Abstract
BackgroundWhole-genome shotgun resequencing of wheat is expensive because of its large, repetitive genome. Moreover, sequence data can fail to map uniquely to the reference genome, making it difficult to unambiguously assign variation. Resequencing using target capture enables sequencing of large numbers of individuals at high coverage to reliably identify variants associated with important agronomic traits. Previous studies have implemented complementary DNA/exon or gene-based probe sets in which the promoter and intron sequence is largely missing alongside newly characterized genes from the recent improved reference sequences.ResultsWe present and validate 2 gold standard capture probe sets for hexaploid bread wheat, a gene and a putative promoter capture, which are designed using recently developed genome sequence and annotation resources. The captures can be combined or used independently. We demonstrate that the capture probe sets effectively enrich the high-confidence genes and putative promoter regions that were identified in the genome alongside a large proportion of the low-confidence genes and associated promoters. Finally, we demonstrate successful sample multiplexing that allows generation of adequate sequence coverage for single-nucleotide polymorphism calling while significantly reducing cost per sample for gene and putative promoter capture.ConclusionsWe show that a capture design employing an “island strategy” can enable analysis of the large gene/putative promoter space of wheat with only 2 × 160 Mbp probe sets. Furthermore, these assays extend the regions of the wheat genome that are amenable to analyses beyond its exome, providing tools for detailed characterization of these regulatory regions in large populations.
Highlights
It is expensive to perform whole-genome sequencing to depths sufficient for confident variant calling, in species with large genome sizes
We have developed a comprehensive putative promoter capture probe set for wheat that takes 2 kbp upstream of the annotated genes and will facilitate global investigation to fully characterize these regulatory regions
We describe 2 new wheat NimbleGen SeqCap EZ probe sets (Roche NimbleGen Inc., Madison, WI, USA), the first tiled across the genic regions of the hexaploid bread wheat genome and the second tiled across the putative promoter regions; we integrate diverse wheat material into the design to allow broad applicability of the probe sets; we validate the capture probe sets using the reference variety Chinese Spring; and we demonstrate the probe sets’ application to diverse wheat accessions by enriching 8 wheat accessions that were generated by the International Maize and Wheat Improvement Center (CIMMYT), Mexico
Summary
It is expensive to perform whole-genome sequencing to depths sufficient for confident variant calling, in species with large genome sizes To reduce this complexity and to make resequencing more cost effective, we can utilize approaches such as restriction site associated DNA sequencing [1], transcriptome sequencing [2], and sequence capture. Conclusions: We show that a capture design employing an “island strategy” can enable analysis of the large gene/putative promoter space of wheat with only 2 × 160 Mbp probe sets. These assays extend the regions of the wheat genome that are amenable to analyses beyond its exome, providing tools for detailed characterization of these regulatory regions in large populations
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