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

<p>Abstract</p> <p>Background</p> <p>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 <it>Vitis vinifera </it>'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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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 <it>loci </it>in grapevine.</p

Analyse et exploitation de séquences Sanger

École thématiqu

BSA-Seq : an efficient method to decipher a complex trait on Poplar, a highly heterozygous diploid genome

The efficiency of the Bulk Segregant Analysis (BSA) had clearly been demonstrated to detect genomic regions and genes involved in diverse traits. It allows large experiments reducing the cost and time and preserving the power of full individual's population analysis. These past few years the combination of BSA and New Generation Sequence (NGS) data (BSA-Seq) gave a new accuracy and depth to the discovery on many traits of interest, mainly on crop and model species. In our study, we applied the BSA-Seq in a heterozygous and diploid genome context. We worked on the progenies derived from an interspecific cross Populus deltoides x Populus trichocarpa in which segregates the resistance to Melampsora larici populina (Mlp) leaf rust. We detected DNA variations with freebayes/0.9.21 and the soft masked genome of Populus trichocarpa Nisqually v3.0 as reference. Comparing DNA variations in between parents and bulks we obtained 27 regions or Quantitative Trait Loci based on NGS analysis (QTL-Seq) which could explain the resistance to Mlp. We first evaluated the strategy retrieving a previously cloned Mlp resistance gene governing the uredinia size in Populus trichocarpa clone 101-74 (RUS). Then we identified genomic markers which should better characterize this locus. So we demonstrated that, in our context, BSA-Seq allows us to improve the fine mapping of a major gene. We think it can be a promising method on a high heterozygous diploid genome as Poplar, to decipher complex trait. Next step is to proceed with it to fine map the other QTL-Seq

High throughput SNPs discovery in grapevine, poplar and tomato

International audienc

BSA-Seq : An efficient tool to characterize loci involved in the Poplar leaf rust resistance.

The efficiency of the Bulk Segregant Analysis (BSA) had clearly been demonstrated to detect genomic regions and genes involved in diverse traits. It allows large experiments reducing the cost and time and preserving the power of full individual's population analysis. These past few years the combination of BSA and New Generation Sequence (NGS) data (BSA-Seq) gave a new accuracy and depth to the discovery on many traits of interest, mainly on crop and model species. In our study, we applied the BSA-Seq to narrow down Populus genomic regions involved in the resistance to Melampsora larici populina (Mlp) leaf rust. We worked on the 1417 progenies derived from an interspecific cross Populus deltoides clone 73028-62 (Pd) x Populus trichocarpa clone 101-74 (Pt) in which segregates qualitative and quantitative Mlp resistances. Four bulks were constituted based on (1) the phenotypes for uredinias size (bulk1: large, bulk2 and bulk3: intermediate, bulk4: small) and (2) the Pt genotypes at the RUS locus governing the uredinia size (bulk1 and bulk3 : [RUS], bulk2 and bulk4 : [rUS]). We used independently the soft masked genomes of Populus trichocarpa Nisqually v3.0 (Ptv3) and Populus deltoides v2.0 (Pdv2) as references to map parents and bulks Illumina reads with the BWAmem/0.7.15 suite and to detect DNA variations with Freebayes/0.9.21. For each common variant position between parents and bulks, we tracked the specific alleles of Pt and/or Pd in the bulks. Respectively from Ptv3 and Pdv2, we identified 13 and 11 regions. We first evaluated the strategy retrieving the previously finally mapped RUS gene, designed new genomic markers from Ptv3 to better characterize this locus and performed the in silico validation. Then we proceed with it to fine map the other regions. Most of them co-locate with QTL and could explain the resistance to Mlp. So we demonstrated that, in our context, even if the reference genome is different to the studied genomes, BSA-Seq allowed us to detect quickly and costly, regions which may be involved in a complex trait and improve the fine mapping of a major gene. We think it can be a promising method on a high heterozygous diploid genome as Poplar, to decipher complex trait. Next step is to identify candidate genes within the regions and better describe the mechanisms of resistance

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

International audienceRop/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 discusse

Sequencing allelic BACs for exploring the level of genome heterozygosity in oak

absen

Toward the deciphering of chromosome structure in Vitis vinifera

International audienceIn the context of the sequencing project for the grapevine reference genome sequence, two genetic maps were completed with SSR markers in order to align the sequence contigs of the Vv12X.0 version of the genome assembly along the chromosomes. For this purpose, two full-sib families, 'Syrah' x 'Grenache' (SxG, 192 individuals) and 'Chardonnay' x 'Bianca' (CxB, 358 individuals) were respectively scored with 223 and 401 SSR. These two maps, supplemented with former data, allowed anchoring over 90% of the reference genome sequence. SNP markers were then defined from 37 anchored but not oriented and 44 not oriented and not anchored scaffolds of sequence. So far 110 new markers have been mapped, allowing anchoring 31 not yet anchored scaffolds (similar to 9 Mb) and orienting 35 scaffolds (similar to 15.5 Mb). In parallel, the recombination rate along the chromosomes is under study, in relation with sequence features (gene/repeat density, %GC, distance to the centromere/telomere). Finally, we are developing a cytogenetic map in Vitis vinifera using BAC-FISH. All these approaches will be combined to study the structure of the Vitis vinifera genome, including the factors affecting recombination rates and the dynamics of the genome evolution

Sequencing allelic BACs for exploring the level of genome heterozygosity in oak

absen

Molecular linkage maps: strategies, resources and achievements

Chapitre 5International audienceThe development of genetic maps in grapevine started in the late 90s. It greatly benefi ted from the development of SSR markers by an international consortium, leading to the construction of integrated genetic maps. Software was improved for facilitating map construction in full sib families. A review of the different strategies that were developed for mapping in grapevine, including mapping populations, software and markers is provided here together with a discussion on their interest and limitations. Thanks to all these resources and experience, genetic mapping is now easy to handle in grapevine and is now widely used for QTL detection, map-based cloning, comparative mapping across species and genome sequence anchoring