V. Laucou

Development of a standard set of microsatellite reference alleles for identification of grape cultivars

In order to investigate the comparability of microsatellite profiles obtained in different laboratories, ten partners in seven countries analyzed 46 grape cultivars at six loci (VVMD5, VVMD7, VVMD27, VVS2, VrZAG62, and VrZAG79). No effort was made to standardize equipment or protocols. Although some partners obtained very similar results, in other cases different absolute allele sizes and, sometimes, different relative allele sizes were obtained. A strategy for data comparison by means of reference to the alleles detected in well-known cultivars was proposed. For each marker, each allele was designated by a code based on the name of the reference cultivar carrying that allele. Thirty-three cultivars, representing from 13 to 23 alleles per marker, were chosen as references. After the raw data obtained by the different partners were coded, more than 97% of the data were in agreement. Minor discrepancies were attributed to errors, suboptimal amplification and visualization, and misscoring of heterozygous versus homozygous allele pairs. We have shown that coded microsatellite data produced in different laboratories with different protocols and conditions can be compared, and that it is suitable for the identification and SSR allele characterization of cultivars. It is proposed that the six markers employed here, already widely used, be adopted as a minimal standard marker set for future grapevine cultivar analyses, and that additional cultivars be characterized by means of the coded reference alleles presented here. The complete database is available at http://www.genres.de/eccdb/vitis/. Cuttings of the 33 reference cultivars are available on request from the Institut National de la Recherche Agronomique Vassal collection (didier.vares@ensam.inra.fr).

Genetic mapping in pea. 1. RAPD-based genetic linkage map of Pisum sativum

Abstract A genetic linkage map of Pisum sativum L. was constructed based primarily on RAPD markers that were carefully selected for their reproducibility and scored in a population of 139 recombinant inbred lines (RILs). The mapping population was derived from a cross between a protein-rich dry-seed cultivar ‘Térèse’ and an increased branching mutant (K586) obtained from the pea cultivar ‘Torsdag’. The map currently comprises nine linkage groups with two groups comprising only 6 markers (n=7 in pea) and covers 1139 cM. This RAPD-based map has been aligned with the map based on the (JI281×JI399) RILs population that currently includes 355 markers in seven linkage groups covering 1881 cM. The difference in map lengths is discussed. For this alignment 7 RFLPs, 23 RAPD markers, the morphological marker le and the PCR marker corresponding to the gene Uni were used as common markers and scored in both populations.

Large-scale parentage analysis in an extended set of grapevine cultivars ( Vitis vinifera L.)

Inheritance of nuclear microsatellite markers (nSSR) has been proved to be a powerful tool to verify or uncover the parentage of grapevine cultivars. The aim of the present study was to undertake an extended parentage analysis using a large sample of Vitis vinifera cultivars held in the INRA “Domaine de Vassal” Grape Germplasm Repository (France). A dataset of 2,344 unique genotypes (i.e. cultivars without synonyms, clones or mutants) identified using 20 nSSR was analysed with FAMOZ software. Parentages showing a logarithm of odds score higher than 18 were validated in relation to the historical data available. The analysis first revealed the full parentage of 828 cultivars resulting in: (1) 315 original full parentages uncovered for traditional cultivars, (2) 100 full parentages confirming results established with molecular markers in prior papers and 32 full parentages that invalidated prior results, (3) 255 full parentages confirming pedigrees as disclosed by the breeders and (4) 126 full parentages that invalidated breeders’ data. Second, incomplete parentages were determined in 1,087 cultivars due to the absence of complementary parents in our cultivar sample. Last, a group of 276 genotypes showed no direct relationship with any other cultivar in the collection. Compiling these results from the largest set of parentage data published so far both enlarges and clarifies our knowledge of the genetic constitution of cultivated V. vinifera germplasm. It also allows the identification of the main genitors involved in varietal assortment evolution and grapevine breeding.

Low Level of Pollen-Mediated Gene Flow from Cultivated to Wild Grapevine: Consequences for the Evolution of the Endangered Subspecies Vitis vinifera L. subsp. silvestris

A parentage and a paternity-based approach were tested for estimation of pollen-mediated gene flow in wild grapevine (Vitis vinifera L. subsp. silvestris), a wind-pollinated species occurring in Mediterranean Europe and southwestern Asia. For this purpose, 305 seedlings collected in 2 years at 2 locations in France from 4 wild female individuals and 417 wild individuals prospected from France and Italy were analyzed using 20 highly polymorphic microsatellite loci. Their profiles were compared with a database consisting of 3203 accessions from the Institut National de la Recherche Agronomique Vassal collection including cultivars, rootstocks, interspecific hybrids, and other wild individuals. Paternity was assigned for 202 (66.2%) of the 305 seedlings, confirming the feasibility of the method. Most of the fertilizing pollen could be assigned to wild males growing nearby. Estimates of pollen immigration from the cultivated compartment (i.e., the totality of cultivars) ranged from 4.2% to 26% from nearby vineyards and from hidden pollinators such as cultivars and rootstocks that had escaped from farms. In an open landscape, the pollen flow was correlated to the distance between individuals, the main pollinator being the closest wild male (accounting for 51.4-86.2% of the pollen flow). In a closed landscape, more complex pollination occurred. Analysis of the parentage of the 417 wild individuals also revealed relationships between nearby wild individuals, but in the case of 12 individuals (3%), analysis revealed pollen immigration from vineyards, confirming the fitness of the hybrid seedlings. These pollen fluxes may have a significant effect on the evolution of wild populations: on the one hand, the low level of pollen-mediated gene flow from cultivated to wild grapevine could contribute to a risk of extinction of the wild compartment (i.e., the totality of the wild individuals). On the other hand, pollen dispersal within the wild populations may induce inbreeding depression of wild grapevines.

Genetic structure in cultivated grapevines is linked to geography and human selection

BackgroundGrapevine (Vitis vinifera subsp. vinifera) is one of the most important and ancient horticultural plants in the world. Domesticated about 8–10,000 years ago in the Eurasian region, grapevine evolved from its wild relative (V. vinifera subsp. sylvestris) into very diverse and heterozygous cultivated forms. In this work we study grapevine genetic structure in a large sample of cultivated varieties, to interpret the wide diversity at morphological and molecular levels and link it to cultivars utilization, putative geographic origin and historical events.ResultsWe analyzed the genetic structure of cultivated grapevine using a dataset of 2,096 multi-locus genotypes defined by 20 microsatellite markers. We used the Bayesian approach implemented in the STRUCTURE program and a hierarchical clustering procedure based on Ward’s method to assign individuals to sub-groups. The analysis revealed three main genetic groups defined by human use and geographic origin: a) wine cultivars from western regions, b) wine cultivars from the Balkans and East Europe, and c) a group mainly composed of table grape cultivars from Eastern Mediterranean, Caucasus, Middle and Far East countries. A second structure level revealed two additional groups, a geographic group from the Iberian Peninsula and Maghreb, and a group comprising table grapes of recent origins from Italy and Central Europe. A large number of admixed genotypes were also identified. Structure clusters regrouped together a large proportion of family-related genotypes. In addition, Ward’s method revealed a third level of structure, corresponding either to limited geographic areas, to particular grape use or to family groups created through artificial selection and breeding.ConclusionsThis study provides evidence that the cultivated compartment of Vitis vinifera L. is genetically structured. Genetic relatedness of cultivars has been shaped mostly by human uses, in combination with a geographical effect. The finding of a large portion of admixed genotypes may be the trace of both large human-mediated exchanges between grape-growing regions throughout history and recent breeding.

Genetic mapping in pea. 2. Identification of RAPD and SCAR markers linked to genes affecting plant architecture

Abstract Random amplified polymorphic DNA (RAPD) markers linked to two morphological markers ( fa and det), three ramosus genes (rms2, rms3 and rms4) and two genes conferring flowering response to photoperiod in pea (sn, dne) were selected by bulk segregant analysis on F2 populations. Two RAPD fragments were cloned and sequenced to generate the two SCAR markers V20 and S2 which are linked to rms3 and dne, respectively. All these genes, except rms2, were previously located on the pea classical linkage map. Rms2 mapped to linkage group IB which contains the afila gene. Precise genetic maps of the regions containing the genes were obtained and compared to the RAPD map generated from the recombinant inbred-lines population of the cross Térèse×K586. This cross was chosen because several mutants were obtained from cultivars Térèse and Torsdag (K586 was derived from Torsdag). This collection of isogenic lines was used for the construction of F2 mapping populations in which polymorphic RAPD markers were already known and mapped. Moreover, the well-known problem in pea of variability in the linkage associations between crosses was avoided. This work contributes to the precise integration between the classical map and the molecular maps existing in pea.

New stable QTLs for berry weight do not colocalize with QTLs for seed traits in cultivated grapevine ( Vitis vinifera L.)

BackgroundIn grapevine, as in other fruit crops, fruit size and seed content are key components of yield and quality; however, very few Quantitative Trait Loci (QTLs) for berry weight and seed content (number, weight, and dry matter percentage) have been discovered so far. To identify new stable QTLs for marker-assisted selection and candidate gene identification, we performed simultaneous QTL detection in four mapping populations (seeded or seedless) with various genetic backgrounds.ResultsFor berry weight, we identified five new QTLs, on linkage groups (LGs) 1, 8, 11, 17 and 18, in addition to the known major QTL on LG 18. The QTL with the largest effect explained up to 31% of total variance and was found in two genetically distant populations on LG 17, where it colocalized with a published putative domestication locus. For seed traits, besides the major QTLs on LG 18 previously reported, we found four new QTLs explaining up to 51% of total variance, on LGs 4, 5, 12 and 14. The previously published QTL for seed number on LG 2 was found related in fact to sex. We found colocalizations between seed and berry weight QTLs only for the major QTL on LG 18 in a seedless background, and on LGs 1 and 13 in a seeded background. Candidate genes belonging to the cell number regulator CNR or cytochrome P450 families were found under the berry weight QTLs on LGs 1, 8, and 17. The involvement of these gene families in fruit weight was first described in tomato using a QTL-cloning approach. Several other interesting candidate genes related to cell wall modifications, water import, auxin and ethylene signalling, transcription control, or organ identity were also found under berry weight QTLs.ConclusionWe discovered a total of nine new QTLs for berry weight or seed traits in grapevine, thereby increasing more than twofold the number of reliable QTLs for these traits available for marker assisted selection or candidate gene studies. The lack of colocalization between berry and seed QTLs suggests that these traits may be partly dissociated.

Identification of mildew resistance in wild and cultivated Central Asian grape germplasm

BackgroundCultivated grapevines, Vitis vinifera subsp. sativa, evolved from their wild relative, V. vinifera subsp. sylvestris. They were domesticated in Central Asia in the absence of the powdery mildew fungus, Erysiphe necator, which is thought to have originated in North America. However, powdery mildew resistance has previously been discovered in two Central Asian cultivars and in Chinese Vitis species.ResultsA set of 380 unique genotypes were evaluated with data generated from 34 simple sequence repeat (SSR) markers. The set included 306 V. vinifera cultivars, 40 accessions of V. vinifera subsp. sylvestris, and 34 accessions of Vitis species from northern Pakistan, Afghanistan and China. Based on the presence of four SSR alleles previously identified as linked to the powdery mildew resistance locus, Ren1, 10 new mildew resistant genotypes were identified in the test set: eight were V. vinifera cultivars and two were V. vinifera subsp. sylvestris based on flower and seed morphology. Sequence comparison of a 620 bp region that includes the Ren1-linked allele (143 bp) of the co-segregating SSR marker SC8-0071-014, revealed that the ten newly identified genotypes have sequences that are essentially identical to the previously identified mildew resistant V. vinifera cultivars: ‘Kishmish vatkana’ and ‘Karadzhandal’. Kinship analysis determined that three of the newly identified powdery mildew resistant accessions had a relationship with ‘Kishmish vatkana’ and ‘Karadzhandal’, and that six were not related to any other accession in this study set. Clustering procedures assigned accessions into three groups: 1) Chinese species; 2) a mixed group of cultivated and wild V. vinifera; and 3) table grape cultivars, including nine of the powdery mildew resistant accessions. Gene flow was detected among the groups.ConclusionsThis study provides evidence that powdery mildew resistance is present in V. vinifera subsp. sylvestris, the dioecious wild progenitor of the cultivated grape. Four first-degree parent progeny relationships were discovered among the hermaphroditic powdery mildew resistant cultivars, supporting the existence of intentional grape breeding efforts. Although several Chinese grape species are resistant to powdery mildew, no direct genetic link to the resistance found in V. vinifera could be established.

Grape varieties (Vitis vinifera L.) from the Balearic Islands: genetic characterization and relationship with Iberian Peninsula and Mediterranean Basin

Ampelographic descriptions, a set of 20 nuclear microsatellite loci (nuSSR), five chloroplast microsatellites (cSSR), as well as historical references have been used to identify 66 accessions of Vitis vinifera L. The plant material included major and minor varieties under risk of extinction, collected in the Balearic Islands, and now conserved in two germplasm repositories site in Spain. The 66 samples analyzed corresponded to 32 different genotypes, several unique genotypes were found, three of them remaining unknown. Some synonyms and homonyms were found in the Mediterranean basin, highlighting that the dispersal of some varieties are related with historical human movements and migrations occurred in three several periods, (1) around seventh century related to Islam expansion, (2) around thirteen to fifteenth centuries and (3) in the nineteenth century related to phylloxera crisis. Some parentages were identified, being the cultivar Callet Cas Concos a key variety in several crosses, confirming the high value of unknown varieties for parentage analysis. Several grouping methods confirm the existence of two gene pools.

Genetic diversity of Moroccan grape accessions conserved ex situ compared to Maghreb and European gene pools

Grape diversity present in Morocco and the part of this diversity used nowadays are poorly documented. In order to choose diversified genotypes, to select them so that their agronomic interest will be tested, a group of 21 autochthonous cultivars preserved in the germplasm collections of SODEA and 18 Moroccan cultivars from “Domaine de Vassal” INRA grape collection was compared to a group of cultivars from neighbouring countries (Algeria and Tunisia), and from a core collection optimizing simple sequence repeat (SSR) allelic diversity of grape. Data from 20 nuclear and 3 chloroplastic SSR markers were obtained for this set of 211 cultivars. A total of 156 alleles (mean of 7.8 alleles per locus) were detected for the nSSRs and 7 alleles for the cpSSR in the Moroccan group. Chlorotype diversity in Moroccan and Algerian group were similar, but slightly lower than in the Tunisian group and the core collection. Similarly, the nSSR diversity was high in the core collection and low in the Moroccan and the Algerian groups compared to the two other groups. Clustering of cultivars based on nSSR data reflected their geographical origin and, to a certain extent, the use of the cultivars. The specificity of the Moroccan plant material was attested by the Bayesian analysis using Structure, while differences of the core collection were clearly revealed both by the Bayesian and a multivariate analysis. These results confirm the differentiation of the material from Maghreb and more specifically of Moroccan material, having evolved independently from Europe.