Finding invisible quantitative trait loci with missing data

Abstract

Evolutionary processes during plant polyploidization and speciation have led to extensive presence-absence variation (PAV) in crop genomes, and there is increasing evidence that PAV associates with important traits. Today, high-resolution genetic analysis in major crops frequently implements simple, cost-effective, high-throughput genotyping from single nucleotide polymorphism (SNP) hybridization arrays; however, these are normally not designed to distinguish PAV from failed SNP calls caused by hybridization artefacts. Here, we describe a strategy to recover valuable information from single nucleotide absence polymorphisms (SNaPs) by population-based quality filtering of SNP hybridization data to distinguish patterns associated with genuine deletions from those caused by technical failures. We reveal that including SNaPs in genetic analyses elucidate segregation of small to large-scale structural variants in nested association mapping populations of oilseed rape (Brassica napus), a recent polyploid crop with widespread structural variation. Including SNaP markers in genomewide association studies identified numerous quantitative trait loci, invisible using SNP markers alone, for resistance to two major fungal diseases of oilseed rape, Sclerotinia stem rot and blackleg disease. Our results indicate that PAV has a strong influence on quantitative disease resistance in B. napus and that SNaP analysis using cost-effective SNP array data can provide extensive added value from 'missing data'. This strategy might also be applicable for improving the precision of genetic mapping in many important crop species.

Keywords: Brassica napus; SNaP; presence-absence variation; quantitative resistance; single nucleotide absence polymorphism.

Figure 1

Schematic representation of allele segregation patterns and frequencies within and across subpopulations for different single nucleotide polymorphism (SNP) types observed in homozygous biparental (a) or multiparental mapping populations (b) and SNP probes excluded from analysis applying standard filtering procedures.

Figure 2

Physical anchoring of single nucleotide absence polymorphism (SNaP) markers (red lines) and Illumina resequencing data for six nested association mapping (NAM) parental lines to Darmor‐bzh and comparison with SNP segregation patterns in 200 NAM lines for (a) four consecutive SNaP markers on chromosome A03, Bn‐A03‐p10962307 (10 076 697 bp), Bn‐A03‐p10962645 (10 077 034 bp), Bn‐A03‐p10962969 (10 077 358 bp), Bn‐A03‐p10964394 (10 078 777 bp) and two consecutive SNaP markers on chromosome A07, Bn‐A07‐p21404492 (22 954 748 bp) and Bn‐A07‐p21406280 (22 956 683 bp).

Figure 3

Alignment of Illumina resequencing data for six nested association mapping parents to the Darmor‐bzh reference and comparison with PCR amplification results for six genes contained within a 30‐kb deletion (position 21 934 109 to 21 964 245 bp on chromosome A03) in the common parent DH5ON (Actin gene and genotype Darmor‐bzh are used as controls, and genotypes with no amplified products are shown in red font).

Figure 4

Genomewide deletion patterns visualized by single nucleotide polymorphism marker distribution with failed calls (red) in six nested association mapping (NAM) parental lines compared to single nucleotide absence polymorphism (SNaP) marker segregation patterns in the 200 B. napus nested association mapping (BnNAM) lines and subpopulations (blue).

Figure 5

Alignment of resequencing data in six parents of the nested association mapping population showing deletions in gene range size (left, in red) compared to single nucleotide absence polymorphism marker–trait association for Sclerotinia stem rot disease resistance in five subpopulations (right). Effect of an absence allele on phenotype in the respective subfamilies showing an association with (a) susceptibility on chromosome A01 at position 873 225 bp and an association with (b) resistance on chromosome A03 at position 21 934 764 bp.