The Use and Limitations of Exome Capture to Detect Novel Variation in the Hexaploid Wheat Genome

Abstract

The bread wheat (Triticum aestivum) pangenome is a patchwork of variable regions, including translocations and introgressions from progenitors and wild relatives. Although a large number of these have been documented, it is likely that many more remain unknown. To map these variable regions and make them more traceable in breeding programs, wheat accessions need to be genotyped or sequenced. The wheat genome is large and complex and consequently, sequencing efforts are often targeted through exome capture. In this study, we employed exome capture prior to sequencing 12 wheat varieties; 10 elite T. aestivum cultivars and two T. aestivum landrace accessions. Sequence coverage across chromosomes was greater toward distal regions of chromosome arms and lower in centromeric regions, reflecting the capture probe distribution which itself is determined by the known telomere to centromere gene gradient. Superimposed on this general pattern, numerous drops in sequence coverage were observed. Several of these corresponded with reported introgressions. Other drops in coverage could not be readily explained and may point to introgressions that have not, to date, been documented.

Keywords: Triticum aestivum; exome capture; exome capture sequencing; introgression; sequence variation; wheat.

FIGURE 1

Read coverage for the accession ‘Bacanora’ after alignment to IWGSC ‘Chinese Spring’ assembly version 1.0. (A) Read coverage across chromosomes tended to be higher toward the telomeres and lower across the centromere. (B) Chromosome 1B shows a clear drop in coverage across the short arm (NB in all plots, chromosome short arms are on the left). (C) Location and density of capture probes across chromosome 1B (data from Gardiner et al., 2019).

FIGURE 2

(A) Average depth of coverage for chromosome 5B in the accessions ‘Bobwhite’ and ‘Pavon 76’; both show a drop in read coverage on the long arm at approximately position 490,000,000–540,000,000. (B) Dendrogram based on the 1,749 Axiom markers mapped to chromosome 5B; the 8 varieties (‘Bobwhite’, ‘Boregar’, ‘KWS Kielder’, ‘Maris Huntsman’, ‘Pavon 76’, ‘Renan’, ‘Riband’, and ‘Watkins 141’) with the drop in read coverage cluster. (C) A sample of the SNP calls across the interval 499,569, 304–534,345,241 highlighting the difference between the two groups (blue and red are the alternative homozygote calls; green indicates heterozygote calls).

FIGURE 3

Sequence coverage across the first 100 Mb of chromosome 2AS. The two accessions, ‘Boregar’ and ‘Renan’, show reduced coverage across the first 25–30 Mb which corresponds with the size of the known introgression from Ae. ventricosa (Robert et al., 1999).

FIGURE 4

(A) Bar graphs showing the number of capture probes that had BLAST hits to ‘Chinese Spring’ chromosome 1BS (IWGSC v1), S. cereale chromosome 1RS (JADQCU000000000 v1 of the cultivar Weining), ‘Chinese Spring’ chromosome 5DS (IWGSC v1), and Ae. tauschii chromosome 5DS (PRJNA341983 assembly of Ae. tauschii subsp. strangulata). The number of probe sequences for chromosomes 1BS and 5DS was 26,985 and 20,253, respectively. The number of probes that produced a hit was 26,222 to ‘Chinese Spring’ 1BS, 8,419 to S. cereale 1RS, 20,082 to ‘Chinese Spring’ 5DS, and 19,872 to Ae. tauschii 5DS. There were more hits than probe sequences as some probes had multiple hits. (B) Box and whisker plots showing the percentage similarity between the probe sequences and their respective targets.

FIGURE 5

Details of the Pm2 gene in ‘Chinese Spring’ and Ae. tauschii: (A) a 3 bp insertion and (B) a 7 bp insertion. Respectively, green and blue bases are ‘Chinese Spring’ reference sequences before and after the indel. Red bases are the insertion (found in both Ae. tauschii and ‘Maris Huntsman’).

FIGURE 6

(A) Pie chart showing the best BLAST hits against a combined Poaceae/S. cereale database for captured reads that didn’t map to the IWGSC ‘Chinese Spring’ assembly v1. (B) Phylogenetic tree (redrawn from Zhou et al., 2017), showing the relationship of the species used in our Poaceae/S. cereale database.