Analysis of Genetic Variation in Polyploid Wheat by Whole-Exome Capture.

The size of the wheat genome precludes the large-scale analysis of genetic variation by direct genome sequencing. However, a recently developed method of sequence capture offers the possibility of reducing the cost of sequencing by enriching genomic libraries with valuable targets including coding and regulatory sequences. A Nimblegen capture assay targeting 110 Mb of gene-harboring regions of the wheat genome was used to characterize nucleotide sequence variation in wheat cultivars and landraces. The coding sequences for assay design were selected based on the analysis of a large collection of transcript sequences including ESTs, full-length cDNAs and 454 transcriptome sequencing data combined with the comparative analysis of wheat (cv. Chinese Spring) and Brachypodium genomes. Homoeologous targets on the wheat exome capture assay were represented by a non-redundant set of oligonucleotide probes designed using the sequences of one of the duplicated gene copies. Enriched genomic libraries for next-generation sequencing were prepared by pooling up to 8 barcoded libraries in a single capture reaction followed by 2x100 bp paired-end sequencing on the HiSeq2000 instrument. Each sample was re-sequenced to achieve the average target coverage of about 50x for reliable SNP detection. About 60-73% of Illumina reads could be mapped to a reference and more than 90% of exonic targets were represented in sequenced libraries. The coverage of homoeologous mutations differentiating wheat subgenomes showed that the majority of duplicated genes were captured with equal efficiency. We used exome re-sequencing data to characterize the patterns of divergence and variation across the wheat genome, estimate the extent of genetic differentiation among wheat populations, and to assess SNP effects on gene function and biological pathways.