Thierry Lacombe

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).

Wine grape (Vitis vinifera L.) color associates with allelic variation in the domestication gene VvmybA1

During the process of crop domestication and early selection, numerous changes occur in the genetic and physiological make-up of crop plants. In grapevine (Vitis vinifera) numerous changes have occurred as a result of human selection, including the emergence of hermaphroditism and greatly increased variation in berry color. This report examines the effect of human selection on variable skin color by examining the variation present in the gene VvmybA1, a transcriptional regulator of anthocyanin biosynthesis. In over 200 accessions of V. vinifera, the insertion of the retroelement Gret1 in the promoter region of VvmybA1 was in strong association with the white-fruited phenotype. This retroelement was inserted at the same location for each individual in which it was present. Additional polymorphisms in the VvmybA1 gene were also strongly associated with red or pink fruited accessions, including variation that was generated by the excision of Gret1 from the promoter of VvmybA1. Differences in nucleotide diversity were observed between the white and pigmented alleles of VvmybA1, suggesting that the white allele arose only once or a limited number of times. Rarely, association of Gret1 with the white fruited phenotype was not observed, suggesting that the white phenotype can also be obtained through mutation in additional genes. These results provide evidence that variation in one transcriptional regulator has generated an allelic series strongly associated with fruit color variation in cultivated grapevine. These findings provide information about the evolution of grapes since domestication and have direct implications for the regulation of fruit and wine quality of this important crop plant.

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

BackgroundThe 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 worlds largest germplasm collection (Domaine de Vassal, INRA Hérault, France), containing 2262 unique genotypes.ResultsSub-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.ConclusionThe 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.

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.

Evolution of the VvMybA gene family, the major determinant of berry colour in cultivated grapevine ( Vitis vinifera L.)

Polymorphisms in the grape transcription factor family VvMybA are responsible for variation in anthocyanin content in the berries of cultivated grapevine (Vitis vinifera L. subsp. sativa). Previous study has shown that white grapes arose through the mutation of two adjacent genes: a retroelement insertion in VvMybA1 and a single-nucleotide polymorphism mutation in VvMybA2. The purpose of this study was to understand how these mutations emerged and affected genetic diversity at neighbouring sites and how they structured the genetic diversity of cultivated grapevines. We sequenced a total of 3225 bp of these genes in a core collection of genetic resources, and carried out empirical selection tests, phylogenetic- and coalescence-based demographic analyses. The insertion in the VvMybA1 promoter was shown to have occurred recently, after the mutation of VvMybA2, both mutations followed by a selective sweep. The mutational pattern for these colour genes is consistent with progressively relaxed selection from constrained ancestral coloured haplotypes to light coloured and finally white haplotypes. Dynamics of population size in the VvMybA genes showed an initial exponential growth, followed by population size stabilization. Most ancestral haplotypes are found in cultivars from western region, whereas recent haplotypes are essentially present in table cultivars from eastern regions where intense breeding practices may have replaced the original diversity. Finally, the emergence of the white allele was followed by a recent strong exponential growth, showing a very fast diffusion of the initial white allele.

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.

A small XY chromosomal region explains sex determination in wild dioecious V. vinifera and the reversal to hermaphroditism in domesticated grapevines

BackgroundIn Vitis vinifera L., domestication induced a dramatic change in flower morphology: the wild sylvestris subspecies is dioecious while hermaphroditism is largely predominant in the domesticated subsp. V. v. vinifera. The characterisation of polymorphisms in genes underlying the sex-determining chromosomal region may help clarify the history of domestication in grapevine and the evolution of sex chromosomes in plants. In the genus Vitis, sex determination is putatively controlled by one major locus with three alleles, male M, hermaphrodite H and female F, with an allelic dominance M > H > F. Previous genetic studies located the sex locus on chromosome 2. We used DNA polymorphisms of geographically diverse V. vinifera genotypes to confirm the position of this locus, to characterise the genetic diversity and traces of selection in candidate genes, and to explore the origin of hermaphroditism.ResultsIn V. v. sylvestris, a sex-determining region of 154.8 kb, also present in other Vitis species, spans less than 1% of chromosome 2. It displays haplotype diversity, linkage disequilibrium and differentiation that typically correspond to a small XY sex-determining region with XY males and XX females. In male alleles, traces of purifying selection were found for a trehalose phosphatase, an exostosin and a WRKY transcription factor, with strikingly low polymorphism levels between distant geographic regions. Both diversity and network analysis revealed that H alleles are more closely related to M than to F alleles.ConclusionsHermaphrodite alleles appear to derive from male alleles of wild grapevines, with successive recombination events allowing import of diversity from the X into the Y chromosomal region and slowing down the expansion of the region into a full heteromorphic chromosome. Our data are consistent with multiple domestication events and show traces of introgression from other Asian Vitis species into the cultivated grapevine gene pool.

Linkage disequilibrium in wild French grapevine, Vitis vinifera L. subsp. silvestris.

Association mapping based on linkage disequilibrium (LD) can provide high resolution for whole-genome mapping of genes underlying phenotypic variation. This field has received considerable attention over the last decade. We present here the first characterization of LD in wild French grapevine, Vitis vinifera L. subsp. silvestris. To assess the pattern and extent of LD, we used a sample of 85 plants from southern France and 36 microsatellite markers distributed over 5 linkage groups. LD was evaluated with independence tests and multiallelic r2, using both unphased genotypic data and reconstructed haplotypic data. LD decayed rapidly, with r2 values decreasing to 0.1 within 2.7 cM for genotypic data and within 1.4 cM for haplotypic data. Compared to the results of a previous study on cultivated grapevine subsp. sativa, where significant LD was found up to 16.8 cM, LD in subsp. silvestris was no longer significant past 1.4 cM. LD was therefore 12 times further extended in cultivated than wild grapevine, even though LD in wild grapevine seemed to extend slightly further than in wild relatives of other crops. Domestication bottlenecks and vegetative propagation are the primary factors responsible for this difference between cultivated and wild grapevine. The rapid decay of LD observed in this study seems promising for future association mapping studies of functional variation in wild V. vinifera grapevine.

Bioarchaeological insights into the process of domestication of grapevine (Vitis vinifera L.) during Roman times in Southern France.

Grapevine (Vitis vinifera), one of the most important fruit species in the Classical Mediterranean world, is thought to have been domesticated first in South-Western Asia, during the Neolithic. However, the domestication process remains largely unknown. Crucial unanswered questions concern the duration of the process (rapid or slow?) and the related geographical area (single or multiple-origins?). Seeds from domesticated grapevine and from its wild ancestor are reported to differ according to shape. Our work aims, first, to confirm this difference and secondly to identify the extent of domestication in the grapes cultivated by Romans in Southern France during the period 50 BCE–500 CE. We had the opportunity to analyze uncharred waterlogged grape pips from 17 archaeological sites. Based on an extended reference sample of modern wild grapevines and cultivars our work shows that both subspecies can be discriminated using simple measurements. The elongation gradient of the pip’s body and stalk may be regarded as an indicator of the strength of the selection pressures undergone by domesticated grapes. Grapevines cultivated during the Roman period included a mix of morphotypes comprising wild, intermediate and moderately selected domesticated forms. Our data point to a relative shift towards more selected types during the Roman period. Domestication of the grapevine appears to have been a slow process. This could result from the recurrent incorporation into cultivation of plants originating from sexual reproduction, when grape cultivation essentially relies on vegetative propagation.