dna barcoding of wild italian dendroflora: first … · generate unique identifiers at either the...

Proceedings of the 52nd Italian Society of Agricultural Genetics Annual CongressPadova, Italy – 14/17 September, 2008ISBN 978-88-900622-8-5

Poster Abstract – F.01

DNA BARCODING OF WILD ITALIAN DENDROFLORA: FIRSTEVIDENCES OF APPLICATION IN OLEACEAE, FAGACEAE ANDACERACEAE

DE NICOLA S.*, FERREIRA R.*, D’AMBROSIO C.*, PIREDDA R.*,**, PUDDU G.*,SIMEONE M.C.*, ATTIMONELLI M.**, SCHIRONE B.*

*) Dipartimento di Tecnologie, Ingegneria e Scienze dell’Ambiente e delle Foreste (D.A.F.),Università della Tuscia, Via S. Camillo de’ Lellis, 01100 Viterbo**) Dipartimento di Biochimica e Biologia Molecolare "E. Quagliariello", Università di Bari,Via Orabona 4, 70125 Bari

DNA barcodes, genetic variation, biodiversity, tree species

DNA barcodes have been proposed as a molecular tool that would provide speciesidentifications and as a way to accelerate the discovery of new species. Barcodes are short segmentsof DNA (<800 bp) that can be used to uniquely assign an unknown specimen to a species,particularly when diagnostic morphological features are absent, or insufficient. The advantage ofthis approach might be particularly relevant to cope with the fundamental crisis facing biodiversity,as a standardized, species-level identification tool for biodiversity assessment, life history andecological studies.

In order to be useful, a DNA barcode sequence must not only easily produce PCRamplifications with universal reaction conditions and primers, but also contain enough variation togenerate unique identifiers at either the species or population levels. Zoologists selected amitochondrial gene (CO1) as the standard barcode for animals, but the slow evolutionary rate ofplant mitochondrial DNA required an alternative solution. The plastid genome shares many of thedesirable attributes, however, the rate of evolution is generally slow, and to find a plastid region thatis sufficiently variable for DNA barcoding is a non-trivial problem.

A number of different chloroplast regions have been proposed, but at present there is nostandard protocol for DNA barcoding of land plants. Different plant barcoding regions have beensuggested.

Aim of our research is to identify the DNA barcode of wild Italian dendroflora. We selectedtwo options among the ones proposed: rpoC1+matK+trnH-psbA and rbcL+trnH-psbA. We havestarted to test their applicability in 3 different plant groups of the Italian wild dendroflora:Oleaceae, Aceraceae and Quercus spp. (Fagaceae). These plants are largely widespread in theItalian peninsula and together collect about 30 (well-)acknowledged tree species, each of whichplays an important role in the conservation of numerous land ecosystems, in the wood trade, and inthe definition of biogeographic processes in the Mediterranean area. Nevertheless, tree speciesidentification may pose serious difficulties, due to the high individual morphological plasticity andto the high levels of inter-specific crossability, which make hard to discriminate closely related taxa,contributing to the production of ambiguous rankings, and giving rise to multiple, often contrasting,taxonomies.

Ease of amplification with universal primers and reaction conditions were determined for eachof the proposed markers in the three plant groups. New primer pairs were developed where needed.Generated sequences where compared and used to discriminate taxa. To this purpose, one to fiveindividuals per 28 species were analysed. To evaluate the extent of intra-specific variation, samplesfrom biogeographic regions other than the Italian peninsula were evaluated also. The level ofsequence divergence, analyzed for each locus, confirmed the non-coding trnH-psbA intergenicspacer as the most variable region and highlighted the potential discriminatory power at level ofspecies discrimination. However, the three plant groups responded differently to the analyses; samespecies of the quercus group were invariant at each of the five loci tested. The possible explanationsfor the lack of sequence variation are several (very low rates of sequence evolution, taxonomicmisidentification, recent divergence or hybridization), and probably a nuclear marker will be neededto correct identification and description of the species considered in the present study.



GENETIC DIVERSITY OF PINUS HALEPENSIS MILL. POPULATIONS INCENTRAL ITALY DETERMINED BY CHLOROPLASTMICROSATELLITES VARIATION

ARMENISE L.*, AGRILLO E.***, FARINA A.**, SIMEONE M.C.*, PAGNOTTA M.A.**,SPADA F.***, SCHIRONE B.*

*) Dipartimento di Tecnologie, Ingegneria e Scienze dell’Ambiente e delle Foreste (D.A.F.),Università della Tuscia, Via S. Camillo de’ Lellis, 01100 Viterbo**) Dipartimento di Agrobiologia e Agrochimica (D.A.B.A.C.), Università della Tuscia,Via S. Camillo de’ Lellis, 01100 Viterbo***) Dipartimento di Biologia Vegetale, Università “La Sapienza”, Piazzale Aldo Moro 5,00185 Roma

Pinus halepensis Mill. is a Mediterranean species, spread discontinuously from Spain andMorocco in the west, to Jordan in the east. In the south, it extends from Israel as scattered relics inthe Judean hills and Galilee, reaching its northern limits in the upper Rhone Valley in France. It isdistributed mainly along the coast and exceptionally inland in Italy, Tunisia and Spain.

Recent studies on the genetic diversity of Aleppo pine using biochemical and DNA markershave demonstrated its low levels of variability according to the hypothesis of a genetic bottleneckduring the past glacial episodes of the Holocene. Most P. halepensis genetic diversity has beenfound in Greek and Spanish populations, suggesting a recent expansion of the species in the last10000 years from these two refugial areas; founder effects, anthropic influence and/or populationdynamics regulated by fire have been claimed to explain the low levels of genetic diversity of thisspecies.

Aleppo Pine is a pioneer drought-resistant species, that can survive in a wide range of climaticand soil conditions, and for these reasons it has been extensively planted all over the Mediterraneanbasin during the last century, especially in those degraded areas where environmental constraintsmake impossible the introduction of other woody species. In the last decades, an increasing use ofthis conifer species, for plantations to restore abandoned lands, meanwhile protecting soils againsterosion and providing spaces for recreation, has probably contributed to over-simplify the geneticstructure of Aleppo Pine and the diversity of the native populations.

One of the most relevant issues in the conservation of a species biodiversity is to correctlydefine its fine genetic structure, to discriminate between introduced and native populations, and toidentify relic stands. In this context, appropriate forestry practices focusing on safeguard of nativepopulations represents a useful tool to preserve a valuable genetic pool, and eventually preserve thegene flow from plantations to wild populations, well-known as genetic pollution phenomenon.

In Central Italy, Aleppo pine range is characterized by a patchy distribution, and the naturalorigin of the populations has long been debated. Nevertheless, the presence of scattered stands inmany ecologically different sites speaks in favour of relictuality and emphasizes their putative roleas a pool of genetic diversity.

Here, eight paternally inherited chloroplast microsatellites (cpSSRs) are used to characterizeten diverse P. halepensis populations sampled in Lazio (Sabina and Aurunci), Umbria (Valnerina)

and Abruzzo (Adriatic coast) in order to: 1) quantify their genetic diversity, 2) assess their geneticstructure, and 3) discriminate between natural and introduced germplasm.

A strong genetic differentiation among populations was found. Correlation between geneticand geografic distance has been identified, and the impact of man-mediated genotype dispersal isdiscussed.



MOLECULAR CHARACTERIZATION OF CURRANT (RIBES SPP.)ACCESSIONS USING SNP AND SSR MARKERS

SORDO M., PALMIERI L., GIONGO L., GRANDO M.S.

Fondazione Edmund Mach, IASMA Research Center, Via Mach 1, 38010 San Michele all'Adige(TN) (Italy)

molecular markers, genotyping, Single Nucleotide Polymorphism, Ribes

Ribes species belong to the Saxifragaceae genus, which is taxonomically fairly isolated, andthe most commercially important species, especially in Northern European countries, are the redcurrant (R. rubrum L.) mainly used for fresh consumption and blackcurrant (R. nigrum L.), used forprocessing. Currants have known a slight production increase in the last years for their healthbiocomponents, highly concentrated both in fresh and in processed products.

In Italy, a small germplasm collection is present at IASMA, and the plant material includescontributions from R. rubrum L., R. sativum Syme, R. petraeum Wulf., R. multiflorum, R.longeracemosum L., R. grossularia L., R. sanguineum Pursh., R. nigrum L. and R. dikuscha Blue.

Single Nucleotide Polymorphisms (SNPs) are the most abundant type of DNA sequencepolymorphism and can be theoretically found within every genomic sequence. Thanks to their highavailability and stability, also compared to Simple Sequence Repeats (SSRs), they can be used asmolecular markers for many purposes, such as cultivar identification and evaluation of geneticdiversity.

The aim of this work is represented by the development of informative molecular markers forcharacterizing the germplasm collection of these Ribes species, that have been phenotyped for sixconsecutive production season in order to obtain a large informative picture of the cultivars andselections, concerning flowering, fruiting, horticultural traits, biotic and abiotic resistances, andhealth compounds.

New SNP markers were detected by re-sequencing several loci amplified from a selection ofRibes accessions. Primers based on Expressed Sequence Tags having homology to known genesinvolved in fruit development were applied and a SNaPshot assays is now being developed for theSNP genotyping.

In order to investigate the genetic relationships among Ribes varieties a set of genomic andESTs derived SSR marker was also tested.



COMPARATIVE HISTOLOGICAL, BIOCHEMICAL AND MOLECULARAPPROACHES TO STUDY CITRUS REPRODUCTIVE BIOLOGY

DISTEFANO G.*, CARUSO M.*, LAS CASAS G.*, LA MALFA S.*, DEL DUCA S.**,GENTILE A.*, TRIBULATO E.*

*) Dipartimento di OrtoFloroArboricoltura e Tecnologie Agroalimentari, Facoltà di Agraria,University of Catania, Via Valdisavoia 5, 95123 Catania (Italy)**) Dipartimento di Biologia Evoluzionistica Sperimentale, University of Bologna, Via Irnerio 42,40126 Bologna (Italy)

mandarin, self-incompatibility, pollen tube, TGase, cDNA-AFLP

In contrast to what is known in other fruit tree species, the self-incompatibility reaction incitrus has not been fully investigated. It is assumed to be of gametophytic system and geneticallycontrolled by the S-locus, which in other species encodes for glycoproteins showing ribonuclease(S-RNases) activity, regulated by several enzymes such as transglutaminase (TGase).

In order to investigate pollen-pistil interaction, the growth of pollen tubes of two clementine(Citrus clementina Hort. ex Tan.) varieties, ‘Comune’ (self-incompatible) and ‘Monreal’ (a‘Comune’ self-compatible mutation) has been analyzed in self- and cross-pollination conditions.The same analysis was carried out on pummelo (Citrus grandis L. Osbeck), a self-incompatibleancestor species. We sequentially examined by histological assays, along two weeks after self andcross pollination, the number and morphology of pollen tubes at various pistil levels and TGaseactivity in the style.

To identify genes involved in pollen-pistil interaction and in self-incompatibility response, aresearch based on cDNA-AFLP technique was carried out to compare transcript profiles betweenthe two clementine varieties and search for differentially expressed genes. RNA was extracted fromemasculated non-pollinated pistils and from self-pollinated pistils four days after pollination.cDNAs of the two varieties were amplified using 43 EcoRI/ MseI primer combinations, and morethan a hundred differentially expressed or polymorphic Transcript Derived Fragments (TDFs) weresuccessfully cloned and sequenced.

Pollen tubes in self-pollinated ‘Comune’ arrested their development in the upper part of thestyle, while in self-pollinated ‘Monreal’ they grew down to the ovary. TGase activity appearedstrongly involved in pollen tube growth inhibition confirming the presence of S-RNase-basedgametophytic self-incompatibility. Among the TDF showing homology with known genes,BLASTX search identified several genes related to reproductive biology such as calcium modulatedproteins, receptor-like kinases, thioredoxins, a histone deacetylase gene, a putative cleavage andpolyadenylation specificity factor, transcription factors and genes related to hormone metabolismand signaling. Moreover, a high percentage of active retrotransposons were identified among theTDFs, which may indicate their possible role in the regulation of self-incompatibility genes asalready reported for other species. Some of the TDFs were analysed through RT-PCR and showeddifferences in their expression patterns.



REGULATION OF CAROTENOID GENE EXPRESSION AND VOLATILECOMPOUND EMISSION IN WHITE- AND YELLOW-FLESHED PEACHGENOTYPES

BRANDI F.*, BAR E.**, MOURGUES F.***, LIVERANI A.*, GIULIANO G.****,LEWINSOHN E.**, ROSATI C.***

*) CRA-Unità di Ricerca per la Frutticoltura-Forlì, Via la Canapona 1 bis, 47100 Forlì (Italy)**) Dept. of Vegetable Crops, Newe Ya'ar Research Center, ARO, P.O. Box 1021, 30095 RamatYishay (Israel)***) ENEA C.R. Trisaia, BAS-BIOTEC-GEN, S.S. 106 km 419+500, 75026 Rotondella (Italy)****) ENEA C.R. Casaccia, BAS-BIOTEC-GEN, Via Anguillarese 301, 00123 Roma (Italy)

carotenoids, Prunus persica, volatile composition, fruit ripening

Carotenoids are ubiquitous plant pigments present in all plant tissues playing a dual role, bothas essential metabolites in photosynthetic tissues and as dispensable colorants in flowers and fruits.Several breeding programs aim at increasing and modifying the carotenoid content of many flowersand fruits for ornamental and nutritional purposes. Carotenoids are also precursors of many volatileorganic compounds (VOCs), which provide peculiar flavours in peach fruits, an important criterionof fruit quality.

The aim of this study is to unravel the regulation mechanisms of carotenoid biosynthesis inpeach genotypes with different mesocarp colour (white vs yellow flesh), by studying geneexpression and metabolite patterns during fruit development in white and yellow cultivars.

This study was performed on the yellow-fleshed ’Redhaven‘ and its white-fleshed mutant’Redhaven Bianca‘. We investigated by qRT-PCR the expression patterns of fifteen genes involvedin the synthesis of isoprenoids (Dxs, IspE, LytB) and the synthesis/degradation of carotenoids (Psy,Pds, Zds, Lcy-b, Lcy-e, Chy-b, Chy-e, Zep, Nced1, Nced2, Ccd1), as well as two genes involved inthe ethylene emission (AccS, AccO) at four stages of late fruit ripening (S3, Breaker 1, Breaker 2,S4). In parallel, the emission patterns of major volatile compounds (VOCs) was also carried out byGC-MS.

Main results show a differential temporal regulation of gene expression between the twogenotypes, and quantitative and qualitative differences in transcript levels for key genes involved incarotenoid synthesis and degradation. The two genotypes also displayed quantitative and qualitativedifferences in VOC emission patterns, with higher levels of carotenoid-derived volatiles in’Redhaven Bianca‘. This result is consistent with a higher activity of carotenoid-specificdioxygenase enzymes which are likely to contribute to the white-fleshed fruit phenotype.



EXPERIMENTAL STRATEGIES FOR THE GENETIC DISSECTION OFQUANTITATIVE TRAITS IN HAZELNUT (CORYLUS AVELLANA)

CATARCIONE G.*,**, RUGINI E.**, VITTORI D.*, DE PACE C.*

*) Dep. Agrobiol. and Agrochem., Univ. of Tuscia, Via S. Camillo de Lellis, 01100 VT (Italy)**) Dep. of Crop Production, Univ. of Tuscia, Via S. Camillo de Lellis, 01100 VT (Italy)

BSA, allele-sharing, genetic analysis, QTL, MAS

Molecular markers useful for assisting selection for phenological and yield trait improvementare not available in hazelnut. Before undertaking DNA-based studies aimed at genetic dissection ofquantitative traits to find useful molecular genetic markers, it is necessary to define the referencepopulation and to infer as much as possible about the genetic basis of a trait by measuring theheritability of the quantitative phenotype in the reference population and the significance of thedetected phenotypic differences. The choice of the reference population dictate the methods usedfor the genetic dissection of quantitative traits. In hazelnut, two types of reference population mightbe used: (1) a large collection of accessions from the cultivated gene pool, or (2) a progeny from across between parents showing a wide range of phenotypic variation.

The first type of population allow genetic dissection through association studies. However,this method is useful only when two allele types at each molecular genetic locus occur over thepopulation range. Previous studies have shown that more than two SSR molecular marker allelescan be found in a large collection of hazelnut accessions, each with its own genetic background,which lower the efficiency of discovering molecular alleles underlying quantitative phenotypictraits.

The second type of population allow the use of the genetic dissection methods of allele-sharing and genetic analysis in the more homogeneous genetic background provided by the allelesof only two parents. We produced an half-sib progeny from intercropping “Tonda Gentile Romana”(TGR) and “Nocchione” (NOCC) accessions. Due to self-pollen incompatibility of the parentalplants, the progeny was considered similar to a full-sib offspring and new homozygote phenotypesfor recessive alleles shared between parents are expected among the 133 individuals of theprogeny. One such novelty was the “evergrowing” phenotype which fail to cease growth and toenter dormancy under the dormancy-inducing (i.e. short days) conditions suitable for the wild-typephenotype. Extreme ends were also found for the distribution of phenotypic values related, amongothers, to the date of vegetative budbreak, date of first male bloom, kernel length, kernel width,seed weight, and eriophyid mite symptoms. Such divergences were significant over the two years ofobservation, and can greatly increase the ability to discover shared molecular alleles within each ofthe progeny group expressing the extreme phenotypic values. Test for differences in the sharedalleles among groups is being carried out using bulk segregant analysis (BSA) at SSR loci. TheSSR-allele that BSA indicated as differing between extreme phenotypic groups, will becomemarkers of flanking quantitative trait loci important in determining phenological, yield and bioticstress resistance traits in hazelnut.



TOWARD CLONING OF THE APPLE COLUMNAR GENE

BALDI P., KOMJANC M., MALNOY M., MAGNAGO P., COSTA F., VELASCO R., SALVI S.

IASMA Research Centre, Via E. Mach 1, 38010 San Michele all’Adige (Italy)

tree architecture, molecular markers, columnar, Malus domestica

In apple breeding programmes tree architecture is considered an important trait, because itinfluences fruit quality, planting density, production and labour requirements. Tree architecture canbe controlled by pruning and using size-controlling rootstocks, but natural tree forms resulting insmall and narrow trees could be desirable to assure a uniform light penetration, high densityplanting and a reduction of pruning interventions. The columnar growth habit in apple presents allthese desirable traits, as it is characterized by short internodes, a thick stem and reduced plant heightand branching. The columnar habit seems to be controlled by a single dominant gene (Co), even ifminor modifier genes can segregate depending of the genetic background. Different apple columnarvarieties are currently available such as “McIntosh Wijcik”, “Telamon” and “Tuscan” andmolecular markers for this character have been developed using segregating population derivedfrom different crosses. The Co gene has been reported to map on the apple linkage group 10, andmolecular markers associated to the columnar habit include SSR markers, RAPD markers andSCAR markers. At FEM-IASMA three columnar segregating populations of 170, 130 and 70individuals respectively are currently available, derived from the crosses “Golden x Wijcik”,“Goldrush x Wijcik” and “Galaxy x Wijcik”. These populations have been phenotypicallycharacterized and tested with some of the available markers in order to associate the columnar habitwith the molecular markers. As the final aim of the work is the cloning of the Co gene, fine andphysical mapping of the columnar region will be necessary. For this purpose, a larger mappingpopulation derived from the cross “ Golden x Wijcik” is being produced. FEM-IASMA has beenrecently funded to sequence the apple genome. This will shortly provide an extremely powerful toolto develop new markers strictly associated to Co and directly characterize the correspondinggenomic region searching for putative candidate genes.



PRELIMINARY SCREENING IN 28 WILD MALUS SPECIES FORIDENTIFICATION OF NEW RESISTANCE GENE ANALOGS

MALNOY M.*, BALDO A.M.**, COSTA F.*, SIAMMOUR A.*, SALVI S.*, VELASCO R.*,ALDWINCKLE H.S.***

*) IASMA Research Centre, Via E. Mach 1, 38010 San Michele all'Adige (TN) (Italy)**) USDA-ARS Plant Genetic Resources Unit, Geneva, NY (USA)***) Cornell University, Department of Plant Pathology, Geneva, NY (USA)

resistance gene, Malus, wild species

Plant R genes are known to confer resistance to a variety of pathogens in a gene-for-genemode. 800 unique apple genome samples were amplified and sequenced from a variety of wildmaterial and rootstocks using degenerate primers for NBS-LRR Resistance Gene Analogs (RGAs).Forty five differents Malus accessions from the USDA apple gerplasm collection at Geneva,representing 28 Malus species (Malus X domestica, asiatica, baccata, florentina, floribunda, fusca,halliana, honanensis, hupehensis, kansuensis, micromalus, ombrophila, orientalis, prattii,prunifolia, pumila, robusta, sargentii, sieboldii, sieversii, sikkimensis, sublobata, sylvestris,transitoria, zumi, yunnanensis, zhaojiaoensis), hybrid white angel, and the rootstock Geneva 41were used in this study. Sequences were screened and cleaned of vector sequences and comparedwith resistance genes previously identified among the Rosaceae and other green plants.Approximately 90 of these matched RGAs previously identified among Malus while an additional200 are similar to RGAs found in other Rosaceae genera and other plant families. Roughly 30% ofthe RGA's identified in this study are TIR-type RGAs, while the remaining 70% are Non-TIR-type.These genes will be used to identify novel sources of disease resistance among the wild apples inthe USDA apple collection



GENETIC TRANSFORMATION OF CHERRY ROOTSTOCK GISELA 6(PRUNUS CERASUS X PRUNUS CANESCENS) USED AS MODEL TOSTUDY GENE FUNCTION IN PRUNUS SPECIES

TESTONE G., NICOLODI C., GIANNINO D.

Institute of Agricultural Biology and Biotechnology - Unit of Rome. National Research Council ofItaly (CNR), Via Salaria km 29.300, 00015 Monterotondo Scalo (Rome) (Italy)

gene function, A. tumefaciens mediated transformation, KNOX genes, GFP

In the post genomic era, the assessment of plant gene function is a mandatory step and geneoverexpression and silencing by stable genetic transformation are widely used approaches. Onemajor drawback in gene transfer to peach fruit tree (Prunus persica) is due to its tediousrecalcitrance to regeneration. Protocols for peach transformations have been reported, though theyare still few, cultivar specific and based on immature embryo manipulation. Our group has beenfocussing on class 1 KNOTTED-like genes from peach, which encode homeodomain transcriptionfactors and regulate meristem identity and cell fate. These genes are tightly conserved in Prunusspecies, hence we approached overexpression of peach KNOPE1 into Gisela 6, a cherry rootstock,which is a genotype suitable to transformation starting from leaf discs. Co-cultivation wasperformed with A. tumefaciens strain EHA105 containing the cassettes CaMV35S:KNOPE1:NOSand CaMV35S:mGFP: CaMV35Ster. Both transgenes behave as visual non destructive markers: theformer causes leaf margin fringing, while the latter is detected by fluorescence microscopy,allowing an easy discrimination between transgenic and escape shoots. Shoot regenerationfrequency and transformation efficiency were estimated 11% and 4%, respectively. We will presentresults on one line overexpressing KNOPE1, which showed leaf margin lobing and vascular systemalteration, and on three independent lines which expressed GFP protein.



SSR AND EST-SSR MARKERS TO ASSESS GENETIC DIVERSITY INEUROPEAN CHESTNUT POPULATIONS

MARTIN M.A.*,**, MATTIONI C.*, CHERUBINI M.*, TAURCHINI D.*, VILLANI F.*

*) Istituto di Biologia Agroambientale e Forestale (IBAF) – CNR, Viale Marconi 2,05010 Porano (TR) (Italy)**) Departamento de Genetica, E.T.S.I.A.M., Edificio Gregor Mendel, Campus de Rabanales,Universidad de Cordoba, ES—14071 Cordoba (Spain)

microsatellites, genetic variation, adaptive genetic variation, Castanea sativa

The genetic diversity is the basis for the adaptability of organisms against environmentalchanges through natural selection. Populations with low genetic variation are more vulnerable topollution, to climate changes and habitat alteration due to human activities. In recent yearsmicrosatellites have become the most used markers for studying population genetic diversity. Thesemarkers, due to their hyper-variability and locus-specific nature are important to measure neutralDNA variation, however they are not useful for measuring the adaptive genetic diversity. Recently,the increased availability of the DNA sequences has given the possibility to develop EST-derivedSSR markers, a new type of functional genomic markers. These markers are present in expressedregions of the genome and are more than three times as transferable across species as comparedwith anonymous SSRs.

Castanea sativa Miller is a widespread species in the Southern Europe. Due to itsmultipurpose characteristics, man has influenced over many centuries its distribution throughpropagation and transplanting of plant material and silvicultural practices. Previous studies, aimedto evaluate genetic and adaptive variation of European chestnut were carried out in a large numberof populations along its whole distribution area. Genetic variation was estimated by ISSR andisozyme markers (Mattioni et al. 2008) and adaptive variation was estimated for traits related toclimate change (water use efficiency, bud burst, bud set, growth) (Lauteri et al. 2004). Theseprevious results showed a high degree of variation within and among populations both at geneticand phenotypic level.

The objective of the present work is to compare the genetic variability and differentiation,based on neutral SSR and EST-SSR markers, of chestnut European populations collected along theMediterranean distribution range and in areas representative of contrasting climate conditions. SixSSR and 10 EST-SSR, markers developed from oak EST data base within EVOLTREE project,were used to assess genetic diversity in 7 natural chestnut populations from Italy, Spain and Greece.All neutral SSRs tested were polymorphic for all the populations analysed whereas 8, out of the tenEST-SSRs tested, resulted polymorphic. Genomic SSRs, showed higher within population genediversity and lower among population variation as compared to the EST-SSRs. As shown fromAMOVA analysis the proportion of variation within populations were 85% and 71% for genomicmicrosatellites and for EST-SSRs respectively, while the variation among population was 15% forneutral SSRs and 29% for EST-SSRs. UPGMA and PCA analysis showed slightly differentgrouping of the populations for the two types of markers. These preliminary results confirm that the

comparison of genomic and EST microsatellite is a useful tool to give a complementary informationto understand the genetic and adaptive diversity in chestnut.



TRIPLOIDY EVENTS IN COMMON FIG (FICUS CARICA L.)

FALISTOCCO E.

Department of Applied Biology, University of Perugia, 06121 Perugia (Italy)

Ficus carica, molecular cytogenetics, rDNA, FISH, unreduced gametes

The common fig (Ficus carica L.), a typical fruit tree of the Mediterranean area, ischaracterized by a complex breeding system which consists of two forms of tree, three functionalfloral forms and the symbiosis with the agaonid Blastophaga psenes, the wasp pollinator. Therelationship between the reproduction of the plant and that of the insect is mutually obligatorybecause the pollen grains are transferred to the female flowers, only by the pollinator. On the otherhand the wasps depend on the figs for their reproductive sites. Although genetic studies on commonfig are recent, they allowed a rapid progress in the knowledge of biodiversity and germplasmcharacterization. Instead, the cytogenetics of the species is still unknown. The results of a researchproject dealing with the study of the biological diversity of F. carica following the cytogeneticapproach are reported. Forty four genotypes, including wild plants, either caprifigs and femaleplants and cultivated common figs were examined. The chromosome count revealed that themajority of plants were 2n=26, as expected, but three were triploid with 2n=39. This result suggeststhat variations of the chromosome number could play a role in the evolution of the fig germplasmand induces to consider the important function of the wild forms as source of variability. Thisappears not only as genetic recombinants but also as polyploid variants. The analysis of pollengrains collected from caprifigs provided evidences in favour of this hypothesis with twice largerpollen grains found, as an indication of unreduced gametes. Moreover, the spreading of the mutantsare facilitated by the vegetative propagation normally used by man in this fruit crop. This studycontributed also to gain knowledge on the cytogenetics of F. carica by means thekaryomorphological analysys and FISH mapping of rDNA sequences.



DEVELOPMENT OF NEW MOLECULAR MARKERS FOR CULTIVARSIDENTIFICATION IN OLEA EUROPAEA L.

INTRIERI M.C., NIEDDU F., GIANNONI A., PARENTI N., LIBRANDI L., BLOISE M.,BOGANI P., MULEO R., BUIATTI M.

Dipartimento di Biologia Evoluzionistica "Leo Pardi", Via Romana 17, 50125 Firenze (FI)

Olea europaea, fluorescent-ISSR, TRAP, cp-SSR, trnT-trnD

The Olea europaea cultivars identification is particularly complex due to the great number oflocal accessions, the synonymy and the ambiguous cultivar assignments. On the other hand,unambiguous characterisation is a crucial cue of modern horticulture, with application in breedingprogrammes, in germplasm collections management and in products traceability. Geneticfingerprinting seems to be the best way for genotyping the specific cultivar and the oil derivingfrom it. To date the SSRs fingerprint is the most used method to identify the cultivar. In addition toSSRs, the molecular identification should be based on the development other kind of unambiguousmarkers and, in the light of the genomic progress, the molecular marker should be targeted onfunctional regions.

In this work 43 cultivar of O. europaea belonging to the germoplasm collection of “IstitutoSperimentale per l’Olivicoltura” have been analysed, with the aim to identify simple and reliablePCR based markers useful in cultivars identification. The molecular markers chosen for our analysisare ISSR (inter simple sequence repeats, Zietkiewicz et al., 1994 Genomics 20:176-183) and TRAP(targeted region amplified polymorphism, Hu and Vick Pl. Mol. Biol. Reporter 21:289–294)techniques and chloroplastic markers. The first method is based on the amplification of genomicDNA by a single primer formed by a microsatellite and a fixed sequence as 3’ or 5’ anchors. Thesecond, TRAP, uses expressed sequence information and a bioinformatics approach to generatepolymorphic markers around targeted candidate gene sequences. In particular we designed primersanchored on genes involved in fatty acid metabolism and expressed during the drupe maturation. Toimprove the resolution power of these markers we set a protocol for the visualisation of the patternsobtained by PCR amplification by means of automated laser fluorescent sequencer (ABI Prism3100). Moreover we used chloroplastic PCR primers trnT-trnD to amplify the correspondingintergenic spacer (Demesure et al. 1995, Mol. Ecol. 4:129-31) and a set of 10 primers for SSR ofthe chloroplastic DNA (Weising and Gardner, 1999, Genome 42-.9-19; Kung and Staub, 2003,TAG 107:757-767). All sequence reactions were made using the AutoRead Sequencing kit.Sequence reaction and the SSR amplicons were run in an automated laser fluorescent sequencer(ABI Prism3100). The intergenic trnT/trnD spacer is able to identify two of the 22 cultivarsanalysed. Only a point mutation is present among the cultivars. The chloroplastic SSR markersanalysed in this work show a sufficient pattern of variation to be able to discriminate somecultivars.

In the second phases of the work we set and verified the repeatability of the fluorescent ISSRand TRAP methods (in fact in general the methods based on random or pseudo-random PCRamplification suffer of a lack of reliability). In our cases both ISSR and TRAP gives good resultsgiving rise to a mean reliability around 95% that is the common standard reported in literature. If

the limits of the protocol set is the small window of fragment dimensions read by the sequencer, onthe other hand the speed and the simplicity make the fluorescent ISSR technique a fast and easymethod to process a large number of samples in few days. Moreover TRAP based on functionallyrelevant loci, may became a method useful to perform a wide screening to find possible functionallyrelevant polymorphisms.



CHARACTERIZATION OF THE ACYL CARRIER PROTEIN GENE INOLIVE

MARIOTTI R.*, BALDONI L.*, ALAGNA F.*, CULTRERA N.G.M.*, SARRI V.**

*) CNR - Institute of Plant Genetics, Perugia, Via Madonna Alta 130, 06128 Perugia**) University of Perugia, Department of Cell and Environmental Biology, Via Elce di Sotto,06123 Perugia

ACP, Olea europaea, oil synthesis, SNP, haplotype

Acyl carrier protein (ACP) is a small acidic protein that acts as an essential cofactor in manybiosynthetic pathways depending on acyl transfer reactions.

In particular, ACP is a very abundant component of the fatty acid synthesis, due to the factthat most of the enzymes require acyl-ACP in their reactions.

Due to the main role played by ACP in the process of oil synthesis in olive fruits, theidentification and characterization of the ACP gene was carried out.

In a first step of the work the complete 763 bp cDNA clone was identified and its expressionduring olive fruit ripening was evaluated.

Recently, the entire olive ACP gene, for a length of 2,920 bp, has been sequenced, in order toevaluate its structure, the number of loci and the number of haplotypes among different cultivars.

Three different loci have been identified, as expected from what observed in other species,each locus carrying different haplotype forms.

The three introns, respectively 729, 1,088 and 575 bp long, hold a high variability.A 1,200 bp fragment, including the first and second intron, was sequenced on a pool of 90

cultivars in order to identify SNP and indel polymorphisms useful for cultivar discrimination and toestablish the relationships between molecular profile and phenotypical behaviour.



NUCLEOTIDE VARIATION ANALYSIS IN THE UPSTREAM REGION OFCHSA AND CHSB GENES FOR CHALCONE SYNTHASE (CHS) IN OLEAEUROPAEA L.

SCIALPI A.**, BOGANI P.*, LUTI S.*, BUIATTI M.*,**

*) Dipartimento di Biologia Evoluzionistica “Leo Pardi”, Via Romana 17/19, 50125 Firenze (Italy)**) Centro Interdipartimentale di Servizi per le Biotecnologie di Interesse Agrario, Chimico edIndustriale (C.I.B.I.A.C.I.), Firenze (Italy)

Olea europaea, chs genes, upstream region, nucleotide variation

In olive (Olea europaea L.) the assessment of genetic variability has been carried out withmorphology-based markers (Barranco et al., 2000) and DNA-based markers. Among these RFLP(Besnard and Bervillè, 2000), RAPD (Belaj et al. 2003), AFLP (Owen et al. 2005), SSR (Bandelj etal. 2004), ISSR (Pasqualone & Caponio, 2001) have been used and different molecular markers(Belaj et al., 2003) have been compared in several studies of genetic characterization of cultivatedolives and also to study the relationships among cultivars, wild forms and related species(Angiolillo et al, 1999; Baldoni et al. 2000). Recently, single nucleotide polymorphism (SNP)-derived markers, identified in coding sequences of different genes, have been developed todiscriminate very similar cultivars (Reale et al., 2006; Consolandi et al., 2007) and for olive oiltraceability and authenticity (Consolandi et al., 2008). In this work we present results concerningthe molecular characterisation of a region upstream the ATG of chsA and chsB genes for Chalconesynthase (CHS), of different bp length, namely 956 base pairs the former and 333 base pairs thelatter, previously isolated in olive in our laboratory (Scialpi et al., 2005). The analysis of nucleotidevariation of these regions was then carried out in eight italian cultivars of Olea europaea L.(Nocellara Belice, Nocellara Etnea, Coratina, Nociara, Carolea, Bosana, Gentile di Chieti andLeccino), in one from Albania (Bardhi i Tirana), and in the wild form O. europaea var. sylvestris.Data obtained from molecular characterisation suggested a regulatory role for both the upstreamregions analysed. Moreover, comparative analysis of sequences of different cultivars showed a highlevel of polymorphism in terms of SNPs and InDels. Genetic relationships established on the baseof nucleotide diversity, using the UPGMA cluster analysis program, showed that cultivars weregrouped into different clusters. In particular, Coratina clusterized with the feral form O. europaeavar. sylvestris, this finding strongly suggesting for this cultivar, a possible introgression from thewild genotype. Finally, results will be discussed concerning the possibility to relate the nucleotidediversity of the upstream-chs sequences, with the modulation of gene activity as previously reportedfor other genes (Thornsberry et al., 2001).

References:Angiolillo et al. (1999). Theor Appl Genet 98: 411–421.Baldoni et al. (2000). Acta Hortic 521:275–284.Bandelj et al. (2004). Euphytica 136: 93–102.Barranco et al. (2000). Consejo Olecola Internacional, Madrid

Belaj et al. (2003). Theor Appl Genet 107:736–744Besnard G, Bervillé A (2000). Life Sciences 323: 173-181.Consolandi et al. (2007). Journal of Biotechnology 129: 565–574.Consolandi et al. (2008). Eur Food Res Technol DOI 10.1007/s00217-008-0863-5Owen et al. (2005). Theor. Appl. Genet. 110 (7): 1169–1176.Pasqualone A. & Caponio F.(2001). Eur. Food Res. Technol. 213: 240–243.Reale et al. (2006). Genome 49 (9): 1193–1205.Scialpi et al. (2005). Proc. SIGA Annual Congress, Potenza Italy – 12/15 SeptemberThornsberry et al. (2001). Nature Genet 28: 286-289.



SNP ANALYSIS OF OLEA EUROPAEA LUPEOL SYNTHASE GENE

SARRI V.*, CECCARELLI M.*, BALDONI L.**, MARIOTTI R.**, CULTRERA N.G.M.**

*) Department of Cell and Environmental Biology, University of Perugia, Via Elce di sotto,06123 Perugia (Italy)**) Institute of Plant Genetics, CNR, Research Division Perugia, Via Madonna Alta 130,06128 Perugia (Italy)

SNPs, Olea europaea, lupeol synthase gene, triterpenoids

Higher plants produce a wide range of non-steroidal triterpenoids. Their functions in plantsare still unknown, but they have important biological properties for human health. Sterols and nonsteroidal triterpenoids are products of the cytoplasmatic acetate/mevalonate pathway, and the 2,3-oxidosqualene is their common precursor. A group of enzymes, called triterpene synthases, utilizesthe oxidosqualene as substrate to produce different triterpenes (Stiti et al., 2007).

Olive (Olea europaea L.) trees synthesize many different sterols and triterpenoids, as theoleanolic acid. Thus, it is of great importance to understand their regulating mechanisms ofsynthesis and the differences about this pathway among cultivars.

Lupeol synthase is a key enzyme for the oleanolic acid synthesis and his cDNA sequence andenzyme function have been previously identified in olive (Shibuya et al., 1999).

The aim of this work was to analyze the Single Nucleotide Polymorphisms (SNPs) of thelupeol synthase gene in olive cultivars to get information about the DNA sequence differencesamong varieties and about the structure and organization of the gene. The obtained data could beutilized to clarify the phylogenetic relationships among cultivar and to establish possiblerelationships between mutational events and different triterpenoid content.

After the identification of the entire genomic sequence of the lupeol synthase gene, a fragmentof 524 base pairs was analysed on 80 olive cultivars. All the amplicons were sequenced andanalyzed: ten SNPs and 14 different aplotypes were identified.

In order to evaluate the presence of multiple lupeol synthase loci in the olive genome, theanalyzed sequences were cloned from some cultivars and sequenced in order to calculate theaplotype number. The results indicate the presence of two different loci of the lupeol synthase genein olive.

References:Stiti N. et al., (2007) Lipids, 42: 55-67Shibuya M. et al., (1999) Eur. J. Biochem., 266: 302-307



MOLECULAR CHARACTERIZATION OF OLIVE (OLEA EUROPEA L.)CULTIVARS FROM ABRUZZO REGION

ALBERTINI E.*, TORRICELLI R.*, RAGGI L.*, BITOCCHI E.**, BATTISTINI A.*,POLLASTRI L.***, DI MINCO G.***, PAPA R.**, VERONESI F.*

*) Dipartimento di Biologia Applicata, Università di Perugia**) Dipartimento di Scienze Ambientali e delle Produzioni Vegetali, Università Politecnica delleMarche, Ancona (Italy)***) ARSSA (Agenzia Regionale per I Servizi di Sviluppo Agricolo) Abruzzo, sede di Pescara

olive, population structure, olive oil, traceability

Olive (Olea europea L. subsp. europaea, 2n=2x=46), an oil-producing tree, is considered themost important fruit crop of the Mediterranean Basin and it is characterized by a large number ofvarieties, most of which behave in a self-incompatible way.

So far, several methodologies have been used to evaluate olive diversity and to characterizeolive germplasm such as leaf, fruit, pit, and growth habits or isozyme analysis. However, a majorlimitation of using these traits as genetic markers in Olive is that they are highly dependent onenvironmental or cultivation factors. In recent years, many papers report the successful assessmentof phylogenetic relationships in the O. europaea complex using RAPD, microsatellite-basedtechnique and AFLPs. Some of these techniques have proved very useful for varietalcharacterization and offers an almost unlimited supply of molecular traits for distinctivefingerprinting of plant materials without prior knowledge of target DNA sequences.

The development of cultivar-specific molecular markers would be highly useful for cultivaridentification, varietal protection, and oil composition determination especially in a view of oiltraceability.

The two main objectives of this study were to evaluate the identity of cultivars cultivated inAbruzzo region by establishing their genetic similarities/diversities and to investigate and analyzegenetic intra-cultivar diversity in Dritta, Gentile di Chieti, Gentile dell’Aquila and Tortiglionecultivars.

A total of 90 olive accessions were genotyped using 12 AFLP primer combinations. A total of1089 amplification products was scored with a percentage of polymorphism ranging from 0.3% to0.76% depending on primer combination. Interestingly, in 14 accessions (Dritta, Gentile di Chieti,Gentile dell’Aquila, Ghiandaro, Monicella, Nebbio, San Felice, Pendolino, Nostrana, Carboncella,Tortiglione, Nostrana, Unknown), at least one accession-specific band was recorded. Moreover, forother 3 bands an accession-absence discriminated one accession from other and could be consideredas informative as a cultivar-specific band (1 band for Gentile dell’Aquila and 2 bands for Leccino).

Moreover, the Pritchard method, implemented in the software STRUCTURE Ver. 2.2, wasused and adapted to dominant molecular markers in order to infer the population structure and, thus,to evaluate the identity of cultivars cultivated in Abruzzo region. Based on the STRUCTURE and∆K results the genetic structure of the whole sample was characterized by eight clusters. UPGMA,PCoA and Structure analysis of population are reported and discussed.

dna barcoding of wild italian dendroflora: first … · generate unique identifiers at either the...

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