208, 33100 Udine
**) Istituto Agrario San Michele all’Adige, via Mach 1, 38010 San Michele TN

grapevine, retroelements, genome structure

One of the fastest ways to get insights into the structure, organization and sequence composition of a eukaryotic genome is to sequence a library of randomly sheared genomic DNA. In order to characterize the genome of grape, V. vinifera (1C = 485 Mbp), two libraries were constructed from the cv. Pinot Noir. Genomic fragments in the size range of 1200-1500 bp were produced by nebulizing genomic DNA, and were then ligated into pCR-Script. The ligation mix was first used to transform E. coli strain DH10 beta (mrcA, mrcB, mrcC, mrr) and produce a random genomic library, and inserts from about 2500 clones were selected for sequencing from both directions. The same ligation mix was then used to transform E. coli strain DH5 alpha (MrcA, MrcB, MrcC, Mrr). The restriction systems for methylated DNA are intact in this strain, so that DNA fragments containing methylated inserts are less likely to survive the cloning process. About 500 clones from this second library (methyl-filtered library) were sequenced from both directions.

Sequences were trimmed and both BlastN and BlastX searches were performed. A total of 2 Mbp of sequence were obtained from the random genomic library, corresponding to about 0.5% of the grape genome. This sample should provide a representative sample of the genome and contain most of the abundant repetitive elements present within it. A sequence was classified as a known element if it had a blast E-value of <10-5. Clusters of related sequences were identified by assembling sequences using the Arachne (Whitehead Institute, MA) genome assembler. Further analysis are currently performed against a proprietary proprietary EST database clustered with 83.000 grape ESTs present into the TIGR database (www.tigr.org/dtb/tgi/vvgi). In the first library about 40% of the sequences were classified, with a preminence of genes (16%) and retroelements (11.5%). About 10% was made up of cpDNA sequences and 2% was classified as mitochondrial. The second library showed a higher percentage of classified sequences, about 53%. Not surprisingly, more than 28% of the sequences were genes, and chloroplast clones accounted for another 16%. We also observed that retroelements were less abundant than in the previous library (3%), while mitochondrial clones had increased to 5.5%.
The comparison between the libraries shows that  repeated sequences like retrotransposons are largely excluded from genomic shotgun libraries by choosing methylation-restrictive E. coli strains, indicating that they must be heavily methylated. In contrast, genes are much more represented in the filtered library than in the random one, being probably hypomethylated. Other basic properties of the grape genome have been estimated from the sequences, such as G+C content, dinucleotide frequencies including that of the CpG one, frequencies of microsatellites. We will also discuss the distribution of specific repeats in the genome, as assessed by their hybridisation to BAC clones and among species of the genus as assessed by hybridisations with labelled total genomic DNA.

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

Poster Abstract - 5.57

DEVELOPMENT AND TRANSFERABILITY OF GRAPE EST-SSR MARKERS SUITABLE FOR MAPPING IN VITIS SPP.

F. SEVINI, R. MARINO, C. SEGALA, M.S. GRANDO

Lab. of Molecular Genetics, Istituto Agrario San Michele all’Adige, via Mach 1, 38010 San Michele a/A, Trento, Italy

Vitis, EST, SSR, allelic variants

Screening of a grapevine EST library of 960 sequences revealed 78 sequences containing mono, di and trinucleotide SSR motifs. Such microsatellites, belonging to expressed regions, should be more informative in mapping loci with a specific genetic function.

Twenty-five of them were selected to design a set of primer combinations and tested on five grape genotypes composed by wild and cultivated Vitis vinifera and American Vitis species. In addition, 24 SSR markers, developed by our group in the frame of the Vitis Microsatellite Consortium (VMC), were also tested for amplification, polymorphism and transferability across the same set of genotypes.

Six EST and 9 VMC derived primer pairs generated polymorphic markers which were used to evaluate the diversity of a larger set of grape accessions. As reported in previous studies, we found that the flanking regions were conserved among different species and the amplification products were in the same size range for all the tested individuals. Allelic variants ranging from a minimum of 3 different alleles (EST2a10 locus) and a maximum of 16 different alleles (VMC 4d9.2 locus) were observed at several loci for different accessions. The allelic frequencies in the Vitis vinifera set resulted very different between cultivated varieties (Vitis vinifera sativa) and wild accessions of Vitis vinifera silvestris, but it was not possible to identify alleles useful to distinguish the two groups of individuals. We could not distinguish between mutants since no one of the developed markers resulted polymorphic within the same variety (i.e. Chardonnay, Pinot). Nevertheless, in some cases we observed more than two alleles at the same locus that could indicate the presence of chimerism.

In comparison to EST derived markers, the SSRs of VMC library resulted much more polimorphic. However, although the low number of tandem repeats we found that these grape ESTs have a suitable variability to allow the mapping of related functional genes.

SESSION V - WORKING GROUP

FODDER CROPS AND AMENITY GRASSES

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

Poster Abstract - 5.58

DETERMINATION OF SAPONINS AS ANTI-NUTRITIONAL COMPOUNDS IN ALFALFA (MEDICAGO SATIVA L.)

B. PINTUS*, A. TAVA*, M. ODOARDI*, A. MARIANI**

*) Istituto Sperimentale Colture Foraggere, Viale Piacenza 29, I-26900 Lodi
**) Istituto di Genetica Vegetale – Sezione di Perugia, CNR, Via Madonna Alta 130, I-06128 Perugia

Medicago sativa L., anti-nutritional compounds, saponins

In alfalfa (Medicago sativa L. 2n = 4x = 32) heterosis is dependent on heterozygosity involving allelic and non-allelic gene interactions. An effective, non-conventional method for maximizing heterozygosity in tetraploid cultivated alfalfa is the use of meiotic mutants producing 2n gametes. Tetraploid plants were produced by unilateral (USP) and bilateral (BSP) sexual polyploidization from diploid (2x) mutants selected for their high production of 2n pollen and 2n eggs. When breeding for productivity in forage species such as alfalfa, one of several factors to be taken into account is the presence of metabolites with an anti-nutritional activity. Among these substances are saponins, because they can affect the quality of green forage and protein concentrates. Based on these considerations, a specific programme of genetic selection was undertaken to obtain new high yield alfalfa varieties with a low saponin content in the aerial parts of the plants. A number of plants obtained by USP and BSP, along with 2x and 4x controls, were evaluated for saponin content and type. A first determination was made by applying a semi-quantitative, micro-hemolytic test to verify the presence and semi-quantitative concentration of biologically active saponins. The test was performed on leaf samples harvested in autumn and spring, in order to estimate possible differences in saponin content among various plant groups and between different growth seasons.

The results of the hemolytic test showed differences among the groups of plants, as well as variations between the two growth seasons. Specifically, the frequency and level of hemolysis decreased in the BSP group from autumn to the following spring, while in the USP group the presence of strongly hemolytic individuals was confirmed. The 4x and 2x controls were only slightly hemolytic. 

The next step of the research consisted in distinguishing among the various sapogenins from plant extracts by quantitative chemical methods, and in determining the content of medicagenic acid, the sapogenin most largely represented in the genus Medicago. Qualitative and quantitative analyses of derivatized sapogenins were carried out by Gas Chromatography and GC-MS. These analyses, still in progress, will allow us to define the possible utilization of medicagenic acid as a chemical marker of the selected genotypes to obtain new high yield and high quality varieties. We will also look for a possible dose effect of the saponins in the tetraploidization process as a result of the doubling of the number of alleles involved in the determination of this metabolite.

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

Poster Abstract - 5.59

KANAMYCIN-RESISTANT ALFALFA HAS A POINT MUTATION IN THE 16S PLASTID rRNA

D. ROSELLINI*, P.R. LAFAYETTE**, P. BARONE*, S. CAPOMACCIO*, F. VERONESI*, W.A. PARROTT**

*) Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali, Università degli Studi di Perugia, Perugia, Italy
**) Center for Applied Genetic Technologies, University of Georgia, Athens, GA 30602, USA

antibiotic resistance, homoplastomy, Medicago sativa L., plastid, Ribosomal RNA 

Kanamycin, an aminoglycoside antibiotic, is used frequently to obtain transgenic plants by transforming plant cells with the nptII gene from E. coli as the selectable marker. Aminoglycoside antibiotics are toxic to prokaryotic cells and chloroplasts because they bind to the 30S ribosomal subunit at the decoding site (A-site) which causes the amino acid incorporation error rate to increase and inhibit ribosome translocation. Kanamycin-sensitive prokaryotic ribosomes, including those of the plastid and mitochondrion, have an A at the 1408 position of the 16S rRNA.  Eukaryotic, kanamycin-insensitive, cytoplasmic ribosomes, have a G at position 1408 (E. coli base numbering). Spontaneous mutations to kanamycin resistance occur in Chlamidomonas reinhardtii (1, 3) but have not been reported to date in higher plants.
In attempting to transform the plastid genome of alfalfa using a kanamycin resistance gene as the selectable marker (2), somatic embryos were obtained after a minimum of two months of culture of leaf explants in the presence of 50 mg l-1 kanamycin. Kanamycin resistance was confirmed by 4 to 5 regeneration cycles on kanamycin-containing media using mature somatic embryos to start each cycle. PCR and Southern analyses demonstrated that the kanamycin resistance gene was not present in the resistant embryos. Resistant plants produced 85% less biomass than controls and produced very few flowers. Their leaves were chlorotic as they formed and slowly became green. When leaf explants from resistant plants were used for regeneration, they produced less callus and embryos than control leaves on both kanamycin and kanamycin-free media; kanamycin presence resulted in slower regeneration and further reduced callus and embryo production, indicating that kanamycin resistance was not complete.

Sequencing of the plastid DNA region corresponding to the decoding site of the16S rRNA in 10 independent resistance events revealed a AC transversion at position 1357 of the 16S plastid rDNA, the same site at which an AG conversion confers kanamycin resistance to C. reinhardtii. 

All the progeny of the resistant plants obtained through somatic embryogenesis in the absence of kanamycin had the mutated phenotype, indicating that the mutation was homoplastomic. 

We propose that kanamycin resistance in alfalfa requires homoplastomy for the described plastid rDNA mutation, which reduces binding affinity of the plastid ribosome for the antibiotic and adversely affects plastid translation.

REFERENCES
1. Bartlett SG, Harris EH, Grabowy CT, Gillham NW, Boynton JE (1979) Ribosomal subunits affected by antibiotic resistance mutations at seven chloroplast loci in Chlamydomonas reinhardtii. Mol Gen Genet 176: 199-208
2. Bateman JM, Purton S (2000) Tools for chloroplast transformation in Chlamydomonas: expression vectors and a new dominant selectable marker. Mol Gen Genet 263: 404-410
3. Harris EH, Burkhart BD, Gillham NW, Boynton JE (1989) Antibiotic resistance mutations in the chloroplast 16S and 23S rRNA genes of Chlamydomonas reinhardtii: correlation of genetic and physical maps of the chloroplast genome. Genetics 123: 281-292.

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

Poster Abstract - 5.60

gfp as a useful reporter for transient expression and stable transformation in alfalfa (Medicago sativa L.)

M. BELLUCCI*, F. DE MARCHIS*, R. MANNUCCI**, S. ARCIONI*

*) Institute of Plant Genetics, Research Division of Perugia, CNR, Via Madonna Alta, 130, 06128 Perugia, Italy
**) Dept Clin. Exp. Med. Sect. Internal Medicine & Oncology, Perugia University Medical School, Policlinico Monteluce, 06122 Perugia, Italy

transgenic alfalfa, green fluorescent protein, transit peptide, plastid targeting, bombardment

Reporter genes coding for proteins that possess an unique activity, or produce a signal that allows the protein to be easily distinguished from other proteins, are essential tools of molecular biology. The use of jellyfish green fluorescent protein (GFP) as a reporter in plant biology research is extensively documented. Transient expression of two green fluorescent protein (GFP) gene constructions was compared in alfalfa (Medicago sativa L.) leaves: the gfp containing the mutation S65C in an essentially mgfp4 background (Reichel et al. 1996) coding for GFP S65C versus the mgfp5-ER (Haseloff et al. 1997) coding for mGFP5. The fluorescent signal was brighter in cells transiently transformed with the gfp S65C gene than with the mgfp5-ER gene. Based on these results, the gfp S65C gene was used to assemble other chimaeric genes. First, a chloroplast-targeted GFP producing green-fluorescent chloroplasts inside transiently transformed cells was constructed. Then, a chloroplast-targeted fusion protein between a maize protein (beta-zein) and GFP was assembled, and this protein accumulated in small aggregates into the chloroplasts of transiently transformed cells. Furthermore, the gfp S65C gene was used to obtain transformed alfalfa plants by particle bombardment. Strong GFP expression was observed in several kinds of tissues from T0 plants derived from a single genotype.

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

Poster Abstract - 5.61

ECTOPIC EXPRESSION OF “BANYULS” INCREASES LEAF TANNIN LEVELS IN LOTUS CORNICULATUS BUT NOT IN MEDICAGO SATIVA

S.M. HESAMZADEH HEJAZI, F. PAOLOCCI, F. DAMIANI

C.N.R. Istituto Genetica Vegetale sez. di Perugia

condensed tannins, forage legumes, genetic transformation, gene expression

Modification of tannin levels in forage tissue of leguminous plants is essential for increasing palatibility and protein assimilation by ruminants.

From a A.thaliana “trasparent testa” (seed coat tannin free) mutant a structural gene supposed to code for LAR, the enzyme that from the reduction of leucoanthocyanidin produces cathechin, was isolated (Devic et al., 1999) Catechin is the starting element for the condensation of monomeric elements of tannins. Such gene after subsequent investigation (Xié et al., 2003; Abrahams et al., 2002) resulted not coding for a LAR (leucoanthocyanidin reductase) but for ANR (anthocyanidin reductase) giving origin to epicathechin, a catechin enantiomer.

From such observation it was supposed that condensed tannins produced in Arabidopsis were different from those of leaf legumes.

From previous transformation in tobacco it was observed that banyuls redirected anthocyanin synthesis to condensed tannins, and flowers lost the characteristic pink color (Xié et al., 2003).

Banyuls was inserted under constitutive promoter in the weak tannin producer Lotus corniculatus genotype S50 (Carron et al., 1994) and in Medicago sativa Regen Sy27 through Agrobacterium transformation. Several transgenic plants were produced from each species. They were tested for transgene copy number and expression. Qualitative and quantitative analysis of condensed tannins was performed on leaves and flowers. Medicago transgenic resulted identical to the mother plant therefore did not show any tannin on leaves, and flowers reacted with tannin specific reagent producing a pink stain not characteristic of tannins, both in transgenic and controls. Leaves of lotus showed increased levels of tannin respect to the starting plant and were high variable for tannin levels in strict relation with transgene copy number. One transgenic did not accumulated any tannins in leaves and contained several copy of banyuls, possibly a silencing mechanism occurred.

Preliminary results of such work indicated that in forage legumes Banyuls can not induce autonomously tannin accumulation neither in leaves nor in flowers however if the tissue is committed for tannin accumulation banyuls can strongly increase such levels. The theoretical consequences of such observation are that a) epicatechin can substitutes cathechin in tannin formation, b) the low level of tannins can derive from the low levels of monomeric elements c) that an enzymatic complex for condensation and accumulation of tannin should be active, otherwise such elements are not accumulated.

Bibliography 
Abrahams et al (2002) Plant Physiol 130: 561-576
Carron et al. (1994) Theor Applied Genet 87:1006-1015
Devic et al. (1999) Plant Journal 19:387-398.
Xié et al. (2003) Science 299:396-399