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

Aegilops caudata L. [syn. Ae. markgrafii (Greuter) Hammer], a diploid wild relative of wheat (2n = 2x = 14, CC), is an important source for new genetic variation for wheat improvement due to a variety of disease resistance factors along with tolerance for various abiotic stresses. Its practical utilisation in wheat improvement can be facilitated through the generation of genome-wide introgressions leading to a variety of different wheat–Ae. caudata recombinant lines. In this study, 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-colour genomic in situ hybridization (mcGISH). Of these, six lines have stable homozygous introgressions from Ae. caudata and will be used for future trait analysis. Through a combination of molecular and cytological analysis of all the recombinant lines, we were able to physically map 182 KASP markers 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 provide a useful genetic resource for wheat improvement with the latter having a wider impact as a tool for detection of introgressions from other Aegilops species into wheat.

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. Wheat is one of the most widely cultivated crops worldwide, contributing about a fifth of the 42 total calories and protein consumed by humans. While wheat production needs to increase to 43 feed the ever-growing population, wheat yields are plateauing due to the relatively small  In the past, the lack of information available for the cytomolecular identification of the C-68 genome chromosomes has hampered the use of the genetic potential of Ae. caudata in wheat 69 pre-breeding programmes. In addition, poor knowledge of syntenic relationships between 70 wheat and Ae. caudata chromosomes has been another obstacle hampering the use of its genetic 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  Genomic DNA was isolated from leaf tissue of 10-day old seedlings in a 96-well plate as 118 described by Thomson and Henry (1995). All back-crossed lines were genotyped alongside 119 five wheat genotypes (Chinese Spring, Paragon, Pavon, Highbury and Alcedo) and the Ae.  For each KASP™ marker, two allele-specific forward primers and one common reverse primer 125 were used (Supplemental Table 1). Genotyping reactions were performed in a ProFlex PCR       In this work, the KASP markers were tested on the Alcedo-Ae. caudata addition lines carrying 178 chromosomes B-G and the assignment of Ae. caudata chromosomes to homoeologous groups 179 was determined based on the distribution of the KASP markers among the addition lines. When 180 a KASP marker detected the presence of Ae. caudata in any one of the addition lines, the 181 marker was assigned to the corresponding C-genome chromosome. As shown in Table 1 Table 2). In total, 40 KASP markers were developed for the C-genome  (Table 1). However, some C-genome chromosomes such as 195 2C and 4C, had markers assigned from non-homoeologous groups as shown in Table 1.

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The KASP markers were also used to genotype a set of 181 wheat-Ae. caudata BCxFy lines to  (Table 2). However, prior to mcGISH analysis, the genome-specific KASP  Based on the molecular marker analysis of the Alcedo-Ae. caudata addition lines (Table 1)  confirmed to be chromosome 3D by FISH analysis (Figure 5a-b). The FISH analysis also 280 showed that one copy of chromosome 1D was missing and hence, the total chromosome 281 number for this line was 41 as mentioned in Table 2. Ae. caudata is known to carry many useful genes that can be used for wheat breeding. Its  found that the C-genome chromosomes carried several inversions and translocations but did 340 not agree on the rearrangements carried by each chromosome. In this study, we used the same 341 set of addition lines for the assignment of the KASP markers to the C-genome chromosomes.

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Genotyping of these addition lines with the KASP markers showed that chromosome B (2C) 343 had markers from wheat homoeologous groups 2/4 assigned to it (Table 1)   We assigned KASP markers that were not assigned to chromosomes B-G to chromosome A 350 and found that they were all homoeologous to the group 1 chromosomes of wheat (Figure 4a (Table 2), of which 6 had stable 364 homozygous recombinant wheat-Ae. caudata chromosomes (Figure 1). These lines will be 365 made available for further trait characterisation studies.

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Previous work investigating wheat-Ae. caudata introgression lines did not attribute the disease 368 resistance QTLs they found in these lines to a specific Ae. caudata chromosome. Gong et al.,  The genotyping results were also validated by mcGISH analysis of the recombinant lines 397 (Figure 2; top).

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Genotyping of these recombinant lines helped to physically order some of the KASP markers  (Table   422 1 and Figure 4f). This may be due to differences in the Ae. caudata accessions used in the two 423 studies.

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Our work also found one marker from wheat homoeologous group 7 assigned to chromosome  The authors declare that the research was conducted in the absence of any commercial or 510 financial relationships that could be construed as a potential conflict of interest.