Combining Exome Capture and Bulked Segregant Analysis for Marker Discovery in Durum Wheat

Next generation sequencing coupled with reduced representation libraries provide the opportunity for novel strategies to localize genes in wheat, which currently lacks a reference genome sequence.  A Nimblegen capture assay targeting 110 Mb of gene-harboring regions of wheat genome was used in conjunction with bulked segregant analysis (BSA) of tetraploid wheat to tag a gene conferring resistance to the orange wheat blossom midge (Sm1).  Sm1 has been mapped previously to wheat chromosome 2BS, but few robust diagnostic markers are available.   Genomic DNA from 14 lines were pooled for each of the resistant and susceptible bulks and parental and bulk DNA samples were captured and sequenced (200 bp paired-end) on an Illumina HiSeq2000. A single lane of data was generated for each bulk and the DNA from the two parents were indexed and sequenced together. Sequence reads were aligned to a wheat exome pseudo-reference (107,000 contigs), with an average coverage of >20X achieved for each  sample.  After filtering for homoeologous variants, read depth and expected allele frequencies, inter-bulk SNPs were identified on 12,000 contigs of the pseudo-reference.  The number of contigs selected for genetic mapping was reduced by localizing only those that mapped to chromosome 2BS of the wheat chromosome survey sequence assembly generated by the International Wheat Genome Sequencing Consortium.  KASPar marker assays were developed from these contigs and over 50 localized within a 0.8 cM interval of Sm1 in two tetraploid recombinant inbred line populations.    Thus, the combination of exome capture and BSA is an effective strategy to tag genes in the unsequenced wheat genome.