URGV - Plant Genomics Research
Unité de Recherche en Génomique Végétale

 

Grapevine and Forest Trees Genomics

	Anne-Françoise Adam-Blondon » DR2 INRA adam@evry.inra.fr +33 1 60 87 45 34	Table of Content                    + » « Projects: Identification of efficient alleles for resistance to disease in grapevine and poplar Establishment of a HAPMAP in the Vitaceae and in Populus for the development of high throughput genotyping tools Tools: Physical and genetic maps in grapevine can be accessed h e r e and     h e r e Grapevine BAC libraries are distributed by C N R G V The grapevine genome can be browsed h e r e or     h e r e A Populus trichocarpa and a Quercus robus BAC libraries have been constructed. The Populus trichocarpa library is public and requests can be addressed to URGV. Publications Former Team Members and Post-Doctoral Fellows
	Patricia Faivre Rampant » CR1 INRA faivre@evry.inra.fr + 33 1 60 87 45 34

Publications

Moroldo M, Paillard S, Marconi R, Legeai F, Canaguier A, Cruaud C, De Berardinis V, Guichard C, Brunaud V, Le Clainche I, Scalabrin S, Testolin R, Di Gaspero G, Morgante M., Adam-Blondon A.F (2008)
A physical map of the heterozygous grapevine ‘Cabernet Sauvignon’ allows mapping candidate genes for disease resistance.
BMC Plant Biol, 8:66
Detailed Paper PMID: 18554400

Paux E, Legeai F, Guilhot N, Adam-Blondon F-F, Alaux M, Salse J, Sourdille P, Leroy P, Feuillet C (2008)
Physical mapping in large genomes : accelerating anchoring of BAC contigs to genetic maps through in silico analysis
Funct Integr Genomics, 8 : 29-32.
Detailed Paper PMID: 18038165

Di Gaspero G, Cipriani G, Adam-Blondon A-F, Testolin R (2006)
Linkage maps of grapevine displaying the chromosomal locations of 420 microsatellire markers and 83 markers for R-gene candidates.
Theor Appl Genet, 114 : 1249-1263
Detailed Paper PMID: 17380315

Jorge V, Dowkiw A, Faivre-Rampant P, Bastien C (2005)
Genetic architecture of qualitative and quantitative Melampsora larici populina leaf rust resistance in hybrid poplar: genetic mapping and QTL detection.
New Phytol, 167: 113-127
Detailed Paper PMID: 15948835

Barker CL, Donald T, Pauquet J,, Ratnaparkhe A,, Bouquet A, Adam-Blondon A-F,, Thomas MR, Dry I (2005)
Genetic and physical mapping of the grapevine powdery mildew resistance gene, Run1, using a bacterial artificial chromosome library.
Theor Appl Genet, 111 : 370-377
Detailed Paper PMID: 15902396

 

Establishment of a HAPMAP in the Vitaceae and in Populus for the development of high throughput genotyping tools

People currently involved: Aurélie Canaguier; Cléa Houel; Isabelle Le Clainche; Isabella Paolucci

National Collaborations: UMR DiaPC (Montpellier), EPGV (CNG, Evry), URGI, UMR S&G University of Evry, UAGPF Orléans; UMR EGV (Bordeaux); UMR BioGeCo (Bordeaux)
International Collaborations: Univ. of Verona (Italy), Univ. British Columbia (Canada), Univ. of Verona (Italy), Univ. of Udine (Italy); USDA Geneva (NY,USA), Cold Spring Harbor (NY, USA); JKI-Geilweilerhof (Germany); University of Bielefeld (Germany)
Financial support: INRA project TPV and PlantReSeq (coord. D. Brunel, EPGV), PHD thesis of Cléa Houel (Univ. of Evry); EU project EVOLTREE, Novel Tree Breeding (coord. C. Plomion, BioGeCo); Trilateral project GRAPERESEQ 2009-2011 (coord. A-F Adam-Blondon).

Two breakthroughs have completely modified the way genetic analysis can be carried out: the high density genotyping methods and the high throughput sequencing technologies. The density of markers that can be developed by re-sequencing is highly dependent on the possibility to quickly re-sequence genome fragments in a well chosen set of genotypes to assess the SNP frequency, which would be used to define accurate and useful panels of markers. Pilot projects are undergoing, aiming at the Sanger re-sequencing of around 1000 gene fragments in core-collections of genotypes in poplar and in grapevine and targeting in particular genes invoved in pathways for disease resistance, wood formation, adaptation to environment and berry size. The new sequencing techniques are decreasing re-sequencing costs but necessitate a step of genome complexity reduction. Our aim is to test the method developed by Hodges et al (2007) for high quality re-sequencing of the exons in grapevine and poplar. Chips for high density and high throughput genotyping will be designed.

PhD 2007-2010 : Cléa Houel
Titre : Association genetics in relation with the pericarpe development in grapevine.

 

Publications

Le Cunff L, Fournier-Level A, Laucou V, Vezzulli S, Lacombe T, Adam-Blondon AF, Boursiquot JM, This P. (2008)
Construction of nested core collections to optimize the exploitation of natural diversity in Vitis vinifera L. subsp sativa.
BMC Plant Biology, 8:31
Detailed Paper PMID: 18384667

Jaillon O, Aury J-M, Noel B, Policriti A, Clepet C, Casagrande A, Choisne N, Aubourg S, Vitulo N, Jubin C, Vezzi A, Legeai F, Hugueney P, Dasilva C, Horner D, Mica E, Jublot D, Poulain J, Bruyere C, Billault A, Segurens B, Gouyvenoux M, Ugarte E, Cattonaro F, Anthouard V, Vico V, Del Fabbro C, Alaux M, Di Gaspero G, Dumas V, Felice N, Paillard S, Juman I, Moroldo M, Scalabrin S, Canaguier A, Le Clainche I, Malacrida G, Durand E, Pesole G, Laucou V, Chatelet P, Merdinoglu D, Delledonne M, Pezzotti M, Lecharny A, Scarpelli C, Artiguenave F, Pé E, Valle G, Morgante M, Caboche M, Adam-Blondon A-F, Weissenbach J, Quétier F, Wincker P (2007)
The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla.
Nature,449 : 463-468
Detailed Paper PMID: 17721507

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Publications

 

A physical map of the heterozygous grapevine 'Cabernet Sauvignon' allows mapping candidate genes for disease resistance.

BMC Plant Biol. 2008 Jun 13;8:66. PMID: 18554400
333   Moroldo M, Paillard S, Marconi R, Fabrice L, Canaguier A, Cruaud C, De Berardinis V, Guichard C, Brunaud V, Le Clainche I, Scalabrin S, Testolin R, Di Gaspero G, Morgante M, Adam-Blondon AF.
UMR de Génomique Végétale, INRA-CNRS-UEVE, 2, Rue Gaston Crémieux, CP5708, 91057 Evry Cedex, France. moroldo@evry.inra.fr

BACKGROUND: Whole-genome physical maps facilitate genome sequencing, sequence assembly, mapping of candidate genes, and the design of targeted genetic markers. An automated protocol was used to construct a Vitis vinifera 'Cabernet Sauvignon' physical map. The quality of the result was addressed with regard to the effect of high heterozygosity on the accuracy of contig assembly. Its usefulness for the genome-wide mapping of genes for disease resistance, which is an important trait for grapevine, was then assessed. RESULTS: The physical map included 29,727 BAC clones assembled into 1,770 contigs, spanning 715,684 kbp, and corresponding to 1.5-fold the genome size. Map inflation was due to high heterozygosity, which caused either the separation of allelic BACs in two different contigs, or local mis-assembly in contigs containing BACs from the two haplotypes. Genetic markers anchored 395 contigs or 255,476 kbp to chromosomes. The fully automated assembly and anchorage procedures were validated by BAC-by-BAC blast of the end sequences against the grape genome sequence, unveiling 7.3% of chimerical contigs. The distribution across the physical map of candidate genes for non-host and host resistance, and for defence signalling pathways was then studied. NBS-LRR and RLK genes for host resistance were found in 424 contigs, 133 of them (32%) were assigned to chromosomes, on which they are mostly organised in clusters. Non-host and defence signalling genes were found in 99 contigs dispersed without a discernable pattern across the genome. CONCLUSION: Despite some limitations that interfere with the correct assembly of heterozygous clones into contigs, the 'Cabernet Sauvignon' physical map is a useful and reliable intermediary step between a genetic map and the genome sequence. This tool was successfully exploited for a quick mapping of complex families of genes, and it strengthened previous clues of co-localisation of major NBS-LRR clusters and disease resistance loci in grapevine.

A reference integrated map for cultivated grapevine (Vitis vinifera L.) from three crosses, based on 283 SSR and 501 SNP-based markers.

Theor Appl Genet. 2008 May 27. PMID: 18504538
332   Silvia Vezzulli(1, @), Michela Troggio(1), Giuseppina Coppola(1), Angelica Jermakow(2), Dustin Cartwright(3), Andrey Zharkikh(3), Marco Stefanini(1), M. Stella Grando(1), Roberto Viola(1), Anne-Françoise Adam-Blondon(4), Mark Thomas(2), Patrice This(5) and Riccardo Velasco(1)
(1)  IASMA Research Centre, San Michele a/Adige, Italy
(2)  CSIRO Plant Industry, Glen Osmond, Australia
(3)  Myriad Genetics Inc., Salt Lake City, UT, USA
(4)  UMR 1165, INRA-CNRS-Université d Evry Génomique Végétale, Paris Cedex, France
(5)  UMR 1097, DIA-PC Equipe génétique Vigne, INRA Supagro, Montepellier, France
(@)  silvia.vezzulli@iasma.it.

We have developed an integrated map from five elite cultivars of Vitis vinifera L.; Syrah, Pinot Noir, Grenache, Cabernet Sauvignon and Riesling which are parents of three segregating populations. A new source of markers, SNPs, identified in ESTs and unique BAC-end sequences was added to the available IGGP reference set of SSRs. The complete integrated map comprises 1,134 markers (350 AFLP((R)), 332 BESs, 169 ESTs, 283 SSRs) spanning 1,443 cM over 19 linkage groups and shows a mean distance between neighbouring loci of 1.27 cM. Marker order was mainly conserved between the integrated map and the highly dense Syrah x Pinot Noir consensus map except for few inversions. Moreover, the marker order has been validated through the assembled genome sequence of Pinot Noir. We have also assessed the transferability of SNP-based markers among five V. vinifera varieties, enabling marker validation across different genotypes. This integrated map can serve as a fundamental tool for molecular breeding in V. vinifera and related species and provide a basis for studies of genome organization and evolution in grapevines.
Supplemental Material
Vezzulli_etal2008_FS1.ppt         Vezzulli_etal2008_FS2.ppt
Vezzulli_etal2008_TS1.xls         Vezzulli_etal2008_TS2.xls

Construction of nested genetic core collections to optimize the exploitation of natural diversity in Vitis vinifera L. subsp sativa.

BMC Plant Biol. 2008 Apr 2;8(1):31 PMID: 18384667
323 Le Cunff L, Fournier-Level A, Laucou V, Vezzulli S, Lacombe T, Adam-Blondon AF, Boursiquot JM, This P.

ABSTRACT: BACKGROUND:
The first high quality draft of the grape genome sequence has just been published. This is a critical step in accessing all the genes of this species and increases the chances of exploiting the natural genetic diversity through association genetics. However, our basic knowledge of the extent of allelic variation within the species is still not sufficient. Towards this goal, we constructed nested genetic core collections (G-cores) to capture the simple sequence repeat (SSR) diversity of the grape cultivated compartment (Vitis vinifera L. subsp. sativa) from the world's largest germplasm collection (Domaine de Vassal, INRA Herault, France), containing 2262 unique genotypes.
RESULTS:
Sub-samples of 12, 24, 48 and 92 varieties of V. vinifera L. were selected based on their genotypes for 20 SSR markers using the M-strategy. They represent respectively 58%, 73%, 83% and 100 % of total SSR diversity. The capture of allelic diversity was analyzed by sequencing three genes scattered throughout the genome on 233 individuals: 41 single nucleotide polymorphisms (SNPs) were identified using the G-92 core (one SNP for every 49 nucleotides) while only 25 were observed using a larger sample of 141 individuals selected on the basis of 50 morphological traits, thus demonstrating the reliability of the approach. CONCLUSION: The G-12 and G-24 core-collections displayed respectively 78% and 88% of the SNPs respectively, and are therefore of great interest for SNP discovery studies. Furthermore, the nested genetic core collections satisfactorily reflected the geographic and the genetic diversity of grape, which are also of great interest for the study of gene evolution in this species.<

Physical mapping in large genomes: accelerating anchoring of BAC contigs to genetic maps through in silico analysis.

Funct Integr Genomics. 2008 Feb;8(1):29-32. Epub 2007 Nov 24. PMID: 18038165
308 Paux E, Legeai F, Guilhot N, Adam-Blondon AF, Alaux M, Salse J, Sourdille P, Leroy P, Feuillet C.
INRA, UMR 1095, ASP, 234, avenue du Brézet, 63100, Clermont-Ferrand, France, catherine.feuillet@clermont.inra.fr.

Anchored physical maps represent essential frameworks for map-based cloning, comparative genomics studies, and genome sequencing projects. High throughput anchoring can be achieved by polymerase chain reaction (PCR) screening of bacterial artificial chromosome (BAC) library pools with molecular markers. However, for large genomes such as wheat, the development of high dimension pools and the number of reactions that need to be performed can be extremely large making the screening laborious and costly. To improve the cost efficiency of anchoring in such large genomes, we have developed a new software named Elephant (electronic physical map anchoring tool) that combines BAC contig information generated by FingerPrinted Contig with results of BAC library pools screening to identify BAC addresses with a minimal amount of PCR reactions. Elephant was evaluated during the construction of a physical map of chromosome 3B of hexaploid wheat. Results show that a one dimensional pool screening can be sufficient to anchor a BAC contig while reducing the number of PCR by 384-fold thereby demonstrating that Elephant is an efficient and cost-effective tool to support physical mapping in large genomes.

The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla

Nature 449, 463-467 (27 Sept. 2007) | doi:10.1038/nature06148; PMID: 17721507
Olivier Jaillon, Jean-Marc Aury, Benjamin Noel, Alberto Policriti, Christian Clepet, Alberto Casagrande, Nathalie Choisne, Sébastien Aubourg, Nicola Vitulo, Claire Jubin, Alessandro Vezzi, Fabrice Legeai, Philippe Hugueney, Corinne Dasilva, David Horner, Erica Mica, Delphine Jublot, Julie Poulain, Clémence Bruyère, Alain Billault, Béatrice Segurens, Michel Gouyvenoux, Edgardo Ugarte, Federica Cattonaro, Véronique Anthouard, Virginie Vico, Cristian Del Fabbro, Michaël Alaux, Gabriele Di Gaspero, Vincent Dumas, Nicoletta Felice, Sophie Paillard, Irena Juman, Marco Moroldo, Simone Scalabrin, Aurélie Canaguier, Isabelle Le Clainche, Giorgio Malacrida, Eléonore Durand, Graziano Pesole, Valérie Laucou, Philippe Chatelet, Didier Merdinoglu, Massimo Delledonne, Mario Pezzotti, Alain Lecharny, Claude Scarpelli, François Artiguenave, M. Enrico Pè, Giorgio Valle, Michele Morgante, Michel Caboche, Anne-Françoise Adam-Blondon, Jean Weissenbach, Francis Quétier & Patrick Wincker

The analysis of the first plant genomes provided unexpected evidence for genome duplication events in species that had previously been considered as true diploids on the basis of their genetics. These polyploidization events may have had important consequences in plant evolution, in particular for species radiation and adaptation and for the modulation of functional capacities. Here we report a high-quality draft of the genome sequence of grapevine (Vitis vinifera) obtained from a highly homozygous genotype. The draft sequence of the grapevine genome is the fourth one produced so far for flowering plants, the second for a woody species and the first for a fruit crop (cultivated for both fruit and beverage). Grapevine was selected because of its important place in the cultural heritage of humanity beginning during the Neolithic period. Several large expansions of gene families with roles in aromatic features are observed. The grapevine genome has not undergone recent genome duplication, thus enabling the discovery of ancestral traits and features of the genetic organization of flowering plants. This analysis reveals the contribution of three ancestral genomes to the grapevine haploid content. This ancestral arrangement is common to many dicotyledonous plants but is absent from the genome of rice, which is a monocotyledon. Furthermore, we explain the chronology of previously described whole-genome duplication events in the evolution of flowering plants.

Linkage maps of grapevine displaying the chromosomal locations of 420 microsatellite markers and 83 markers for R-gene candidates.

Theor Appl Genet. 2007 May;114(7):1249-63. Epub 2007 Mar 23. PMID: 17380315
Di Gaspero G, Cipriani G, Adam-Blondon A-F, Testolin R (2006)

Genetic maps functionally oriented towards disease resistance have been constructed in grapevine by analysing with a simultaneous maximum-likelihood estimation of linkage 502 markers including microsatellites and resistance gene analogs (RGAs). Mapping material consisted of two pseudo-testcrosses, 'Chardonnay' x 'Bianca' and 'Cabernet Sauvignon' x '20/3' where the seed parents were Vitis vinifera genotypes and the male parents were Vitis hybrids carrying resistance to mildew diseases. Individual maps included 320-364 markers each. The simultaneous use of two mapping crosses made with two pairs of distantly related parents allowed mapping as much as 91% of the markers tested. The integrated map included 420 Simple Sequence Repeat (SSR) markers that identified 536 SSR loci and 82 RGA markers that identified 173 RGA loci. This map consisted of 19 linkage groups (LGs) corresponding to the grape haploid chromosome number, had a total length of 1,676 cM and a mean distance between adjacent loci of 3.6 cM. Single-locus SSR markers were randomly distributed over the map (CD = 1.12). RGA markers were found in 18 of the 19 LGs but most of them (83%) were clustered on seven LGs, namely groups 3, 7, 9, 12, 13, 18 and 19. Several RGA clusters mapped to chromosomal regions where phenotypic traits of resistance to fungal diseases such as downy mildew and powdery mildew, bacterial diseases such as Pierce's disease, and pests such as dagger and root-knot nematode, were previously mapped in different segregating populations. The high number of RGA markers integrated into this new map will help find markers linked to genetic determinants of different pest and disease resistances in grape.

Molecular characterization and expression analysis of the Rop GTPase family in Vitis vinifera.

J Exp Bot. 2007;58(10):2641-52. Epub 2007 Jun 19. PMID: 17578867
309 Abbal P, Pradal M, Sauvage FX, Chatelet P, Paillard S, Canaguier A, Adam-Blondon AF, Tesniere C.
UMR1083, Sciences pour l'Oenologie, AgroM, INRA, Université Montpellier 1, F-34000, Montpellier, France.

Rop/Rac GTPases are plant-specific signalling proteins with multiple roles, some of which have implications in plant development and in hormone signal transduction. Using expressed sequence tag (EST) and gene database analyses, members of the Rop family were characterized for the first time in a perennial species (Vitis vinifera). The grapevine genome was found to contain seven expressed VvRops. The phylogenetic analyses indicated that VvRops could be distributed into four groups, as described in the literature for model plants. Genetic mapping was successfully performed for five VvRops, which were localized on independent linkage groups. Conserved and divergent regions were identified on the protein sequences. The results of VvRop expression obtained by real-time quantitative reverse transcription-PCR analyses indicated that all the organs investigated displayed VvRop expression, however with different patterns. Whereas no total organ specificity for VvRop expression could be evidenced, VvRop9 displayed high expression in developing berries only. During berry development, the transcript profile was generally similar for all the VvRops, i.e. displaying a peak early in the herbaceous phase followed by a decline towards veraison and thereafter. Western blotting gave a similar expression profile for VvRop proteins. Response to growth regulators was generally specific to each VvRop. The potential involvement of specific VvRops in grapevine development is discussed.

An integrated SSR map of grapevine based on five mapping populations.

Theor Appl Genet. 2006 Aug;113(3):369-82. Epub 2006 Jun 24. PMID: 16799809
026 Doligez A;Adam-Blondon A-F; Cipriani G; Di Gaspero G; Laucou V; Merdinoglu D; Meredith C; Riaz S; Roux C; This P (2006)
INRA, UMR DGPC 1097, équipe Génétique Vigne, bâtiment 6, 2 place Viala, 34060, Montpellier Cedex 1, France. doligez@ensam.inra.fr

A grapevine (mainly Vitis vinifera L., 2n = 38) composite genetic map was constructed with CarthaGene using segregation data from five full-sib populations of 46, 95, 114, 139 and 153 individuals, to determine the relative position of a large set of molecular markers. This consensus map comprised 515 loci (502 SSRs and 13 other type PCR-based markers), amplified using 439 primer pairs (426 SSRs and 13 others) with 50.1% common markers shared by at least two crosses. Out of all loci, 257, 85, 74, 69 and 30 were mapped in 1, 2, 3, 4 and 5 individual mapping populations, respectively. Marker order was generally well conserved between maps of individual populations, with only a few significant differences in the recombination rate of marker pairs between two or more populations. The total length of the integrated map was 1,647 cM Kosambi covering 19 linkage groups, with a mean distance between neighbour loci of 3.3 cM. A framework-integrated map was also built, with marker order supported by a LOD of 2.0. It included 257 loci spanning 1,485 cM Kosambi with a mean inter-locus distance of 6.2 cM over 19 linkage groups. These integrated maps are the most comprehensive SSR-based maps available so far in grapevine and will serve either for choosing markers evenly scattered over the whole genome or for selecting markers that cover particular regions of interest. The framework map is also a useful starting point for the integration of the V. vinifera physical and genetic maps.
The maps may be viewed at (Unité de Recherche Génomique Info - URGI)

Anchoring a large set of markers onto a BAC library for the development of a draft physical map of the grapevine genome.

Theor Appl Genet. 2006 May 18; PMID: 16708229
031 Lamoureux D, Bernole A, Le Clainche I, Tual, S, Thareau V, Paillard S, Legeai F, Dossat C, Wincker P, Oswald M, Merdinoglu D, Vignault C, Delrot S, Caboche M, Chalhoub B, Adam-Blondon A-F (2006)

Five hundred and six EST-derived markers, 313 SSR markers and 26 BAC end-derived or SCAR markers were anchored by PCR on a subset of a Cabernet Sauvignon BAC library representing six genome equivalents pooled in three dimensions. In parallel, the 12,351 EST clusters of the grapevine UniGene set (build #11) from NCBI were used to design 12,125 primers pairs and perform electronic PCR on 67,543 nonredundant BAC-end sequences. This in silico experiment yielded 1,140 positive results concerning 638 different markers, among which 602 had not been already anchored by PCR. The data obtained will provide an easier access to the regulatory sequences surrounding important genes (represented by ESTs). In total, 1,731 islands of BAC clones (set of overlapping BAC clones containing at least one common marker) were obtained and 226 of them contained at least one genetically mapped anchor. These assigned islands are very useful because they will link the genetic map and the future fingerprint-based physical map and because they allowed us to indirectly place 93 ESTs on the genetic map. The islands containing two or more mapped SSR markers were also used to assess the quality of the integrated genetic map of the grapevine genome.
The results can be accessed here

Colour variation in red grapevines (Vitis vinifera L.): genomic organisation, expression of flavonoid 3′-hydroxylase, flavonoid 3′,5′-hydroxylase genes and related metabolite profiling of red cyanidin-/blue delphinidin-based anthocyanins in berry skin.

BMC Genomics. 2006 Jan 24;7:12. PMID: 16433923
Castellarin SD, Di Gaspero G, Marconi R, Nonis A, Peterlunger E, Paillard S, Adam-Blondon A-F, Testolin R (2006)
Dipartimento di Scienze Agrarie e Ambientali, University of Udine, via delle Scienze 208, 33100 Udine, Italy. castellarin.simone@spes.uniud.it

BACKGROUND: Structural genes of the phenyl-propanoid pathway which encode flavonoid 3'- and 3',5'-hydroxylases (F3'H and F3'5'H) have long been invoked to explain the biosynthesis of cyanidin- and delphinidin-based anthocyanin pigments in the so-called red cultivars of grapevine. The relative proportion of the two types of anthocyanins is largely under genetic control and determines the colour variation among red/purple/blue berry grape varieties and their corresponding wines. RESULTS: Gene fragments of VvF3'H and VvF3'5'H, that were isolated from Vitis vinifera 'Cabernet Sauvignon' using degenerate primers designed on plant homologous genes, translated into 313 and 239 amino acid protein fragments, respectively, with up to 76% and 82% identity to plant CYP75 cytochrome P450 monooxygenases. Putative function was assigned on the basis of sequence homology, expression profiling and its correlation with metabolite accumulation at ten different ripening stages. At the onset of colour transition, transcriptional induction of VvF3'H and VvF3'5'H was temporally coordinated with the beginning of anthocyanin biosynthesis, the expression being 2-fold and 50-fold higher, respectively, in red berries versus green berries. The peak of VvF3'5'H expression was observed two weeks later concomitantly with the increase of the ratio of delphinidin-/cyanidin-derivatives. The analysis of structural genomics revealed that two copies of VvF3'H are physically linked on linkage group no. 17 and several copies of VvF3'5'H are tightly clustered and embedded into a segmental duplication on linkage group no. 6, unveiling a high complexity when compared to other plant flavonoid hydroxylase genes known so far, mostly in ornamentals. CONCLUSION: We have shown that genes encoding flavonoid 3'- and 3',5'-hydroxylases are expressed in any tissues of the grape plant that accumulate flavonoids and, particularly , in skin of ripening red berries that synthesise mostly anthocyanins. The correlation between transcript profiles and the kinetics of accumulation of red/cyanidin- and blue/delphinidin-based anthocyanins indicated that VvF3'H and VvF3'5'H expression is consistent with the chromatic evolution of ripening bunches. Local physical maps constructed around the VvF3'H and VvF3'5'H loci should help facilitate the identification of the regulatory elements of each isoform and the future manipulation of grapevine and wine colour through agronomical, environmental and biotechnological tools.

Genetic architecture of qualitative and quantitative Melampsora larici populina leaf rust resistance in hybrid poplar: genetic mapping and QTL detection.

New Phytol. 2005 Jul;167(1):113-27. PMID: 15948835
111 Jorge V., Dowkiw A., Faivre Rampant P., Bastien C. 2005.
Unité Amélioration, Génétique et Physiologie Forestières, Institut National de la Recherche Agronomique, F-45166 Olivet Cedex, France. jorge@orleans.inra.fr

In order to elucidate the genetic control of resistance to Melampsora larici-populina leaf rust in hybrid poplars, a Populus deltoides x P. trichocarpa F(1) progeny was analysed for qualitative and quantitative rust resistances. This progeny was evaluated for three components of quantitative resistance (latent period, uredinia number and uredinia size) to seven M. larici-populina strains in controlled conditions, and for one component of field susceptibility (rust colonization on the most infected leaf). One qualitative resistance locus inherited from P. deltoides, R(1), was localized on the genetic map. It segregates 1 : 1 in the F(1) progeny and is effective against four of the studied strains. QTL analysis was performed separately on R(1) and r(1) genotype subsets. An additional detection was conducted on the entire F(1) progeny for the three strains able to overcome R(1) and for MAX2. A total of nine QTLs were detected. Two had large, broad-spectrum effects. One (R(US)) is inherited from the P. trichocarpa parent; the other is inherited from P. deltoides and colocalized with R(1). Seven QTLs had only limited and specific effects. Significant interaction effects were detected mainly between the two major QTLs. Implications of these results for durable resistance breeding strategies, and possible benefits from the Populus genome sequence, are discussed.

Genetic and physical mapping of the grapevine powdery mildew resistance gene, Run1, using a bacterial artificial chromosome library.

Theor Appl Genet. 2005 Jul;111(2):370-7. Epub 2005 May 18. PMID: 15902396
108 Barker CL, Donald T, Pauquet J,, Ratnaparkhe A,, Bouquet A, Adam-Blondon A-F, Thomas MR, Dry I (2005)

Resistance to grapevine powdery mildew is controlled by Run1, a single dominant gene present in the wild grapevine species, Muscadinia rotundifolia, but absent from the cultivated species, Vitis vinifera. Run1 has been introgressed into V. vinifera using a pseudo-backcross strategy, and genetic markers have previously been identified that are linked to the resistance locus. Here we describe the construction of comprehensive genetic and physical maps spanning the resistance locus that will enable future positional cloning of the resistance gene. Physical mapping was performed using a bacterial artificial chromosome (BAC) library constructed using genomic DNA extracted from a resistant V. vinifera individual carrying Run1 within an introgression. BAC contig assembly has enabled 20 new genetic markers to be identified that are closely linked to Run1, and the position of the resistance locus has been refined, locating the gene between the simple sequence repeat (SSR) marker, VMC4f3.1, and the BAC end sequence-derived marker, CB292.294. This region contains two multigene families of resistance gene analogues (RGA). A comparison of physical and genetic mapping data indicates that recombination is severely repressed in the vicinity of Run1, possibly due to divergent sequence contained within the introgressed fragment from M. rotundifolia that carries the Run1 gene.

Construction and characterization of BAC libraries from major grapevine cultivars.

Theor Appl Genet. 2005 May;110(8):1363-71. Epub 2005 Apr 16. PMID: 15834699
061 Adam-Blondon A-F, Bernole A, Faes G, Lamoureux D, Pateyron S, Grando MS, Caboche M, Velasco R, Chalhoub B (2005)
Unité Mixte de Recherches sur les Génomes des Végétaux, INRA, 2 rue Gaston Crémieux, 5708 91 057, Evry Cedex, France. adam@evry.inra.fr

Genome projects were initiated on grapevine (Vitis vinifera L., 2n=38, genome size 475 Mb) through the successful construction of four bacterial artificial chromosome (BAC) libraries from three major cultivars, Cabernet Sauvignon (Cabernet S), Syrah and two different clones of Pinot Noir (Pinot N). Depending on the library, the genome coverage represented 4.5-14.8 genome equivalents with clones having a mean insert size of 93-158 kb. BAC pools suitable for PCR screening were constructed for two of these BAC libraries [Cabernet S and Pinot N clone (cl) 115] and subsequently used to confirm the genome coverage of both libraries by PCR anchoring of 74 genetic markers sampled from the 19 linkage groups. For ten of these markers, two bands on separate BAC pools were differentiated that could correspond either to different alleles or to a duplication of the locus being studied. Finally, a preliminary assessment of the correspondence between genetic and physical distances was made through the anchoring of all the markers mapped along linkage group 1 of the V. vinifera genetic map. A pair of markers, 2.1 cM apart, anchored the same BAC clones, which allowed us to estimate that 1 cM corresponded in this particular region to a maximum length of 130 kb.

Development and characterization of a large set of microsatellite markers in grapevine(Vitis vinifera L.) suitable for multiplex PCR.

Molecular Breeding, 2005 15 : 349-366
070 Merdinoglu D, Butterlin G, Bevilaqua L, Chiquet V, Adam-Blondon A-F, Decroocq S (2005)

Despite their numerous advantages, the use of microsatellites as genetic markers could be limited because of the low number of loci that can be simultaneously analysed per experiment. To increase the information per simple sequence repeat (SSR) assay in the grapevine, we developed a large set of new markers suitable for multiplexing and multi-loading. We produced microsatellite motif-enriched genomic libraries containing preferentially large size inserts which allowed us to design primers generating a wide range of allele sizes in a very standard and unique PCR condition. Three hundred and fifty clones were sequenced and 190 of them (54%) contained microsatellite motifs with suitable flanking regions for primer design. We developed 169 new SSR markers giving suitable signal with fluorescent-based DNA detection. The total number of alleles detected varied from 1 to 8 per locus with an average of 3.5 and the mean expected heterozygosity was 0.544 (range: 0 0.86). Sixty-eight loci (40%) were perfect types, 73 (43%) were imperfect and 28 (17%) were compound or imperfect-compound. The number of alleles generated by perfect and imperfect type loci was positively correlated to the length of the microsatellite motif. Forty-six multiplex sets based on 125 selected loci were developed. Considering their allele size range, up to four PCR multiplex were pooled together for multi-loading. The 169 SSR loci developed in this study represent a new and informative set of markers easy to combine for multiplexing and multi-loading according to the needs of any user and suitable for large scale genetic analyses in grapevine.

Mapping 245 SSR markers on the Vitis vinifera genome: a tool for grape genetics.

Theor Appl Genet. 2004 Sep;109(5):1017-27. Epub 2004 Jun 4. PMID: 15184982
167 Adam-Blondon A-F, Roux C, Claux D, Butterlin G, Merdinoglu D, This P, (2004)

The aim of the present work was to develop a microsatellite marker-based map of the Vitis vinifera genome (n=19), useful for genetic studies in this perennial heterozygous species, as SSR markers are highly transferable co-dominant markers. A total of 346 primer pairs were tested on the two parents (Syrah and Grenache) of a full sib population of 96 individuals (S x G population), successfully amplifying 310 markers. Of these, 88.4% markers were heterozygous for at least one of the two parents. A total of 292 primer pairs were then tested on Riesling, the parent of the RS1 population derived from selfing (96 individuals), successfully amplifying 299 markers among which 207 (62.9%) were heterozygous. Only 6.7% of the markers were homozygous in all three genotypes, stressing the interest of such markers in grape genetics. Four maps were constructed based on the segregation of 245 SSR markers in the two populations. The Syrah map was constructed from the segregations of 177 markers that could be ordered into 19 linkage groups (total length 1,172.2 cM). The Grenache map was constructed with the segregations of 178 markers that could be ordered into 18 linkage groups (total length 1,360.6 cM). The consensus S x G map was constructed with the segregations of 220 markers that were ordered into 19 linkage groups (total length 1,406.1 cM). One hundred and eleven markers were scored on the RS1 population, among them 27 that were not mapped using the S x G map. Out of these 111 markers, 110 allowed to us to construct a map of a total length of 1,191.7 cM. Using these four maps, the genome length of V. vinifera was estimated to be around 2,200 cM. The present work allowed us to map 123 new SSR markers on the V. vinifera genome that had not been ordered in a previous SSR-based map (Riaz et al. 2004), representing an average of 6.5 new markers per linkage group. Any new SSR marker mapped is of great potential usefulness for many applications such as the transfer of well-scattered markers to other maps for QTL detection, the use of markers in specific regions for the fine mapping of genes/QTL, or for the choice of markers for MAS.

Identification of Resistance Gene Analogs linked to a powdery mildew resistance locus in grapevine

Theor Appl Genet. 2002 Mar;104(4):610-618. PMID: 12582665
135 Donald T, Pellerone F, Adam-Blondon A-F, Bouquet A, Thomas M, Dry I (2002)
CSIRO Plant Industry, Horticulture Unit, PO Box 350, Glen Osmond SA 5064, Australia. ian.dry@pi.csiro.au

Oligonucleotide primers, designed to conserved regions of nucleotide binding site (NBS) motifs within previously cloned pathogen resistance genes, were used to amplify resistance gene analogs (RGAs) from grapevine. Twenty eight unique grapevine RGA sequences were identified and subdivided into 22 groups on the basis of nucleic acid sequence-identity of approximately 70% or greater. Representatives from each group were used in a bulked segregant analysis strategy to screen for restriction fragment length polymorphisms linked to the powdery mildew resistance locus, Run1, introgressed into Vitis vinifera L. from the wild grape species Muscadinia rotundifolia. Three RGA markers were found to be tightly linked to the Run1 locus. Of these markers, two (GLP1-12 and MHD145) cosegregated with the resistance phenotype in 167 progeny tested, whereas the third marker (MHD98) was mapped to a position 2.4 cM from the Run1 locus. The results demonstrate the usefulness of RGA sequences, when used in combination with bulked segregant analysis, to rapidly generate markers tightly linked to resistance loci in crop species.

 

Former Team Members and Post-Doctoral Fellows

Former Members of the Team

Anne Bernole	2000 - 2003	Research Assistant (IE, CDD Génoplante)
Réjane Boulay	2005	Master of Science
Magella Drouet	2006	Research Assistant (AI, INRA, CDD ANR)
Eléonore Durand	Mar 2007 - Sept 2007	Master of Science
Julie Jousseaumes	2007	Research Assistant (AI, CDD ANR)
Sarah Tual	2004	Master of Science

Post-Doctoral Fellows

Frank Chopin	ATER, Group ODG-Grapevine	Sep 06 - Sep 07	Characterisation of the sequence variation in grapevine
Didier Lamoureux	Group ODG-Grapevine	Dec 03 - Dec 04	Anchoring a large set of markers on Cabernet S BAC clones
Marco Moroldo	Group ODG-Grapevine	Apr 06 - Apr 08	Physical mapping of genes involved in non host resistance in Grapevine
Sophie Paillard	Group ODG-Grapevine	Feb 04 - Feb 06	Responsible for the physical mapping of grape wine