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

Abstract

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.

Figure 1

Alignment of the contig 207 along the reference genetic map (A) and the 8.4× genome assembly (B). The genetic markers are indicated on the left of LG 5 (vertical black bar on the left; distances in cM) with different colours. The BAC clones, indicated by vertical bars on the right of A and B, are positioned into the physical contig according to FPC calculations (A) or according to the alignment of their end sequences on the 8.4× genome assembly (Vertical black bar on the left of B; distances in Mbp). Their respective colours correspond to the genetic marker they carry.

Figure 2

Distribution of grapevine homologues to non-host, host, and defence signalling genes. The 19 grapevine chromosomes were drawn according to the orientation and the genetic distances of the Vitis reference map [37]. Gene localisation was inferred based on the integration of the physical and genetic data. The position of the 27 homologues to non-host and disease-resistance signalling genes (black horizontal ticks) are given only for the physical contigs of 'Cabernet Sauvignon' identified by PCR screening. The position of the grapevine analogues to NBS-LRR class resistance genes are indicated with red boxes and the RLK class resistance genes with green boxes.

Figure 3

Abundance of non-host, host, and defence signalling genes. The number of BAC contigs containing resistance related genes and anchored on each linkage group is reported for non-host and signalling genes and for two families of host resistance genes, NBS-LRR, and Pto-like kinases.