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

Authors: GREWAL, SURBHI - University Of Nottingham; JOYNSON, RYAN - Earlham Institute; COOMBES, BENEDICT - Earlham Institute; HALL, ANTHONY - Earlham Institute; Fellers, John; YANG, CAI-YUN - University Of Nottingham; SCHOLEFIELD, DUNCAN - University Of Nottingham; ASHLING, STEPHEN - University Of Nottingham; HUBBART-EDWARDS, STELLA - University Of Nottingham; ISAAC, PETER - Idna Genetics Ltd; KING, IAN - University Of Nottingham; KING, JULIE - University Of Nottingham

Submitted to: Journal of Theoretical and Applied Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/20/2021
Publication Date: 3/25/2022
Citation: Grewal, S., Joynson, R., Coombes, B., Hall, A., Fellers, J.P., Yang, C., Scholefield, D., Ashling, S., Hubbart-Edwards, S., Isaac, P., King, I., King, J. 2022. Chromosome-specific KASP markers for detecting Amblyopyrum muticum segments in wheat introgression lines. Journal of Theoretical and Applied Genetics. https://doi.org/10.1002/tpg2.20193.
DOI: https://doi.org/10.1002/tpg2.20193

Interpretive Summary: Wheat is limited in the amount of available genetic resources for breeders to use in the improvement of varieties. Wild relatives of wheat represent a pool that can be tapped for new genes. Unfortunately, there are significant genome differences in the relatives that make it difficult to cross pollinate with wheat. Also, it is difficult to know which of the progeny from the pollinations actually have genetic material from the wild species. In this work, new genome sequencing technology was used to sequence the genome of the wheat relative Amblyopyrum muticum and a new computer analysis pipeline was developed to compare this genome with wheat to find useful differences. Single nucleotide changes specific to Am. muticum were found and a polymerase chain reaction assay was developed for 335 new genetic markers which were added to a previous collection of markers. With the newly completed set, it is now possible to not only identify large fragments of the Am. muticum genome in wheat, but also be able to find smaller fragments that cannot be identified through classical chromosome staining. The validation of this new pipeline will help streamline and reduce effort in moving genes from other wild relatives into wheat and thus open up new sources of important traits.

Technical 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 use genome-specific SNP-based Kompetitive allele-specific PCR (KASP) markers to track wild species introgressions. Previous attempts to develop codominant KASP markers have involved both exome and PCR-amplicon-based sequencing. 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 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 SNP per 60 Mbp, 335 SNP assays were validated. Together with previously derived Am. muticum KASP markers from 90K chip assays, 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 recombinant events or substituted with Am. muticum chromosomes and the higher density of markers allowed detection of new small introgressions in these DH lines.