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

Abstract

Aegilops caudata L. [syn. Ae. markgrafii (Greuter) Hammer], is a diploid wild relative of wheat (2n = 2x = 14, CC) and a valuable source for new genetic diversity for wheat improvement. It has a variety of disease resistance factors along with tolerance for various abiotic stresses and can be used for wheat improvement through the generation of genome-wide introgressions resulting in different wheat-Ae. caudata recombinant lines. Here, we report the generation of nine such wheat-Ae. caudata recombinant lines which were characterized using wheat genome-specific KASP (Kompetitive Allele Specific PCR) markers and multi-color genomic in situ hybridization (mcGISH). Of these, six lines have stable homozygous introgressions from Ae. caudata and will be used for future trait analysis. Using cytological techniques and molecular marker analysis of the recombinant lines, 182 KASP markers were physically mapped onto the seven Ae. caudata chromosomes, of which 155 were polymorphic specifically with only one wheat subgenome. Comparative analysis of the physical positions of these markers in the Ae. caudata and wheat genomes confirmed that the former had chromosomal rearrangements with respect to wheat, as previously reported. These wheat-Ae. caudata recombinant lines and KASP markers are useful resources that can be used in breeding programs worldwide for wheat improvement. Additionally, the genome-specific KASP markers could prove to be a valuable tool for the rapid detection and marker-assisted selection of other Aegilops species in a wheat background.

Generation of Doubled Haploid Wheat-Triticum urartu Introgression Lines and Their Characterisation Using Chromosome-Specific KASP Markers

“…Genotyping of homozygous introgression lines with chromosome-specific KASP markers also allows the potential identification of the wheat chromosome recombined with the T. urartu segment ( Grewal et al, 2020a , b ) due to the absence of the wheat-specific allele in homozygous lines which will instead only show the presence of the wild-relative-specific allele for a marker that lies within the introgression. Since the lines characterised in this work are DH lines, they are expected to be homozygous for every introgression from T. urartu and thus, the chromosome-specific KASP markers should be able to identify the wheat chromosome recipient of the introgression.…”

“…All DH lines shown to have an introgression from T. urartu in the array genotyping were subsequently genotyped with KASP markers after a round(s) of self-fertilisation. The genotyping procedure was as described by Grewal et al (2020b) . In summary, the genotyping reactions were set up using the automated PIPETMAX ® 268 (Gilson, United Kingdom) and performed in a ProFlex PCR system (Applied Biosystems by Life Technology) in a final volume of 5 μl with 1 ng genomic DNA, 2.5 μl KASP reaction mix (ROX), 0.068 μl primer mix and 2.43 μl nuclease free water.…”

“…The protocol for genomic in situ hybridization (GISH) and preparation of chromosome spreads was as described in Kato et al (2004) , King et al (2017) , and Grewal et al (2020b) . In summary, genomic DNA was isolated from the three putative diploid progenitors of bread wheat, i.e., T. urartu (A genome), Ae.…”

“…The use of the new technologies at the Nottingham BBSRC Wheat Research Centre (WRC) has allowed the transfer of hundreds of introgressions from a variety of wild relatives into wheat, e.g., Amblyopyrum muticum ( King et al, 2017 ), Thinopyrum bessarabicum ( Grewal et al, 2018b ), Ae. speltoides ( King et al, 2018 ), T. urartu ( Grewal et al, 2018a ), Triticum timopheevii ( Devi et al, 2019 ), Thinopyrum elongatum ( Baker et al, 2020 ), and Aegilops caudata ( Grewal et al, 2020b ). However, without identifying what genetic variation is carried by the introgressions, the lines produced will remain quite simply as ‘seeds in a packet’ of unknown agronomic potential.…”

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

“…The Kompetitive allele-specific PCR (KASP) platform has been demonstrated to be a flexible, efficient and cost-effective system for genotyping of introgression lines using wild relative genome-specific SNPs (Bansal et al 2020;Grewal et al 2020b;Han et al 2020). However, hexaploid wheat's polyploid genome makes it complicated to develop codominant interspecific SNPs.…”