A high-density SNP genotyping array for Brassica napus and its ancestral diploid species based on optimised selection of single-locus markers in the allotetraploid genome

Abstract

The Brassica napus Illumina array provides genome-wide markers linked to the available genome sequence, a significant tool for genetic analyses of the allotetraploid B. napus and its progenitor diploid genomes. A high-density single nucleotide polymorphism (SNP) Illumina Infinium array, containing 52,157 markers, was developed for the allotetraploid Brassica napus. A stringent selection process employing the short probe sequence for each SNP assay was used to limit the majority of the selected markers to those represented a minimum number of times across the highly replicated genome. As a result approximately 60 % of the SNP assays display genome-specificity, resolving as three clearly separated clusters (AA, AB, and BB) when tested with a diverse range of B. napus material. This genome specificity was supported by the analysis of the diploid ancestors of B. napus, whereby 26,504 and 29,720 markers were scorable in B. oleracea and B. rapa, respectively. Forty-four percent of the assayed loci on the array were genetically mapped in a single doubled-haploid B. napus population allowing alignment of their physical and genetic coordinates. Although strong conservation of the two positions was shown, at least 3 % of the loci were genetically mapped to a homoeologous position compared to their presumed physical position in the respective genome, underlying the importance of genetic corroboration of locus identity. In addition, the alignments identified multiple rearrangements between the diploid and tetraploid Brassica genomes. Although mostly attributed to genome assembly errors, some are likely evidence of rearrangements that occurred since the hybridisation of the progenitor genomes in the B. napus nucleus. Based on estimates for linkage disequilibrium decay, the array is a valuable tool for genetic fine mapping and genome-wide association studies in B. napus and its progenitor genomes.

Fig. 1

GenomeStudio images showing representative SNP cluster patterns across Brassica napus genotypes. One cluster representing one parental allele is coloured in red (AA), the second in blue (BB), and heterozygote genotypes in purple. a Shows a genome-specific SNP marker in B. napus, almost 60 % of the SNP loci show this clear separation of the expected three genotypes. b SNP locus likely resulting from hybridisation of two segregating homoeologous loci reveals five clusters and an excess of heterozygotes. c and d Show a SNP locus called automatically by the software and after manual adjustment of the cluster profile, respectively. e SNP locus where one parental allele shows no hybridisation (presence/absence marker)

Fig. 2

GenomeStudio images showing representative SNP cluster patterns in the different Brassica species for the marker Bn-scaff_23108_p362932. One parental allele is coloured in red (AA), the second in blue (BB), and heterozygotes in purple. The SNP marker is polymorphic, but not genome-specific in B. napus, a resulting in condensed clusters due to the detection of the homoeologous locus on the other genome. In the diploid B. rapa, b this marker is polymorphic and shows widely distributed clusters (no second homoeologous locus detected, typical diploid pattern). In B. oleracea material, this marker is monomorphic

Fig. 3

Physical distribution of SNP loci across the B. napus genome. The SNP loci were aligned to the genome of spring-type DH12075 based on BLAT scores, with the numbers of SNP loci per 125 Kb window indicated on the y axis for each chromosome

Fig. 4

Relationship between the physical and genetic positions of the SNP loci in B. napus. The inner circle represents the genetic map which is flanked to the outside by the physical position in the spring-type DH12075 and to the inside by the physical position in the winter-type Darmor bzh. The green lines connecting across the centre of the circle represent those loci that are genetically positioned to an alternate (mostly homoeologous) position compared to their physical coordinates in the genome sequence

Fig. 5

Alignment of the genetic map for linkage group N10 of B. napus with the genome sequence of the equivalent chromosome in the two independent B. napus assemblies and in B. rapa