Chromosome‐specific KASP markers for detecting Amblyopyrum muticum segments in wheat introgression lines

Abstract

Many wild‐relative species are being used in prebreeding programs to increase the genetic diversity of wheat (Triticum aestivum L.). Genotyping tools such as single nucleotide polymorphism (SNP)‐based arrays and molecular markers have been widely used to characterize wheat–wild relative introgression lines. However, due to the polyploid nature of the recipient wheat genome, it is difficult to develop SNP‐based Kompetitive allele‐specific polymerase chain reaction (KASP) markers that are codominant to track the introgressions from the wild species. Previous attempts to develop KASP markers have involved both exome‐ and polymerase chain reaction (PCR)‐amplicon‐based sequencing of the wild species. But chromosome‐specific KASP 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 (Boiss.) Eig and development of a de novo SNP discovery pipeline that generated ∼38,000 SNPs in unique 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 diagnostic for Am. muticum in a wheat background. 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.

Introgression of the Triticum timopheevii genome into wheat detected by chromosome-specific KASP markers

“…Advanced backcrossed populations of wheat- T. timopheevii introgression lines developed by Devi et al, 2019, segregating for various T. timopheevii segments, were propagated through backcrossing and self-fertilisation and genotyped at every generation with chromosome-specific KASP markers (Grewal et al, 2020a; Grewal et al, 2021; Grewal et al, 2022) as shown in Figure 1. Plants carrying segments of various sizes and from different linkage groups were selected to create a panel of overlapping T. timopheevii introgressions from each of its linkage groups in the wheat background.…”

“…Simultaneously to this work, KASP markers were being developed to characterise doubled haploid wheat ILs from T. urartu and Am. muticum (Grewal et al, 2021; Grewal et al, 2022). These two groups of chromosome-specific KASP assays consisting of 224 markers developed for T. urartu and 425 markers developed for Am.…”

“…Leaf tissue samples were collected and freeze-dried in a deep-well plate and ground in a TissueLyser II (QIAGEN) for 4-6 minutes at a frequency of 25 Hz. Genomic DNA for sequencing and genotyping was extracted as described in Grewal et al, 2022.…”

“…A graphical representation of physical positions of the four sets of chromosome-specific KASP markers, across all wheat chromosomes, used in this study to characterise wheat- T. timopheevii introgression lines. Sets 1, 2 and 3 were developed in previous works (Grewal et al, 2022; Grewal et al, 2021; Grewal et al, 2020a) and Set 4 was developed in this study to fill in the gaps between markers from the previous sets. Circles depict KASP markers that specifically detect A t subgenome segments, squares depict markers that detect G subgenome segments and triangles represent markers that detect both subgenomes.…”

“…For genotyping during the breeding pipeline, four sets of KASP markers were used. The first three were developed during previous studies (Grewal et al, 2022; Grewal et al, 2021; Grewal et al, 2020a) and the fourth was developed during this work using chromosome-specific SNPs between wheat and T. timopheevii , discovered as described above.…”