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.

Key message One hundred and thirty four introgressions from Thinopyrum elongatum have been transferred into a wheat background and were characterised using 263 SNP markers. Abstract Species within the genus Thinopyrum have been shown to carry genetic variation for a very wide range of traits including biotic and abiotic stresses and quality. Research has shown that one of the species within this genus, Th. elongatum, has a close relationship with the genomes of wheat making it a highly suitable candidate to expand the gene pool of wheat. Homoeologous recombination, in the absence of the Ph1 gene, has been exploited to transfer an estimated 134 introgressions from Th. elongatum into a hexaploid wheat background. The introgressions were detected and characterised using 263 single nucleotide polymorphism markers from a 35 K Axiom ® Wheat-Relative Genotyping Array, spread across seven linkage groups and validated using genomic in situ hybridisation. The genetic map had a total length of 187.8 cM and the average chromosome length was 26.8 cM. Comparative analyses of the genetic map of Th. elongatum and the physical map of hexaploid wheat confirmed previous work that indicated good synteny at the macro-level, although Th. elongatum does not contain the 4A/5A/7B translocation found in wheat.

27For future food security it is important that wheat, one of the most widely consumed crops 28 in the world, can survive the threat of abiotic and biotic stresses. New genetic variation is 29 currently being introduced into wheat through introgressions from its wild relatives. For 30 trait discovery, it is necessary that each introgression is homozygous and hence stable. 31Breeding programs rely on efficient genotyping platforms for marker-assisted selection 32 (MAS). Recently, single nucleotide polymorphism (SNP) based markers have been made 33 available on high-throughput Axiom ® SNP genotyping arrays. However, these arrays are 34 inflexible in their design and sample numbers, making their use unsuitable for long-term 35 MAS. SNPs can potentially be converted into Kompetitive allele-specific PCR (KASP™) 36 assays which are comparatively cost-effective and efficient for low-density genotyping of 37 introgression lines. However, due to the polyploid nature of wheat, KASP assays for 38 homoeologous SNPs can have difficulty in distinguishing between heterozygous and 39 homozygous hybrid lines in a backcross population. To identify co-dominant SNPs, that 40 can differentiate between heterozygotes and homozygotes, we PCR-amplified and 41 sequenced genomic DNA from potential single-copy regions of the wheat genome and 42 compared them to orthologous copies from different wild relatives. A panel of 620 43 chromosome-specific KASP assays have been developed that allow rapid detection of wild 44relative segments and provide information on their homozygosity and site of introgression 45 in the wheat genome. A set of 90 chromosome-nonspecific assays was also produced that 46 can be used for genotyping introgression lines. These multipurpose KASP assays represent 47 a powerful tool for wheat breeders worldwide. 48 49