Rapid identification of homozygosity and site of wild relative introgressions in wheat through chromosome‐specific KASP genotyping assays

Abstract

Summary For future food security, it is important that wheat, one of the most widely consumed crops in the world, can survive the threat of abiotic and biotic stresses. New genetic variation is currently being introduced into wheat through introgressions from its wild relatives. For trait discovery, it is necessary that each introgression is homozygous and hence stable. Breeding programmes rely on efficient genotyping platforms for marker‐assisted selection (MAS). Recently, single nucleotide polymorphism (SNP)‐based markers have been made available on high‐throughput Axiom® SNP genotyping arrays. However, these arrays are inflexible in their design and sample numbers, making their use unsuitable for long‐term MAS. SNPs can potentially be converted into Kompetitive allele‐specific PCR (KASP™) assays that are comparatively cost‐effective and efficient for low‐density genotyping of introgression lines. However, due to the polyploid nature of wheat, KASP assays for homoeologous SNPs can have difficulty in distinguishing between heterozygous and homozygous hybrid lines in a backcross population. To identify co‐dominant SNPs, that can differentiate between heterozygotes and homozygotes, we PCR‐amplified and sequenced genomic DNA from potential single‐copy regions of the wheat genome and compared them to orthologous copies from different wild relatives. A panel of 620 chromosome‐specific KASP assays have been developed that allow rapid detection of wild relative segments and provide information on their homozygosity and site of introgression in the wheat genome. A set of 90 chromosome‐nonspecific assays was also produced that can be used for genotyping introgression lines. These multipurpose KASP assays represent a powerful tool for wheat breeders worldwide.

Development of Wheat-Aegilops caudata Introgression Lines and Their Characterisation Using Genome-Specific KASP Markers

“…In a previous study, we reported the generation of 183 KASP markers polymorphic between hexaploid wheat and Ae. caudata accession 2090001 (Grewal et al, 2020). Although, the physical location of these markers on the wheat genome was reported, the study did not address the physical location of these markers on the Ae.…”

“…caudata chromatin in a wheat background. In this research, a total of 182 KASP markers, developed in a previous study (Grewal et al, 2020), were assigned to specific Ae. caudata chromosomes (Table 1), providing a new rapid detection approach for the quick selection and identification of wheat- Ae.…”

“…The wheat (Alcedo)- Ae. caudata B-G addition were also used as control lines to verify the specificity of the KASP markers (Grewal et al, 2020) to each of the Ae. caudata chromosomes.…”

“…Preparation of the root-tip metaphase chromosome spreads, the protocol for mcGISH and the image capture was as described in (Grewal et al, 2020). In summary, genomic DNAs from young leaves of the three putative diploid progenitors of bread wheat, i.e.…”

Scale development and utilization of universal PCR-based and high-throughput KASP markers specific for chromosome arms of rye (Secale cereale L.)

“…Ma et al [34] developed the co-dominant KASP markers that were able to detect Agropyron cristatum chromosome based on the RNA-seq data. Recently, Grewal et al [57] developed chromosome-specific KASP genotyping assays for ten wild relative species based on the probe sequences on the Axiom® Wheat-Relative Genotyping Array. These markers that have been developed will provide powerful tools for wheat breeders worldwide.…”