Physical mapping in highly heterozygous genomes: a physical contig map of the Pinot Noir grapevine cultivar

Abstract

Background: Most of the grapevine (Vitis vinifera L.) cultivars grown today are those selected centuries ago, even though grapevine is one of the most important fruit crops in the world. Grapevine has therefore not benefited from the advances in modern plant breeding nor more recently from those in molecular genetics and genomics: genes controlling important agronomic traits are practically unknown. A physical map is essential to positionally clone such genes and instrumental in a genome sequencing project.

Results: We report on the first whole genome physical map of grapevine built using high information content fingerprinting of 49,104 BAC clones from the cultivar Pinot Noir. Pinot Noir, as most grape varieties, is highly heterozygous at the sequence level. This resulted in the two allelic haplotypes sometimes assembling into separate contigs that had to be accommodated in the map framework or in local expansions of contig maps. We performed computer simulations to assess the effects of increasing levels of sequence heterozygosity on BAC fingerprint assembly and showed that the experimental assembly results are in full agreement with the theoretical expectations, given the heterozygosity levels reported for grape. The map is anchored to a dense linkage map consisting of 994 markers. 436 contigs are anchored to the genetic map, covering 342 of the 475 Mb that make up the grape haploid genome.

Conclusions: We have developed a resource that makes it possible to access the grapevine genome, opening the way to a new era both in grape genetics and breeding and in wine making. The effects of heterozygosity on the assembly have been analyzed and characterized by using several complementary approaches which could be easily transferred to the study of other genomes which present the same features.

Figure 1

Effect of heterozygosity on the assembly of the physical map. a| and b| CB Maps of two real contigs of the grape physical map ('a' corresponds to contig number 69 and 'b' to contig number 354) showing the so called 'scissoring effect', one of the main consequences of heterozygosity. On both contigs a polymorphic SNP marker has been physically mapped by BAC-pooling first, followed by PCR determination of the BAC clones containing each of the two alleles. The clones on which one allele maps have been highlighted in red and the ones on which the other allele maps in blue. It is interesting to observe that the two alleles of the same SNP marker map onto different clones which are not overlapping each other. The problem which affects the assembly of these two contigs has been called 'scissoring effect'. Basically, the clones within a contig tend to split apart, with the ones belonging to one allele at one extremity and the ones belonging to the other allele at the other extremity. As a consequence, the affected contigs show an expansion in the CB map size. c| and d| Two plots depicting simulated contigs from the in silico simulation at 42% sequence divergence, where the clones belonging to one allele have been highlighted in orange and the ones belonging to the other allele in blue. In 'c' the 'scissoring effect' is particularly evident on the right extremity, while the other part of the contig shows a more complex situation. Indeed, the scissoring effect can be locally more or less important and this is thought to be related to the variation in the levels of heterozygosity along the contig/chromosome. This situation is particularly consistent with the pictures 'a' and 'b'. The contig displayed in 'd' shows instead a different situation, being made up by clones which belong only to one allele (in orange). This scenario is consistent with the cases of allele testing in which the two alleles hybridize onto separate contigs (see text).

Figure 2

Comparison of the physical contig 50 (CB units 0-1,000) with linkage group 17 (10-20 cM interval in the genetic map) and the cluster 9651 (2-3 Mb) from the genome sequence assembly of Pinot noir (CMap viewer available at http://genomics.research.iasma.it). Correspondences among marker loci in the genetic map, BAC clones in the physical contig, and marker positions in the sequence are displayed with solid lines.