A novel approach to develop wheat chromosome-specific KASP markers for detecting Amblyopyrum muticum segments in doubled haploid introgression lines

Abstract

Many wild relative species are being used in pre-breeding programmes to increase the genetic diversity of wheat. Genotyping tools such as single nucleotide polymorphism (SNP)-based arrays and molecular markers have been widely used to characterise wheat-wild relative introgression lines. However, due to the polyploid nature of the recipient wheat genome, it is difficult to develop SNP-based KASP markers that are codominant to track the introgressions from the wild species. Previous attempts to develop KASP markers have involved both exome- and PCR-amplicon-based sequencing of the wild species. But chromosome-specific KASPs assays have been hindered by homoeologous SNPs within the wheat genome. This study involved whole genome sequencing of the diploid wheat wild relative Amblyopyrum muticum and development of a SNP discovery pipeline that generated ~38,000 SNPs in single-copy wheat genome sequences. New assays were designed to increase the density of Am. muticum polymorphic KASP markers. With a goal of one marker per 60 Mbp, 335 new KASP assays were validated as functional. Together with assays validated in previous studies, 498 well distributed chromosome-specific markers were used to recharacterize previously genotyped wheat-Am. muticum doubled haploid (DH) introgression lines. The chromosome specific nature of the KASP markers allowed clarification of which wheat chromosomes were involved with recombination events or substituted with Am. muticum chromosomes and the higher density of markers allowed detection of new small introgressions in these DH lines.

Whole genome sequencing uncovers the structural and transcriptomic landscape of hexaploid wheat/Ambylopyrum muticum introgression lines

“…Using this approach, we confirm the existence of 100% of segments previously identified with KASP genotyping (Grewal et al, 2021). However, we were able to resolve the locations of segment junctions to a much higher resolution than previous methods, due to the limited marker density available for KASP genotyping and the inability of GISH to resolve segments below ~ 20Mbp.…”

“…However, we were able to resolve the locations of segment junctions to a much higher resolution than previous methods, due to the limited marker density available for KASP genotyping and the inability of GISH to resolve segments below ~ 20Mbp. In addition, we were able to uncover two previously unreported segments that have been subsequently validated by KASP genotyping (Grewal et al, 2021); a 17.39Mbp on the telomere of chr7D of DH195 and a 22.68Mbp segment on the telomere of chr5D in DH121. We also identified a new 3.99Mbp segment on chr6D of DH15 that we subsequently validated using 2 KASP markers, WRC1873 and WRC1890 (Table S3).…”

“…The current approach for studying synthetic introgression lines prior to deployment in breeding programmes relies on cytogenetic and genotyping techniques, namely GISH and KASP (12,18). De novo discovery of SNPs to produce higher density KASP markers has improved the resolution and as demonstrated here, recent KASP genotyping was able to detect most of the introgressed segments identified with our approach.…”

“…The current approach for studying synthetic introgression lines prior to deployment in breeding programmes relies on cytogenetic and genotyping techniques, namely GISH and KASP (Grewal et al, 2021; King et al, 2019). De novo discovery of SNPs to produce higher density KASP markers has improved the resolution but are insufficient for unpicking the precise size and location of segments and will likely miss small segments without the guidance of WGS data to identify areas in which additional markers should be deployed.…”

“…muticum introgression lines (Fellers et al, 2020) has revealed resistances to leaf, stem and stripe rust not observed in the parental wheat lines and thus likely conferred by introgressed genes. KASP genotyping to identify segments has been conducted on many of these lines (Grewal et al, 2021). Through this analysis, we have pinpointed introgression segment junctions to a higher resolution than previously possible, in many cases within a single pair of reads, demonstrating segments of variable size that overlap between introgression lines, which explains some differences in resistance phenotype seen between lines.…”