Integrating genomic resources to present full gene and putative promoter capture probe sets for bread wheat

Abstract

Background Whole-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. Results We 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. 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. 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.

Strategies for reducing per‐sample costs in target capture sequencing for phylogenomics and population genomics in plants

“…Sequencing using other platforms is also possible but has been less well explored (e.g., Ion Torrent Proton [Thermo Fisher Scientific]; Lesur et al., ). Larger projects will be able to take advantage of lower per‐sample costs of the higher‐throughput platforms (e.g., NovaSeq 6000 [Illumina]; Gardiner et al., ). In the future, new developments in high‐throughput sequencing with longer read lengths (e.g., NovaSeq S Prime flow cell 2 × 250 bp) or single‐molecule sequencing (e.g., PacBio [Pacific Biosciences, Menlo Park, California, USA], Wolf et al., ; MinION [Oxford Nanopore Technologies, Oxford, United Kingdom], Bethune et al., ; Li et al., ) could facilitate the recovery of flanking regions, but at this time these newer sequencing platforms remain relatively unexplored for target capture projects (but see Gilpatrick et al., ; Kovaka et al., ; Payne et al., ).…”

Limited haplotype diversity underlies polygenic trait architecture across 70 years of wheat breeding

“…Single Sequence Repeat (SSR) markers [20]. We sequenced 15 founders after enrichment for (a) genic regions and (b) putative promoters using a capture probe-set [21] at average coverage of 22.94x of the targets (Supplementary Table 1). The remaining founder, Holdfast, was sequenced by WGS, but to ensure consistency across founders, we restricted our attention to the capture targets, for which coverage in Holdfast was 15.8x.…”

“…The other founders were sequenced after capture using two recently designed probe sets targeting promoter and genic regions, respectively [21].…”

“…For compatibility with the bam file format, we split each chromosome in the reference genome at the halfway point before alignment. We called variants from the founder sequences within the high confidence gene, promoter and 5' UTR regions targeted by the capture probes [21] using GATK (version 4.0.8.0) [66] HaplotypeCaller and GenotypeGVCFs (options --interval-padding 100 --minimum-mapping-quality 30). We analysis, we used the genotype calls in the founders as a reference panel and outputted the estimated ancestry dosages of each founder at each position in each RIL using the outputHaplotypeProbabilities and output_haplotype_dosages options.…”

Study of a region on wheat chromosome 5BS by targeted sequencing

“…In 2019, two gold standard capture probe sets for a gene and a putative promoter capture of hexaploid bread wheat were presented and validated, which are designed using recently developed genome sequence and annotation resources. Researchers 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 low-confidence genes and associated promoters (Gardiner et al, 2019). Our results suggest that sequence capture is a reliable approach to study and annotate of the chromosome region in both the gene space and non-repetitive intergenic regions.…”