Introgression of the Triticum timopheevii Genome Into Wheat Detected by Chromosome-Specific Kompetitive Allele Specific PCR Markers

Abstract

Triticum timopheevii (2n = 28, AtAtGG) is a tetraploid wild relative species with great potential to increase the genetic diversity of hexaploid wheat Triticum aestivum (2n = 42, AABBDD) for various important agronomic traits. A breeding scheme that propagated advanced backcrossed populations of wheat-T. timopheevii introgression lines through further backcrossing and self-fertilisation resulted in the generation of 99 introgression lines (ILs) that carried 309 homozygous segments from the At and G subgenomes of T. timopheevii. These introgressions contained 89 and 74 unique segments from the At and G subgenomes, respectively. These overlapping segments covered 98.9% of the T. timopheevii genome that has now been introgressed into bread wheat cv. Paragon including the entirety of all T. timopheevii chromosomes via varying sized segments except for chromosomes 3At, 4G, and 6G. Homozygous ILs contained between one and eight of these introgressions with an average of three per introgression line. These homozygous introgressions were detected through the development of a set of 480 chromosome-specific Kompetitive allele specific PCR (KASP) markers that are well-distributed across the wheat genome. Of these, 149 were developed in this study based on single nucleotide polymorphisms (SNPs) discovered through whole genome sequencing of T. timopheevii. A majority of these KASP markers were also found to be T. timopheevii subgenome specific with 182 detecting At subgenome and 275 detecting G subgenome segments. These markers showed that 98% of the At segments had recombined with the A genome of wheat and 74% of the G genome segments had recombined with the B genome of wheat with the rest recombining with the D genome of wheat. These results were validated through multi-colour in situ hybridisation analysis. Together these homozygous wheat-T. timopheevii ILs and chromosome-specific KASP markers provide an invaluable resource to wheat breeders for trait discovery to combat biotic and abiotic stress factors affecting wheat production due to climate change.

Identification of Fusarium Head Blight Resistance in Triticum timopheevii Accessions and Characterization of Wheat-T. timopheevii Introgression Lines for Enhanced Resistance

“…The development of a panel of introgressions of Tim_P95-99.1-1 into the wheat variety Paragon has been reported previously ( Devi et al, 2019 ). These lines were advanced and a selection of 32 homozygous introgression lines from this panel ( King et al, 2022 ; Table 2 ) were screened for FHB resistance by spray inoculation. Twenty-five of these introgression lines were tested in 2020 and 29 lines in 2021.…”

“…Twenty-five of these introgression lines were tested in 2020 and 29 lines in 2021. Each line was subjected to genotyping using chromosome-specific KASP markers ( King et al, 2022 ). These introgression lines contain a variety of segments from each of the two subgenomes present in T. timopheevii (A t and G) ( Table 2 ).…”

“…Development of wheat/ T. timopheevii introgression lines was as outlined in Devi et al (2019) and King et al (2022) . Briefly, Paragon ph1/ph1 (obtained from the GRU) was used as the female parent in a cross with T. timopheevii P95-99.1-1 to generate F 1 interspecific hybrids.…”

“…When genotyping of these plants showed the number of introgressions present to be three or less, the plants were then self-fertilized. Chromosome-specific KASP markers, polymorphic between wheat and T. timopheevii have been developed at the WRC and 480 of these KASP markers have been used to characterize a panel of homozygous wheat/ T. timopheevii introgression lines including those investigated in this work ( King et al, 2022 ).…”

“…Fluorescence in situ hybridization analysis of wheat- T. timopheevii introgression lines was carried out as described in King et al (2022) . Root metaphase spreads of chromosomes were hybridized with probes pSc119.2 ( McIntyre et al, 1990 ) and pAs.1 ( Rayburn and Gill, 1986 ) that were nick-labeled ( Rigby et al, 1977 ) with Alexa Fluor 488-5-dUTP (green) and Alexa Fluor 594-5-dUTP (red), respectively.…”