SNiPlay: a web-based tool for detection, management and analysis of SNP (C900)

The rapidly increasing amount of re-sequencing and genotyping data generated by large-scale genetic diversity projects requires the development of integrated bioinformatics tools able to efficiently manage, analyze, and combine genetic data with the genome's structure and with external data available. In this context, we developed SNiPlay, a flexible, user-friendly and integrative web-based tool dedicated to polymorphism discovery and analysis. It integrates: 1) a pipeline, freely accessible through the internet, combining existing software with new tools to detect SNPs and to compute different kinds of statistical indices and graphical layouts for SNP data. From Sanger sequencing traces, multiple sequence alignments or genotyping data given as input, SNiPlay detects SNPs and insertion/deletion events. In a second time, it sends sequences and genotyping data into a series of modules in charge of various post-processes: physical mapping to a reference genome, annotation (genomic position, intron/exon location, synonymous/non-synonymous substitutions), SNP frequency determination in user-defined groups, haplotype reconstruction and networking, linkage disequilibrium evaluation, and diversity analysis (Pi, Watterson's Theta, Tajima's D). 2) a database storing polymorphisms, genotyping data and sequences of grapevine produced by nationally-funded public projects. It allows one to retrieve SNPs using various filters (such as genomic position, missing data, polymorphism type, allele frequency), to compare SNP patterns between populations, and to export genotyping data or sequences in various formats. SNiPlay is available at: http://www.sniplay.cirad.fr/. (Texte intégral

Grape selector: a Shiny application for grapevine breeding

Both variety and clonal selection programs require the study of many traits. Selecting the best accession becomes a challenge when the number of accessions and traits being studied increases. Grape selector is a user-friendly tool that allows finding the best compromises according to a set of criteria (selection index). This tool can be used with phenotypic data at the variety or clone scale. The application was tested with existing phenotypic data from a breeding program and provided consistent selection with the one done manually

Marqueurs moléculaires utilisables en sélection chez la canne à sucre : cas de la résistance à la rouille brune

Les cultivars modernes de canne à sucre (Saccharum spp., 2n=100 à 130) sont hautement polyploïdes, aneuploïdes et d'origine interspécifique. Un gène majeur (Bru1) conférant la résistance à la rouille brune, causée par le champignon Puccinia melanocephala, a été identifié chez le cultivar R570. Nous avons analysé 380 cultivars modernes et clones en cours de sélection, représentatifs de la diversité mondiale, à l'aide de 22 marqueurs moléculaires regroupés dans un segment de 8,2cM génétiquement lié à Bru1 chez R570. Nos résultats révèlent un fort déséquilibre de liaison dans la région autour de Bru1 et de fortes associations entre la plupart des marqueurs et la résistance à la rouille. Deux marqueurs PCR flanquant le segment portant Bru1 sont complètement associés et sont présents uniquement chez les clones résistants. Ces marqueurs constituent des marqueurs moléculaires diagnostics de Bru1. 86% des 194 clones de canne à sucre résistants possèdent les marqueurs diagnostics de Bru1, révélant que ce gène constitue la principale source de résistance à la rouille brune chez les cultivars modernes. Les marqueurs PCR diagnostics de Bru1 devraient être particulièrement utiles pour identifier des cultivars possédant des sources de résistance alternatives à Bru1 afin de diversifier la base génétique de la résistance à la rouille brune dans les programmes de sélection. (Résumé d'auteur

High quality phased assembly of grape genome offer new opportunities in chimera detection

In perennial plants and especially those propagated through cuttings, several genotypes can coexist in a single individual, thus leading to chimeras. When the variant induces a noticeable phenotype modification, it can lead to a new cultivar. Viticulture already took economic advantage of this natural phenomenon: for instance, the berry skin of ‘Pinot gris’ derived from ‘Pinot noir’ by the selection of a chimera. Chimeras could also impact other crucial traits without being visually identified. Periclinal chimera where the variant has entirely colonized a cell layer is the most stable and can be propagated through cuttings. In grapevine, two functional cell layers are present in leaves, L1 and L2. However, lateral roots are formed from the L2 cell layer only. Thus, comparing DNA sequences of roots and leaves could allow chimera detection. In this study we used new generation Hifi long reads sequencing and recent bioinformatics tools applied to ‘Merlot’ to detect periclinal chimeras. Sequencing of ‘Magdeleine Noire des Charentes’ and ‘Cabernet franc’, the parents of ‘Merlot’, allowed haplotype resolved assembly. Pseudomolecules were built with few contigs, in some occasions only one per chromosome. This high resolution allowed haplotype comparison. Annotation from PN40024 was transferred to all pseudomolecules. Through variant detection, periclinal chimeras were found on both haplotypes. These results open new perspectives on chimera detection, which is an important resource to improve cultivars through clonal selection or breed new ones. Detailed results will be presented and discussed

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

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 Hérault, 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

Characterization of the Bru1 (brown rust resistance) locus ; Distribution in modern sugarcane cultivars : [Abstract W242]

The genome of modern sugarcane cultivars is highly polyploid, aneuploid, of interspecific origin, and contains 10 Gb of DNA. Its size and complexity represent a major challenge for the isolation of agronomically important genes. A durable major rust resistance gene (Bru1) identified in the cultivar R570 is the focus of a map-based cloning approach. To overcome constraints associated with high polyploidy, we developed genomic strategies including diploid/polyploid syntenic shuttle mapping with two model diploid species (sorghum and rice) and haplotype-specific chromosome walking. Their applications allowed us (i) to develop a high-resolution map including markers at 0.28 and 0.14 cM on both sides and 13 markers cosegregating with Bru1 and (ii) to develop a physical map of the target haplotype that still includes two gaps at this stage due to the discovery of an insertion specific to this haplotype. Several BAC clones representing different hom(oe)ologous haplotypes were sequenced. Their sequences are used to continue the characterization of this resistance locus. Several markers surrounding Bru1 in R570 were surveyed in 405 international cultivars that were phenotyped for rust resistance in Réunion or Guadeloupe Island. The results revealed that Bru1 is present in most of the resistant cultivars. Only 13% of them did not display the Bru1 haplotype. They represent alternative sources of resistance to the rust pathogen. Two of the PCR markers developed in the course of this mapping are in perfect linkage disequilibrium with Bru1 and thus can be used as diagnostic for the presence of Bru1 in modern sugarcane cultivars. (Texte intégral

Molecular, genetic and transcriptional evidence for a role of VvAGL11 in stenospermocarpic seedlessness in grapevine

<p>Abstract</p> <p>Background</p> <p>Stenospermocarpy is a mechanism through which certain genotypes of <it>Vitis vinifera </it>L. such as Sultanina produce berries with seeds reduced in size. Stenospermocarpy has not yet been characterized at the molecular level.</p> <p>Results</p> <p>Genetic and physical maps were integrated with the public genomic sequence of <it>Vitis vinifera </it>L. to improve QTL analysis for seedlessness and berry size in experimental progeny derived from a cross of two seedless genotypes. Major QTLs co-positioning for both traits on chromosome 18 defined a 92-kb confidence interval. Functional information from model species including <it>Vitis </it>suggested that <it>VvAGL11</it>, included in this confidence interval, might be the main positional candidate gene responsible for seed and berry development.</p> <p>Characterization of <it>VvAGL11 </it>at the sequence level in the experimental progeny identified several SNPs and INDELs in both regulatory and coding regions. In association analyses performed over three seasons, these SNPs and INDELs explained up to 78% and 44% of the phenotypic variation in seed and berry weight, respectively. Moreover, genetic experiments indicated that the regulatory region has a larger effect on the phenotype than the coding region. Transcriptional analysis lent additional support to the putative role of <it>VvAGL11's </it>regulatory region, as its expression is abolished in seedless genotypes at key stages of seed development. These results transform <it>VvAGL11 </it>into a functional candidate gene for further analyses based on genetic transformation.</p> <p>For breeding purposes, intragenic markers were tested individually for marker assisted selection, and the best markers were those closest to the transcription start site.</p> <p>Conclusion</p> <p>We propose that <it>VvAGL11 </it>is the major functional candidate gene for seedlessness, and we provide experimental evidence suggesting that the seedless phenotype might be caused by variations in its promoter region. Current knowledge of the function of its orthologous genes, its expression profile in <it>Vitis </it>varieties and the strong association between its sequence variation and the degree of seedlessness together indicate that the D-lineage MADS-box gene <it>VvAGL11 </it>corresponds to the <it>Seed Development Inhibitor locus </it>described earlier as a major locus for seedlessness. These results provide new hypotheses for further investigations of the molecular mechanisms involved in seed and berry development.</p

Major cause of somatic polymorphism in clonal grape cultivars : W702

Clonal variation is considered as an effective contribution to breeding programs of vegetatively propagated species with major agronomical interest such as banana, coffee and grape. After several propagation cycles, stable and heritable phenotypic variations appear giving rise to a phenotypic variation known as "clonal diversity". Clonal diversity is very important for wine-growers because it allows preserving cultivars identity in the strict respect of Appellation wines specifications. The most parsimonious hypothesis explaining clonal diversity is the accumulation of somatic mutations. Using the power of NGS, we provided the first broad description of polymorphism in different clones of a single grapevine cultivar (Pinot) and then compared the clonal polymorphism in 4 different cultivars: Pinot, Syrah, Grenache and Sultanine. Three types of polymorphism (SNPs, Indels, mobile elements) were observed. As expected, the polymorphism at cultivars level was much higher than at clonal level. The insertion polymorphism generated by mobile elements represented the highest mutational event with respect to clonal variation while SNP and indel polymorphisms were of higher importance for cultivar diversity. Among mobile element with a high polymorphism level, four were analyzed and polymorphisms were confirmed at different diversity levels: inter-species, inter-cultivars, inter-clones and between organs/tissues of a single individual. We confirm the high instability of these elements between clones and between tissues in single individuals. Clone identification through molecular methods would be of high significance for the wine industry. SNP or small indels mutations are less frequent but more stable than structural variation and could have much potential for accurate identification. (Résumé d'auteur

Dissecting genetic architecture of grape proanthocyanidin composition through quantitative trait locus mapping

<p>Abstract</p> <p>Background</p> <p>Proanthocyanidins (PAs), or condensed tannins, are flavonoid polymers, widespread throughout the plant kingdom, which provide protection against herbivores while conferring organoleptic and nutritive values to plant-derived foods, such as wine. However, the genetic basis of qualitative and quantitative PA composition variation is still poorly understood. To elucidate the genetic architecture of the complex grape PA composition, we first carried out quantitative trait locus (QTL) analysis on a 191-individual pseudo-F1 progeny. Three categories of PA variables were assessed: total content, percentages of constitutive subunits and composite ratio variables. For nine functional candidate genes, among which eight co-located with QTLs, we performed association analyses using a diversity panel of 141 grapevine cultivars in order to identify causal SNPs.</p> <p>Results</p> <p>Multiple QTL analysis revealed a total of 103 and 43 QTLs, respectively for seed and skin PA variables. Loci were mainly of additive effect while some loci were primarily of dominant effect. Results also showed a large involvement of pairwise epistatic interactions in shaping PA composition. QTLs for PA variables in skin and seeds differed in number, position, involvement of epistatic interaction and allelic effect, thus revealing different genetic determinisms for grape PA composition in seeds and skin. Association results were consistent with QTL analyses in most cases: four out of nine tested candidate genes (<it>VvLAR1</it>, <it>VvMYBPA2</it>, <it>VvCHI1</it>, <it>VvMYBPA1</it>) showed at least one significant association with PA variables, especially <it>VvLAR1 </it>revealed as of great interest for further functional investigation. Some SNP-phenotype associations were observed only in the diversity panel.</p> <p>Conclusions</p> <p>This study presents the first QTL analysis on grape berry PA composition with a comparison between skin and seeds, together with an association study. Our results suggest a complex genetic control for PA traits and different genetic architectures for grape PA composition between berry skin and seeds. This work also uncovers novel genomic regions for further investigation in order to increase our knowledge of the genetic basis of PA composition.</p