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

Abstract

22Aegilops caudata L. [syn. Ae. markgrafii (Greuter) Hammer], a diploid wild relative of wheat 23 (2n = 2x = 14, CC), is an important source for new genetic variation for wheat improvement 24 due to a variety of disease resistance factors along with tolerance for various abiotic stresses. 25 Its practical utilisation in wheat improvement can be facilitated through the generation of 26 genome-wide introgressions leading to a variety of different wheat-Ae. caudata recombinant 27 lines. In this study, we report the generation of nine such wheat-Ae. caudata recombinant lines 28 which were characterized using wheat genome-specific KASP (Kompetitive Allele Specific 29 PCR) markers and multi-colour genomic in situ hybridization (mcGISH). Of these, six lines 30 have stable homozygous introgressions from Ae. caudata and will be used for future trait 31 analysis. Through a combination of molecular and cytological analysis of all the recombinant 32 lines, we were able to physically map 182 KASP markers onto the seven Ae. caudata 33 chromosomes, of which 155 were polymorphic specifically with only one wheat subgenome. 34Comparative analysis of the physical positions of these markers in the Ae. caudata and wheat 35 genomes confirmed that the former had chromosomal rearrangements with respect to wheat, 36 as previously reported. These wheat-Ae. caudata recombinant lines and KASP markers 37 provide a useful genetic resource for wheat improvement with the latter having a wider impact 38 as a tool for detection of introgressions from other Aegilops species into wheat. 39 40 Introgression of Aegilops caudata into wheat 71 diversity in wheat breeding. Over the past decades, however, efforts have been made to study 72 the molecular organisation of the Ae. caudata genome and its homology with wheat 73 homoeologous groups. Friebe et al. (1992) showed that Ae. caudata has a highly asymmetric 74 karyotype distinct from the metacentric and submetacentric chromosomes of most Triticeae 75 species suggesting the distortion of chromosome collinearity compared to wheat. Fluorescence 76 in situ hybridisation (FISH) and molecular marker analysis of flow-sorted C-genome 77 chromosomes confirmed genome rearrangements (Molnár et al., 2016; Molnár et al., 2015). A 78 set of wheat (cv. Alcedo)-Ae. caudata addition lines B-G (Schubert and Blüthner, 1992; 79 Schubert and Blüthner, 1995) have also been characterised extensively in previous studies 80 using cytogenetic markers (Friebe et al., 1992), isozyme analysis (Schmidt et al., 1993), Simple 81 Sequence Repeat (SSR) markers (Gong et al., 2017; Niu et al., 2018; Peil et al., 1998), FISH 82 with cDNA probes (Danilova et al., 2017), Conserved Orthologous Sequence (COS) and PCR-83 based Landmark Unique Gene (PLUG) markers (Gong et al., 2017), and sequential FISH and 84 genomic in situ hybridization (GISH) (Niu et al., 2018). Many of these studies found that the 85 Introgression of Aegilops caudata into wheat Alcedo-Ae. caudata addition lines carried several inversions and translocations although ...

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

“…Previous studies have reported chromosome‐specific KASP markers between wheat and Am. muticum (Grewal et al., 2020a) and other wild relative species (Grewal et al., 2020b; Grewal et al. 2021), which have been used for genotyping wheat–wild relative introgression lines.…”

“…The genotyping procedure was as described by Grewal et al. (2020b). Briefly, the genotyping reactions were set up using the automated PIPETMAX ® 268 (Gilson) 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, 0.068 μl primer mix, and 2.43 μl nuclease‐free water.…”

“…Preparation of the root‐tip metaphase chromosome spreads, the protocol for multi‐color genomic in situ hybridization (mcGISH) and the image capture was as described in Grewal et al. (2020b). Briefly, genomic DNA from T. urartu (to detect the A‐genome), Aegilops speltoides Tausch (to detect the B‐genome), Aegilops tauschii Coss.…”

“…The Kompetitive allele‐specific polymerase chain reaction (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 co‐dominant interspecific SNPs.…”