LOX ACTIVITY AND CAROTENOIDS CONTENT OF SELECTED … fileC.R.A. Centro Ricerche per la Cerealicoltura, S.S. 16 km 675, 71100 Foggia, Italy – [email protected] lipoxygenase activity,

Proceedings of the 50th Italian Society of Agricultural Genetics Annual CongressIschia, Italy – 10/14 September, 2006ISBN 88-900622-7-4

Poster Abstract – B.01

LOX ACTIVITY AND CAROTENOIDS CONTENT OF SELECTED DURUMWHEAT CULTIVARS: IMPORTANCE OF GENOTYPE AND IMPACT OFPASTA PROCESSING

V. DE SIMONE, D.B.M. FICCO, D. TRONO, G. SCHIAVONE, L. CATTIVELLI, P. DE VITA

C.R.A. Centro Ricerche per la Cerealicoltura, S.S. 16 km 675, 71100 Foggia, Italy –[email protected]

lipoxygenase activity, carotenoids, durum wheat, pasta processing

Bright yellow pasta colour is the result of the natural carotenoid pigments present in the seeds,of their residual contents after the storage of grain or semolina and after milling, and is also affectedby their oxidative degradation by lipoxygenase (LOX) activity during pasta processing. With themain objective to explore and try to improve the nutritional value of durum wheat and durum wheatbased products by a multidisciplinary approach involving germplasm evaluations, molecularstrategies and transformation processes, ninety-four cultivars and breeding lines of durum wheat(Triticum durum Desf.) were screened to quantify the variation of carotenoids content and LOXactivity by means of spectrophotometric assays. The seed samples for each genotype were harvestedfrom plants grown in the same location (Foggia, Italy) in 2004-2005 growing-season. Resultsshowed that there was great variability of carotenoids content and LOX activity suggesting thepossibility to select cultivars possessing high yellow-coloured seeds with a reduced LOX activity.On the basis of this screening we selected four durum wheat cultivars contrasting for endogenouscarotenoids and LOX activity: cv. Primadur with High Carotenoid Content (HCC) and HighLipoxygenase Activity (HLA), cv. Cosmodur with High Carotenoid Content (HCC) and LowLipoxygenase Activity (LLA), cv. Trinakria with Low Carotenoid Content (LCC) and HighLipoxygenase Activity (HLA), cv. Creso with Low Carotenoid Content (LCC) and LowLipoxygenase Activity (LLA), with the aim to investigate, in the present study, their endogenousrole and susceptibility during the pasta processing. The results reported in the poster suggest thatdifferent level of endogenous LOX activity and carotenoids content have differential effects onpasta colour and its degradation during pasta processing. Pasta colour could be improved bybreeding cultivars for a high level of carotenoids and a low LOX activity, in order to preserve thispotential during milling and pasta processing.



USE OF DNA MARKERS TO IDENTIFY DURUM WHEAT CULTIVARS INTHE PRODUCTION OF ALTAMURA PDO BREAD

A. PASQUALONE*, C. MONTEMURRO**, W. SABETTA**, R. SIMEONE**

*) “Agro-food Industries” section of Management of Agro-zootechnic and Forestry Systems’department (Pro.Ge.S.A.) – University of Bari, Via Amendola 165/a, 70126 Bari, Italy**) “Genetic and Breeding” section of Agro-forestry and enviromental Biology and Chemistrydepartment (Di.B.C.A.) – University of Bari, Via Amendola 165/a, 70126 Bari, Italy

Altamura PDO bread, semolina, Triticum turgidum L. var. durum, microsatellites, cultivaridentification

Altamura bread is a very popular type of bread produced in Southern Italy starting from durumwheat re-milled semolina. Because of its special organoleptic characteristics and resistance tostaling a protected denomination of origin (PDO) mark was awarded to this bread at European level.According to the official requirements of the mark, the technology process has to be carried out by aprolonged sponge-dough fermentation procedure based on sourdough, and special attention to thevarietal composition of raw materials (durum wheat re-milled semolina deriving from cultivarsAppulo, Arcangelo, Duilio and Simeto, alone or in combination, at least 80% of the total) and totheir origin has to be fulfilled.

In this work, in order to enable the check of the above-mentioned varietal composition, amethod of analysis based on DNA microsatellites has been set up. At this purpose, has beenessential prior to investigate about the quality of DNA, in terms of integrity, in the examinedproducts. A good quality DNA, indeed, is important to obtain reliable and reproducible resultsensuring the set up of accurate and precise DNA based methods. DNA was thus extracted fromstarting semolina of the cultivars Appulo, Duilio, Arcangelo and Simeto, and from doughs both rawand cooked at different temperatures (from 80 to 250°C). The direct electrophoretic exam of theextracted DNA showed the presence of genomic DNA in semolina and doughs cooked attemperatures below 150°C. Contemporarily, the presence of degraded DNA was detected in doughsstarting from 80°C treatments. The dough which underwent severe treatments (200 and 250°C) justshowed degraded DNA with the shortest fragments having dimensions of about 150 bp.

As a consequence, it is advisable to perform amplifications using primers of short sequences, inorder to have the best probability to obtain good amplification levels even in presence of highlydegraded DNA. A set of 10 microsatellite primers was thus considered, since these molecularmarkers lead to amplicons of about 200 bp, with the aim of searching the most polymorphic primersamong the examined cultivars. The obtained results indicated that basing on the exam of threeselected DNA microsatellite sequences it was possible to identify univocally each of the fourcultivars foreseen by the official recipe of Altamura bread. This method will be useful for checksduring both milling and processing into certified PDO bread.



STUDIES ON DURUM AND BREAD WHEAT LINES WITH LOW-AMYLOSE STARCH

F. SESTILI*, A. SALIOLA BUCELLI*, E. BOTTICELLA*, B. MARGIOTTA**,M. URBANO**, D. LAFIANDRA*

*) Department of Agrobiology and Agrochemistry, University of Tuscia, Via San Camillo de Lellissnc, 01100 Viterbo, Italy - [email protected]**) Institute of Plant Genetics – CNR, Bari (Italy), Via Amendola 165/a, 70010 Bari

starch, wheat, waxy, amylose

Starch is the major storage constituent and it is composed of two different glucan polymers:amylose and amylopectin. Amylose is a linear polymer of α-1,4 linked glucose residues with veryfew α-1,6 branches and it is about 20-30% of total starch; whereas amylopectin exists as shorterchains of α-1,4 linked glucose residues that are connected by α-1,6 glycosidic linkages resulting ina branched structure and constitutes the remaining 70-80% of starch. The relative amounts ofamylose and amylopectin influence the physical-chemical properties of starches. By altering levelsof key enzymes for the regulation of starch synthesis, it is possible to generate novel starches withnew unique properties. The enzymes directly involved in amylopectin biosynthesis are starchsynthases (SSs) and starch branching enzymes (BEs). The waxy protein is a granule-bound starchsynthase responsible for amylose synthesis; in bread wheat (genomic formula AABBDD) threedifferent isoforms are present which are encoded by three genes designated as Wx-A1, Wx-B1 andWx-D1 located, respectively, on chromosome arms 7AS, 4AL and 7DS. In durum wheat (AABB)two isoforms are present, associated to the Wx-A1 and Wx-B1 loci.

Partial waxy mutant lines, characterised by the lack of one or two waxy proteins, have beenidentified by extensive electrophoretic analysis of durum and bread wheat. Crossing of thesematerials has permitted the combination of different null alleles detected both in a bread wheat line(N11) and in a durum wheat cultivar (Svevo) with the production of the entire set of partial linesalong with the total waxy. Comparison of partial waxy lines (single and double null) with wild-typegenotype has showed that amylose content is not linearly proportional to the number of thefunctional waxy genes. In fact there is a difference of 1-7% amylose content between wild-type andpartial waxy mutants, whereas the total waxy genotype presents a drastic reduction of amylosecontent (0-1% of total starch). To investigate if the functional waxy gene compensated for the lackof other isoforms by producing more transcript and consequently more enzyme or increasingenzyme activity, a set of analyses has been performed on developing endosperms, both at thetranscript and protein level.

In order to achieve this, gene specific primers have been identified and used for RT-PCRanalyses on wheat kernels collected at different development stages.

The effects of the different waxy mutations on starch-pasting properties have been assessed byRVA (Rapid Visco Analyzer) analysis.



QUANTITATIVE DETECTION OF FUSARIUM IN GRAIN OF WHEATPLANTS INFECTED AT DIFFERENT DEVELOPMENTAL STAGES

C. MORCIA*, P. FACCIOLI*, V. ROSSI**, G. DELOGU*, V. TERZI*

*) C.R.A., Istituto Sperimentale per la Cerealicoltura, Via San Protaso 302, 29017 Fiorenzuolad’Arda, Italy – [email protected]**) Istituto di Entomologia e Patologia vegetale, Università Cattolica del Sacro Cuore, Via EmiliaParmense 84, 29100 Piacenza, Italy

Fusarium graminearum, Fusarium culmorum, Real time PCR detection, mycotoxin, wheat

Several DNA-based methods have recently been developed to detect and quantify the presenceof mycotoxigenic fungi from complex substrates. In particular, real-time PCR based analyses enablethe tracking of fungal nucleic acids in wide range of samples, allowing quantitative diagnosis ofpathogen levels in different plant tissues during the growing season, in grains after harvest, in foodand feed (Terzi et al., in press). Both generic PCR assays as well as species-specific PCR assaysthat target Fusarium species associated with FHB in small grain cereals have been developed.

In this work three common wheat varieties (Bilancia, Centauro and Sagittario) and three durumwheat varieties (Duilio, Simeto and San Carlo) were artificially inoculated in different stage ofdevelopment with strains of Fusarium graminearum and Fusarium culmorum isolated from seedscollected in bread wheat fields in Italy (Emilia-Romagna) and PCR-characterised for chemotype.

A PCR real time approach has been used to monitor and quantify the presence of Fusariumgraminearum and culmorum in mature grain tissues of these plants to evaluate the opportunity ofusing this analytical tool for early diagnosis.

TERZI V. MORCIA C., FACCIOLI P., FACCINI N., ROSSI V., CIGOLINI M.,CORBELLINI M., SCUDELLARI D., DELOGU G. 2006. Fusarium DNA traceability along thebread production chain. International Journal of Food Science and Technology, in press



IDENTIFICATION AND VALIDATION OF CANDIDATE GENES FORBARLEY MUTANTS ALTERED IN PLANT ARCHITECTURE: ASYNTENY-BASED APPROACH

S. CIANNAMEA*, L. NICOLOSO***, L. ROSSINI**, F. SALAMINI*, C. POZZI*

*) Fondazione Parco Tecnologico Padano, Località Cascina Codazza, Via Einstein, 26900 Lodi,Italy**) Dipartimento di Produzione Vegetale, Università degli Studi di Milano, Via Celoria 2,20133 Milan, Italy***) Università degli Studi di Milano, Via Celoria 2, 20133 Milan, Italy

barley, synteny, candidate gene (CG), Single Nucleotide Polymorphism (SNP)

In our laboratory, a collection of barley mutants affected in plant height (brachytic, brh;slender dwarf, sld4), shoot and inflorescence branching (absent lower laterals, als; branched1,brc1; uniculm, cul; double seed 1, dub1; many noded dwarf, mnd6; six rowed-spike 1, vrs1),development of leaves (liguleless, lig) and leaf-like organs (calcaroides, cal; short awn, lks;suppressors of Hooded, suK; third outer glume, trd; triple awned lemma, trp), has provided astarting point for genetic dissection of plant development.

Linkage analysis led to locate 40 of the corresponding loci in a molecular map of the barleygenome. Based on map position, rice syntenous regions were revealed for 23 of these loci and theirannotation led to the identification of candidate genes (CGs) for the mapped barley mutants. Thisanalysis was conducted especially for those rice mutants which are phenotypically similar to ourbarley mutants. There are evidences that the barley genomic regions hosting the liguleless andbranched 1 (brc1, lig) loci are colinear with the rice regions containing the Liguleless and frizzypanicle genes, respectively. The branched 1 mutant is of particular interest because additional earsdevelop from the main inflorescence and the rachilla is converted into rachis. This phenotype hassimilarities with branched silkless1 (bd1) of maize, and frizzy panicle (fzp) of rice which areaffected in the transition from spikelet to floral meristem identity.

Partial sequence of the gene putatively controlling the brc 1 locus (HvFZP) was obtained byPCR from genomic DNA using degenerate primers designed on the BD1 and FZP genes. Toconfirm that the barley hortologous of FRIZZY PANICLE is the CG of the barley brc 1 locus, asingle nucleotide polymorphism (SNP) analysis is being conducted on populations derived from thecross between the mapping parents of the PxN map and the mutant: homozygous recessive anddominant F2 plants are being used to investigate cosegregation between the brc-1 phenotype and theHvFZP gene.



GENETIC VARIABILITY IN TWO-ROWED BARLEY FOR IN VIVOβ-GLUCAN DEGRADATION

A. GIANINETTI, B. FERRARI, F. FINOCCHIARO, P. FRIGERI, A. M. STANCA

CRA - Experimental Institute for Cereal Research, Fiorenzuola d’Arda (PC), Italy

β-glucanase, β-glucan solubilase

Two-rowed barley genotypes, varieties and advanced lines have been studied for enzymic β-glucan degradation. Malt β-glucan content is an important quality feature and, in turn, it dependsupon barley β-glucan content as well as on β-glucanase activity during modification. Anotherenzyme, β-glucan solubilase, has been repeatedly, but unsatisfactorily, suggested to precede β-glucan depolymerization by β-glucanase. Actually, neither solubilase has been univocallyidentified, nor solubilizing activity has been uncontroversially proven to be different from that of β-glucanase itself.

Our approach was the following: barley genotypes were characterized for parameters related tomalting quality; some of these genotypes were selected for their wide differences and monitored forthe degradation of β-glucans and the development of β-glucan-degrading enzymes during malting(a dedicate assay for the measurement of β-glucan solubilization activity was developed). Abiphasic model for β-glucan degradation implying sequential action of solubilase and β-glucanasewas compared to a monophasic model that assumes all β-glucans are essentially depolymerized byβ-glucanase. The comparison was performed by formulating these models in terms of in vivokinetics, so that confirmatory regression analysis could be used to test their fitting to the observeddata.

Results showed β-glucan degradation is mostly monophasic, notwithstanding the role of asmall fraction of ‘masked’ β-glucans in malting quality remains unclear. However, the genotype-dependent kinetic rate constant (indicating β-glucan degradability), in addition to β-glucanaseactivity, is suggested to play a relevant role in malting quality.

We identified the variety Scarlett as the best one for this trait; consequently, Scarlett isconfirmed to be at top level for malting quality, but even one of our advanced lines, Fior 7054, hadhigh values of β-glucan solubilase.



DIFFERENTIAL PROCESSING OF LOW MOLECULAR WEIGHTGLUTENIN SUBUNITS MET- AND SER- TYPES AT THEIR N-TERMINALEND

P. FERRANTE*, R. D’OVIDIO*, D. LAFIANDRA*, A. CERIOTTI**, W.H. VENSEL***,D.D. KASARDA***, S. MASCI*

*) Tuscia University, Dept. of Agrobiology and Agrochemistry, Viterbo, 01100, Italy**) IBBA, CNR, 20133, Milan, Italy***) U.S. Dept. of Agriculture, A.R.S., W.R.R.C., Albany, 94710, U.S.A.

wheat, low molecular weight glutenin subunits, N-terminal processing, Nicotiana benthamiana

Gluten is a protein complex that confers unique visco-elastic properties to wheat doughs and isof critical importance in determining end product quality. Gluten is composed by gliadins andglutenins. Gliadins are monomeric proteins in which cysteines, if present, form only intra-moleculardisulphide bonds and are classified into α/β-, γ- and ω-gliadins according to their mobility in lacticacid PAGE. Glutenins form polymers whose subunits are joined together by disulphide bonds. Afterchemical reduction, they release high and low molecular weight glutenin subunits (HMW-GS andLMW-GS, respectively) whose amount, structure and interactions into the polymeric networkstrongly influence technological properties of wheat doughs. Whereas functional and structuralproperties of HMW-GS have been extensively investigated, LMW-GS are much less characterizeddue to their greater number and heterogeneity. On the basis of their N-terminal amino acidsequences, LMW-GS have been classified into two main groups, namely LMW-s (or LMW-Ser)and LMW-m (or LMW-Met) types. LMW-s are the most common and their amino acid sequencesstart with SHIPGL-; conversely LMW-m show more various sequences represented byMETSHIPGL-, METSRIPGL-, METSCIPGL-.

It has been hypothesized that the substitution of a threonine at position 23 of the immaturepolypeptide by an asparagine residue could determine a differential processing at the N-terminalend of LMW-s type sequences, that might generate the cleavage of the peptide MEN by anasparaginyl endoprotease. In order to investigate the correctness of this hypothesis, we haveexpressed two LMW-GS in Nicotiana benthamiana by using an episomal vector based on PVX(Potato Virus X). The two LMW-GS expressed are represented by wild types LMW-m and LMW-s,along with the mutated forms at position 23. In particular, in the LMW-m type, the threonine atposition 23 was substituted by an asparagine and in the LMW-s type the asparagine was converselyreplaced by a threonine. Preliminary results give indication that the N-terminal amino acid sequenceof the mutated LMW-m type (T23N) might be SCISGLERWQ- whereas the sequence of the wildtype LMW-m is METSCISGLE-, thus supporting the hypothesis that the presence of an asparaginein position 23 causes a differential processing at the N-terminal end of the mature polypeptide. Thiswork is still in progress and we are currently purifying the wild type and mutated LMW-s type inorder to determine their N-terminal amino acid sequence.



REDOX CONTROL OF GLUTENIN SUBUNIT ASSEMBLY IN TOBACCOPROTOPLASTS

A. LOMBARDI, C. CASTELLAZZI, D. ROSIELLO, A. CERIOTTI

Consiglio Nazionale delle Ricerche, Istituto di Biologia e Biotecnologia Agraria, Via Bassini 15,20133 Milano, Italy

wheat, protein assembly, endoplasmic reticulum, glutenin subunits, polymerization

The maintenance of a correct redox state in the endoplasmic reticulum (ER) is essential toallow efficient disulphide bond formation and reshuffling, protein folding and protein degradation.Here we show that the polymerization pattern of a low-molecular-weight glutenin subunit (LMW-GS) expressed in tobacco leaf protoplasts is influenced by treatments that can potentially affect theER redox state. Treatment with a reducing agent was sufficient to reduce interchain disulphidebonds in vivo and the effect could be readily reverted by restoring oxidizing conditions. Conversely,treatment with an oxidizing agent promoted polymer formation. This indicates that the ER redoxstate plays a crucial role in determining the polymerization state of the LMW-GS. To assess the roleof cytosol in the control of the ER redox state, we analyzed the polymerization state of LMW-GSin microsomes isolated from tobacco leaf protoplasts. While cytosol removal led to the formation oflarge aggregates, addition of reduced glutathione was sufficient to restore the initial polymerizationpattern. These results raise the possibility that a flux of reducing equivalents from the cytosol to theER, possibly in the form of reduced glutathione, is essential to establish a correct redox state and toset the initial polymerization pattern of LMW-GS.



FUNCTIONAL MAPS FOR DEFECTIVE KERNEL MUTANTS IN MAIZE

R. VALSECCHI*, L. PASINI*, F. SALAMINI**, A. MAROCCO*

*) Institute of Agronomy, Università Cattolica S. Cuore, Via E. Parmense 84, Piacenza, Italy, 29100- [email protected]**) Parco Tecnologico Padano, Via Haussmann 7, Lodi, Italy, 26900

AFLP markers, bulk segregant analysis, de mutants, Zea mays

The cereal caryopsis is specialized to convert assimilate solutes rapidly to provide acarbohydrate and protein reserve for the germinating seed. The endosperm tissue has in the courseof this specialization process acquired distinctive pattern of gene expression. Our efforts haveconcentrated on the role of two mutant types, a set of viable reduced endosperm mutant (the declass), and miniature-like mutants. Mutants were obtained by selfing plants of open pollinatedmaize varieties or from mutagenized and random tagging. Allelism tests showed the majority ofmutants were not linked indicating that the phenotype can be caused by mutations in manyunrelated genes. AFLP markers linked to individuals alleles were identified. For this purpose F3families in which the mutations segregated in crosses with the mapping parents were used. Toscreen a large number of primer combinations, the bulked segregant analysis was carried out. Thenew AFLP markers were integrated into the intermated B73XMo17 genetic map. The maize mapwill be enriched of AFLP markers and mutant loci for which developmental mutants are described.



FOOD GENOMICS: AFLPS APPLIED TO OLIVE CULTIVARS AND OILS

S. PAFUNDO*, M. BUSCONI**, C. AGRIMONTI*, C. FOGHER**, N. MARMIROLI*

*) Division of Genetics and Environmental Biotechnology, Department of Environmental Sciences,University of Parma, Parco Area delle Scienze 11/A, 43100 Parma, Italy**) Istituto di Botanica e Genetica vegetale, Univesità Cattolica S. Cuore, Via E. Parmense 84,Piacenza, Italy

AFLPs, olive cultivar, olive oils, food traceability

Determining the origin and authenticity of olive oils with methods based on DNA analysisrepresent an attractive choice. This study reported the molecular analyses made on some olivecultivars with Amplified Fragments Length Polymorphisms (AFLPs) and the characterization of thederived olive oils with the same molecular markers. It was reported also a comparison within theAFLP profile of a cultivar and that of its correspondent monovarietal olive oil. Statistical analyseson reproducibility of results obtained from olive oils in different conditions were reported.



FOOD GENOMICS, APPLICATION TO OLIVE OIL TRACEABILITY

S. PAFUNDO*, M. VIETINA*, D. LOI*, L. PALMIERI**, C. AGRIMONTI*, E. MAESTRI*,P. DONINI***, N. MARMIROLI*

*) Division of Genetics and Environmental Biotechnology, Department of Environmental Sciences,University of Parma, Via G.P. Usberti 11/A, 43100 Parma, Italy**) SafeCrop Centre – Istituto Agrario di San Michele all’Adige, Via Mach 1, 38010 S. Micheleall’Adige (TN), Italy***) NIAB, Huntigdon Road, CB3 0LE, U.K.

food genomics, molecular markers, olive oil, traceability

In Food Genomics, DNA analyses with molecular markers has offered the shortcut towards thegenomic comprehension of complex organisms. The availability of micro-DNA extraction methodscoupled with selective amplification of the smaller extracted fragments with molecular markerscould equally bring to a breakthrough in Food Genomics: the identification of origin andauthenticity of foodsback to their original components. Molecular markers as Simple SequenceRepeats (SSRs), Single Nucleotide Polymorphisms (SNPs), Amplified fragment lengthpolymorphisms (AFLPs), Sequence Characterized Amplified Regions (SCARs) have been appliedin order to trace olive oil, using both qualitative andquantitative PCR. Nevertheless, the directapplication of some of these markers in the analysis of DNA extracted from food matrices iscomplicated by the proprieties of the DNA extracted: high degradation and richness in inhibitors ofenzymatic reactions, so it was important to study their possible use and their limits.



MOLECULAR AND CHEMICAL CHARACTERISATION OF “COLLINA DIBRINDISI” PDO OLIVE OIL

A. PASQUALONE*, C. MONTEMURRO**, C. SUMMO*, F. CAPONIO*, R. SIMEONE**,A. BLANCO**

*) Department of Management of Agro-zootechnic and Forestry Systems’ (Pro.Ge.S.A.), section ofAgro-food Industries, University of Bari, Via Amendola 165/a, 70126 Bari, Italy**) Department of Agro-forestry and Environmental Biology and Chemistry, section of Geneticsand Breeding, University of Bari, Via Amendola 165/a, 70126 Bari Italy

Olea europaea L., microsatellites, PDO mark, virgin olive oil

Apulia is the Italian region with the highest olive oil production. Part of this production isrepresented by oils that, owing to their typicality, have obtained marks of protected denomination oforigin (PDO) at European level. These products have a higher economic value with respect to thecorresponding conventional foodsftuffs so that it is important to avoid possible mixtures orsubstitutions of raw materials and to set up effective methods to enable checks during processing.DNA analysis enables genome fingerprinting with consequent identification of differentindividuals. In the agro-food industry this can have interesting applications for the identification ofspecies and cultivars of both raw materials and processed food, very useful in case of PDOproducts.

Previous researches applied the analysis of microsatellite markers to the DNA extracted fromvirgin olive oils obtained from single cultivars (Pasqualone et al., 2004). In this work, we appliedmicrosatellite analysis to the characterisation of “Collina di Brindisi” PDO olive oil, one of theApulian oils which typicality has been recognised at European level. Besides, the results of themolecular analysis were compared to those of chemical analyses.

Six samples of Collina di Brindisi PDO oil were collected from three different oil mills,together with six samples of monovarietal oils used for the preparation of the PDO mix (Leccino,Ogliarola salentina, Coratina, Frantoio, Picholine, Cellina di Nardò) according to the officialproduction process. After oil centrifugation and DNA extraction from the remaining cell residues,the amplification reactions were carried out and eight microsatellite primer pairs taken fromliterature (Carriero et al., 2002; Cipriani et al., 2002) were tested. Good amplification levels wereobtained even starting from filtered clear oils. A preliminary phase of identification was carried outon the monovarietal oils with comparison to an olive DNA data base composed of sixty olivecultivars native to Italy, Spain, France and Greece (Montemurro et al., 2005). It indicated that inone case the putative cultivar identification effected in the olive mill was wrong (a sample that theolive miller attributed to Leccino was actually a Ogliarola salentina oil), thus remarking the need ofmore reliable and sophisticated means of cultivar checking. After that, the PDO oil characterisationproceeded by analysis of eight microsatellites. We expected a complex electrophoretic pattern dueto the overlapping of the bands to the single cultivars. The obtained results showed, indeed, that theelectrophoretic pattern of PDO oil was mainly consistent with that of Ogliarola salentinamonovarietal oil. This was probably due to the fact that, in this kind of oil, this cultivar has to be

present in amounts higher than 70%, so that it is the prevalent cultivar and very often is almostalone. So, the eventual absence of the Ogliarola salentina pattern in case of oils sold with theCollina di Brindisi PDO mark can reveal a fraud.

Finally, the chemical analyses (both parameters foreseen by the current rules such as freeacidity, peroxide value, UV spectrophotometric constants, sterols, and minor compounds analysissuch as total phenol content) solely indicated the belonging to the extra virgin olive oil category butdid not enable to evidence any significant difference between Collina di Brindisi PDO oil and otheroils belonging to the same commercial class because of a great similarity in the law parameters andan important environmental effect on the minor compounds.



MICROSATELLITE AND AFLP MARKERS TO IDENTIFY ITALIAN ANDGREEK VIRGIN OLIVE OILS FROM SINGLE CULTIVARS

A. PASQUALONE*, C. MONTEMURRO**, P. KALAITZIS***, S. BIHMIDINE***,S. SPANIOLAS****, G. TUCKER****, R. SIMEONE**, A. BLANCO**

*) Department of Management of Agro-zootechnic and Forestry Systems’ (Pro.Ge.S.A.), section ofAgro-food Industries, University of Bari, Via Amendola 165/a, 70126 Bari, Italy**) Department of Agro-forestry and Environmental Biology and Chemistry, section of Geneticsand Breeding, University of Bari, Via Amendola 165/a, 70126 Bari, Italy***) Department of Horticultural Genetics and Biotechnology – Mediterranean AgronomicInstitute, Chania, Greece****) Division of Nutritional Sciences, School of Biosciences, University of Nottingham, SuttonBonington Campus, Leicestershire, LE12 5RD, UK

Olea europaea L., microsatellites, AFLP, cultivar identification, virgin olive oil

Up to now, various categories of DNA-based markers have been employed in Olea europaea L.with cultivar identification purposes but in few cases they have been used to analyse the olive oils.Microsatellites were found to be able to effectively amplify the degraded DNA extracted from oils(Pasqualone et al., 2004, 2005). AFLP markers are characterised by a high efficiency andinformativeness as well as by good reproducibility, and they show a higher number of polymorphicbands per amplification with respect to microsatellites. They have been already used with cultivaridentification purposes in O. europaea L., but using DNA extracted from leaves. The aim of thiswork was to compare the effectiveness of microsatellite and AFLP markers to identify Italian andGreek virgin olive oils from single cultivars.

Olive oils obtained from 10 single cultivars native to Italy (Cellina di Nardò, Leccino,Pendolino, Ogliarola barese, Toscanina, Frantoio, Cima di Melfi, Picholine, Coratina andNocellara del Belice) and 6 from Greece (Throboulia, Kalamon, Koroneiki, Mastoidis, Adramytiniand Valanolia) were analysed by means of microsatellite and AFLP markers. Eight microsatelliteprimer pairs taken from literature (Sefc et al., 2000; Carriero et al., 2002; Cipriani et al., 2002) andsix AFLP primer combinations were used. After oil centrifugation and DNA extraction from theremaining cell residues, the amplification reactions were carried out. The obtained results showedreproducible electrophoretic patterns in case of the use of microsatellite primers while in case ofAFLP some lack of reproducibility were encountered in the upper part of the gels contaning thehigher molecular weight bands. This was probably due to the high degradation level of startingDNA, since the olive oil processing technology involves strong mechanical stresses. Consideringthe zone of the AFLP gels lower than 800 bp, a good reproducibility was observed. On that basis,the mean polymorphism of AFLP markers was 38 polymorphic bands/total bands %. Regardingmicrosatellites, ten primer pairs (already selected during previous researches as being highlypolymorphic, Montemurro et al., 2005) lead to a total number of polymorphic band of 20. Thisremarked the usefulness of AFLP even thought the gels obtained by means of this markers have tobe carefully read exclusively in their lower zone.



TRACEABILITY OF OLIVE OIL USING MICROSATELLITES

M. LA MURA, I. FORONI., R. RAO

Department of Soil, Plant and Environmental Sciences, School of Biotechnology, University ofNaples “Federico II”, Via Università 100, 80055 Portici, Italy - [email protected]

traceability, olive oil, SSR molecular marker

Recently, the demand for higher food safety has aroused a great interest in the determination oforigin and authenticity of agro-food product, especially for extra virgin olive oil protected bydesignation of origin (PDO), that are highly related to the cultivars employed and the environmentalconditions of growth. Rebuilding the history and following the product in every single ring of thechain is a good tool to protect the consumers, safeguarding the quality of olive oil. Traceability canbe successfully performed by SSR molecular markers that permit to trace DNA even whenextracted from complex matrix such as olive oil.

The aim of this study was to evaluate the possibility of tracing the genetic origin ofmonovarietal virgin olive oils using DNA microsatellites. Five SSR primer pairs, were used tocharacterize the DNA extracted from leaves, drupes, and oils of Pisciottana cultivar. Amplificationproducts were separated on 2% agarose gel and on ABI PRISM 3100 genetic analyzer automatedsequencer (Applera).

The allelic profile of DNA extracted from the tissues and oil resulted identical thus confirmingthat traceability in olive agro food-chain is achievable with a reduced number of SSR loci.



MONOCLONAL ANTIBODIES AS MARKERS OF MATURATIONPROCESS IN GRAPE

M. MARSONI*, C. VANNINI*, A.S. NEGRI**, L. ESPEN**, A. ORLANDI***, M. NOLLI***,M. BRACALE*

*) Dipartimento Ambiente Salute e Sicurezza, Università dell’Insubria, Via H. Dunant 3,21100 Varese**) Dipartimento di Produzione Vegetale, Università degli Studi di Milano, Via Celoria 2,20133 MI***) ARETA International, Via R.Lepetit 34, 21040 Gerenzano (VA)

maturation, grape, product quality monoclonal antibodies, proteomics

Grape maturation is a complex process that begins with véraison and continues according to thegenetic varietal and clonal characteristics, the climatic conditions and the eco-physiological state ofthe vines. Whereas biochemical events involved in primary metabolism have been characterised indetail, the ones tied to secondary metabolism, in particular if concerning the synthesis of productswith a role in the organoleptic characteristics of the mature grape, remain largely unknown. Duringthe last few years, the techniques used to study the molecular and biochemical aspectscharacterising various physiological processes have evolved enormously. Grapevine is alsoconcerned by this advance in knowledge and there are consequently powerful tools andtechnologies available for the characterisation and management of the quality of wine-production.

We searched for monoclonal antibodies aimed at proteins whose expressions are subject tovariation during the process of grape maturation. In particular, we concentrated on the proteins ofthe grape skin since in that site most of the processes involved in fruit ripening are localized. Theproteins obtained by phenolic extraction from the skin of Barbera, taken at the moment of véraison(immature skin) and also seven weeks later (mature skin), were used for the immunization of Balb/cmice. Differential ELISA screening was carried out in order to select a panel of specific monoclonalantibodies for both maturation stages. We are currently verifying the specificity of the selectedantibodies by bidimensional electrophoresis. The antigens of greater interest will be identifiedthrough mass spectrometry.

The obtained results will allow the definition of a panel of new generation molecular markersto use in the identification of inter- and/or intra-varietal differences in ripening grape, thereforeverifying the possibility of creating a useful qualitative and quantitative diagnostic system for thecertification of the product quality. It is to be emphasised that, to this date, many studies have beenaimed at the analysis of wines, whereas less attention has been directed towards identifyingparameters useful for defining grape quality.



FUNCTIONAL GENOMICS APPROACHES FOR THE STUDY OF AROMADETERMINATION IN PEACH (PRUNUS PERSICA)

A. VECCHIETTI*, C. ORTUGNO**, F. BARALE**, B. LAZZARI*, O. AMBROSINO*,M. OSNATO**, C. POZZI*

*) Parco Tecnologico Padano - CERSA, Via Einstein, Località Cascina Codazza, 26900 Lodi, Italy**) Dipartimento di Produzione Vegetale, Università di Milano, Via Celoria 2, 20133 Milano, Italy

peach, volatiles, EST, secondary metabolism

Peach flavour consists of a huge variety of volatile compounds. Esters, alcohols, aldehydes,terpens, lactones, C6 compounds are the main components of the peach aroma and their relativeabundance is a fingerprint of a particular variety.

From GC/MS chromatogram of ripe peach fruit shikimic acid derivates (eugenol, isoeugenol,chavicol, methyl benzoate) are present in a high concentration. An enzyme involved in eugenolbiosynthesis belongs to the family of O-methyl transferase. O-methyl transferase (OMT) enzymescatalyse the transfer of a methyl group to an hydroxyl group of an acceptor molecule with theformation of its methyl ether derivates.

Among other volatiles compounds produced during ripening, lactones are a quantitativelyimportant part of the peach aroma. Lactones are supposed to derive from fatty acid degradation andan epoxide hydrolase (EPOX) is suggested to be involved in their production. Here we report thecloning, the expression and the mapping of genes coding for OMTs and EPOXs. Moreover wereport the primary data analysis of ESTs derived from peel cDNA library.



EXPRESSION IN MEDICAGO TRUNCATULA OF PHASEOLIN MUTATEDFORMS WITH DIFFERENT SUBCELLULAR LOCALIZATION

M. ARCHINTI*, B. BERTONCIN**, E. BARIZZA**, F. LO SCHIAVO**, A. VITALE*

*) Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche, Via Bassini 15,20133 Milano**) Università di Padova, Dipartimento di Biologia, Via Ugo Bassi 58/B, Padova

Medicago truncatula, plant transformation, storage proteins

Medicago truncatula is a forage legume. It is closely related to the world's major foragelegume, alfalfa. Unlike alfalfa, which is a tetraploid, obligate outcrossing species, M. truncatula hasa simple diploid genome (two sets of eight chromosomes) and can be self-pollinated.

M. truncatula has been chosen as a model species for genomic studies in view of its smallgenome, fast generation time, high transformation efficiency. Tight syntenic relationships havebeen established between M. truncatula, alfalfa, and pea, as well as Arabidopsis.

We have developed a method for the transformation and regeneration of M. Truncatulatransgenic plants, based on Agrobacterium-mediated embryogenic callus transformation.

We have thus expressed, under constitutive promoter, three different mutant forms ofphaseolin, Phaseolus vulgaris’ major storage protein. In transgenic tobacco leaves, these proteinsare located within different site of the secretory pathway and, consequently, accumulated at verydifferent levels according to the proteolytic activity in their final compartment. We show that intransgenic M. truncatula leaves the reporter proteins are all accumulated at high level. The proteinpatterns observed in protein-blotting experiments suggest that they reach their expected subcellularcompartments. Expression and subcellular localization studies are in progress. Our analysisindicates that M. truncatula is a good system for the production of heterologous proteins; furtherstudies are required to establish the advantages in the use of this plant as a bioreactor.



BREEDING OF THE ALFALFA STEM MORPHOLOGY FOR QUALITYTRAITS

C. SCOTTI, G. GNOCCHI, M. CARELLI, B. PINTUS, A. URSINO, M. ODOARDI

C.R.A. - Istituto Sperimentale Colture Foraggere, Viale Piacenza 29, 26900 Lodi, Italy –[email protected]

leaf /stem ratio, short internodes, M. sativa, synthetics vs hybrids, protein content

The protein production capability in alfalfa is mainly related to the leaf/stem ratio at cutting:the tolerance to early cutting (5% blooming), the modification of stem morphology towards a highernumber of shorter internodes and the uncoupling rate of growth and rate of development are thedifferent approaches used in the breeding programs of ISCF Lodi to act on leaf/stem ratio.

The results obtained on stem morphology by means of positive selection for plant dry matter(DM) and divergent selection for the average internode length, applied through two cycles ofselfing are reported. The parental populations were represented by S1 progenies of plants fromsomatic hybridisation M. sativa x M. falcata crossed to non-inbred M. sativa of different origins.

The S2 individuals selected with short and long internode length (SI and LI respectively) werepolycrossed to obtain Syn1 and Syn2 generation synthetics; besides, five simple hybrids S2xS2 wereobtained in the SI material. The Syn2 experimental synthetics (1600 plants/synthetic), the simplehybrids (760 plants) and four tester varieties (640 plants) were then grown in miniplots at thedensity of 400 plants m2, with not limiting irrigation. Five cuts were done in the sowing year 2004and in the 1st productive year 2005; stem morphology (total stem height at the 1st reproductive node,number of vegetative and reproductive nodes) and DM yield were studied at individual plant basisin cuts 2 to 4 (2004) and 1 to 5 (2005). A subsample of individuals homogeneous for total stemheight (>x + 1s) and biological stage was used for leaf and stem separation and for chemicalanalyses of DM constituents: crude protein, determined by the Dumas method (Kirsten, 1983) andfiber fractions of the stems, according to Goering and Van Soest (1970).

The two synthetics SI and LI resulted to differ significantly for stem morphology: the numberof vegetative internodes was higher in SI compared to LI in cuttings 2 and 4 (2004) and 3-5 (2005),the differences ranging from 0.5 to 1.1 vegetative internode. Total stem height and DM yield werealso higher in SI in comparison with LI in cuttings 3-5 (2005). Both the synthetics differed from thetester cultivars for vegetative internode lenght in all the cuts studied and for number of vegetativeinternodes in cuts 2, 3 (2004) and 3-5 (2005). When the subsample homogeneous for stem heightwas considered, the differences between the SI and LI synthetics were maintained with a similarrange (0.5 – 1.07) in the number of vegetative internodes; such a difference corresponded to asignificant increase in leaf/stem ratio in SI synthetic compared to LI in cuttings 2 and 3 (2005): 0.77vs 0.70 on average. Chemical analyses indicated that crude protein percentage of leaves and stemswas higher in LI synthetic compared to SI; however, total protein production per plant wassignificantly superior in SI material because of the higher amount of leaves. The comparison of thetwo synthetics for the percentage of fiber fractions indicated a higher lignification in SI material inpresence of an NDF content of similar value.

The interest of the experimental synthetics in comparison with the commercial cultivars forhigher leaf/stem ratio and lower earliness is confirmed, the divergent selection applied appears tohave been successful in modifying the number of vegetative internodes and consequently theleaf/stem ratio and the amount of protein produced per plant. The stem morphology of SI synthetic,however, has brought to a higher lignin content, whose role in feeding value has to be evaluated.



COMPLEX TRAITS ANALYSIS OF METABOLIC NETWORKS INVOLVEDIN NUTRITIONAL AND ORGANOLETIC TOMATO QUALITY

P. CARLI*, M. R. ERCOLANO*, M. DE MARTINO*, A. DI MATTEO*, A. BARONE*,V. FOGLIANO**, L. FRUSCIANTE*

*) Dep. of Soil, Plant and Environmental Sciences, University of Naples “Federico II”,Via Università 100, 80055 Portici, Italy**) Dep. of Food Science, University of Naples “Federico II”, Via Università 133, 80055 Portici,Italy

tomato, quality traits, biochemical parameters, large data set, correlation analysis

Italy is one of the most important suppliers of tomato products in the world and is fundamentalthat our productions can satisfy consumer requests for higher quality and healthy food, which wouldto preserve environmental and public health. In fact, tomato contributes significantly to humandietary intake of antioxidant compounds, vitamins and essential minerals, although recent criticismsof tomato quality have encouraged efforts to further improve it...Among the chief quality targets oftomato, are nutritional value and taste of the fruit. Breeding strategies aimed to improve thesetargets require a definition of the major parameters that contribute to their definition andquantification. At his purpose, it is important to identify the most important metabolites to beanalyzed.. Technological developments have considerably extended our ability to describe complexbiological systems, facilitating the analysis of metabolites, even though the interpretation of largegenomic data sets obtained through the implementation of these technologies is troublesome.

In order to explore the genetic basis of tomato fruit biochemistry, in the present work we haveused a high-throughput metabolite profiling protocol, and we have performed whole-plantphenotype characterization and sensory analyses. We phenotyped 38 tomato breeding lines forimportant nutritional traits and 6 traditional Italian ecotypes of tomatoes for taste attributes.

In particular, the tomato accessions were grown in the open field and were analysed foragronomic, biochemical and molecular traits. On ripe fruits sugar, organic acid content, carotenoids,vitamins, total phenolic were determined through HPLC and spectrometry analysis. In addition,considering that tomato fruit is rich of free aminoacids a Maldi-TOF analysis was carried out fortheir quantification. An index of nutritional quality of tomatoes based on the antioxidant contentwas developed to evaluate the antioxidant component of tomatoes. On traditional Italian ecotypes,besides agronomic, molecular and biochemical analyses, a sensory evaluation was also performedthrough a panel taste. Analysis of variance (ANOVA) was performed on data at a significance levelof P<0.05, to compare compounds determining quality of fruit and a Pearsoncorrelation analysis for all possible spectrum of combinations among metabolites, agronomic traitsand/or sensory attributes was also applied. Moreover, a PCA approach was carried out to establishmetabolic determinants of tomato taste. The phenotypic characterization is being integrated in acartographic network based on correlation analysis that reveals the associations between phenotypeand independent metabolic, including links with metabolites of nutritional and organolepticimportance. In fact, t it is really difficult to display such a large data set in a truly quantitativemanner. In the future, we will analyze the correlation network to obtain a less cursory

discrimination of which traits are highly associated by using an algorithm that identifies functionalmodules within complex networks and thereby simplifies their interpretation.



PRELIMINARY CHARACTERIZATION OF EMS TOMATO MUTANTSSELECTED FOR CHANGES IN FRUIT COLOUR

F. CARRIERO*, C. D’AMBROSIO*, G. GIOVINAZZO**, A. D'INTRONO**,A. PETROZZA*, G. SOZIO*, F. CELLINI*

*) Metapontum Agrobios, S.S. Jonica 106, km 448.2, 75010 Metaponto (MT), Italy [email protected]**) ISPA-CNR, Sez. Lecce Via Monteroni, 73100 Lecce, Italy

tomato, fruit, flavonoids, carotenoids, mutant

To create tomato mutant collections for reverse genetic studies (TILLING), we treated RedSetter tomato seeds with two different Ethylmethanesulfonate (EMS) concentration (0.7% and 1%).A total of 13000 M2 plants, grown in open field, were phenotypically scored for their mutant traits.

Two tomato M2 families showed altered colour fruits. Precisely the mutant lines producedfruits that were orange, externally, while their flesh was yellow instead than red.

In order to characterize the colour mutant lines, molecular and biochemical studies wereundertaken to check the pigment biosynthesis pathways. Total RNA was extracted from mutant andcontrol tomato fruits, reverse transcribed and analyzed by RT-PCR with primers complementary toregions of some genes of the carotenoid (Psy-1, ZDS, PDS, Lyc) and flavonoid (PAL, CHS, CHI)pathways. The qualitative molecular analysis showed differences in gene expression levels not onlybetween colour mutant and control lines (Red Setter) but also between the two EMS mutant tomatogenotypes.

Interestingly, the molecular results were confirmed and supported by the biochemical ones thatshowed quantitative differences in secondary metabolites, such as lycopene, beta-carotene(carotenoid pathway), rutin, naringenin and kaemferol (flavonoid pathway), between mutant andcontrol fruits.

Our preliminary molecular and biochemical results demonstrated that the two M2 tomato lines,even if phenotypically identical, differed in their carotenoid and flavonoid contents.

Further investigations will be undertaken to better characterize the mutant lines not onlymolecularly (quantitative RT-PCR) and biochemically but also for their protein contents by meansof proteomic studies.



REPLACEMENT OF CYSTEINE RESIDUES OF A PROTEIN BODY-FORMING PROTEIN INCREASES SOLUBILITY AND CAUSESSECRETION

A. POMPA, A. VITALE

Istituto di Biologia e Biotecnologia Agraria, CNR, Milano

storage proteins, protein bodies, protein traffic

Seed prolamins of a number of cereals accumulate in the endoplasmic reticulum (ER) as verylarge insoluble oligomers termed protein bodies (PB). The mechanisms of prolamin ER retentionand PB formation are still poorly understood. We have previously shown that zeolin, a fusionbetween phaseolin (a vacuolar storage protein) and part of g-zein (a PB- forming protein), formsinsoluble PBs in the ER of transgenic tobacco leaves, and that treatment of protoplasts withreducing agents increases zeolin solubility and leads to its partial secretion. We now show thatreducing agents do not decrease the affinity of zeolin for the ER chaperone BiP, indicating thatbinding to BiP is not a major causing event for the ER retention of zeolin. Conversely, replacementof the six Cys residues of the g-zein domain with Ser residues by site-directed mutagenesis makesthe mutated zeolin fully soluble even in the absence of reducing agents and causes quantitativesecretion. These results point to disulfide bonds as a key structural feature for the formation ofinsoluble PBs and their ER retention.

Research supported by the EU Integrated Project “Pharma-Planta” (LSHB-CT-2003-503565)and the EU Research Training Networks Contract “BioInteractions” (HPRN-CT-2002-00262)



DEVELOPMENT OF A SYSTEM FOR GENETIC TRACEABILITY OF FISHSPECIES AND DERIVING FOODS BY THE IMPLEMENTATION OF AMICROARRAY PLATFORM

M. SPIAZZI, A. FERRARINI, M. DELLEDONNE

Department Scientific and Technologic, Faculty of Science, University of Verona, Strada Le Grazie15, 37134 Verona, Italy - [email protected]

traceability, food, fish, APEX, microarray

Recent emergencies regarding animal derived foods have underlined the necessity of thedevelopment and adoption of a suitable system for controlling every single step of food productionfrom animal breeding to the final product. Moreover, the identification of species is important tofight against food swindle and to guarantee meats origin. For what concern fish food industry, fewgenetic methods have been developed and applied, although the rising economical importance offish foods. This is also more important as European Community in the last decade has approvedmany norms on food traceability in order to guarantee food safeness.

The use of molecular techniques based on specie specific DNA sequences allows an absolutelyreliable identification of species even in final transformed products which are impossible to analyzewith conventional methods based on phenotypic approaches. In 2003, Paul D. Hebert demonstratedthat the nucleotidic sequence of a single gene is sufficient to differentiate and to identify themajority of animal species. This approach, based on the analysis of the mitocondrial gene cox1coding the Cythochrome oxydase subunit I, is called DNA barcoding as the polymorphisms of thenucleotidic sequence of a specific region of this gene can be assimilated to a bar code by whichevery specie is idendified by an highly conserved sequence or by a group of sequences.

Usually DNA barcoding approach is implemented by sequencing the fragment of interest (ascox1) and by comparing the sequences with DNA bar codes available. But this require the use of asequencer and allows the analysis of a single fragment per time, while for discriminatingphylogenetically very close individuals would be more suitable to analyze different sequencescorresponding to more genes. For these reasons we implemented an oligo microarray platform todetect specie specific sequences based on solid-phase APEX. The technology use immobilizedprobes of 20-30 nucletides length designed on species specific sequences. Most of the species ofeconomical interest in Italy will be barcoded and the sequences will be collected in a specificdatabase. These data will then be used to construct a microarray for fish species identification andfood traceability.

Documents

LOX ACTIVITY AND CAROTENOIDS CONTENT OF SELECTED … fileC.R.A. Centro Ricerche per la Cerealicoltura, S.S. 16 km 675, 71100 Foggia, Italy – [email protected] lipoxygenase activity,