Aegilops caudata L. [syn. Ae. markgrafii (Greuter) Hammer], is a diploid wild relative of wheat (2n = 2x = 14, CC) and a valuable source for new genetic diversity for wheat improvement. It has a variety of disease resistance factors along with tolerance for various abiotic stresses and can be used for wheat improvement through the generation of genome-wide introgressions resulting in different wheat-Ae. caudata recombinant lines. Here, 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-color genomic in situ hybridization (mcGISH). Of these, six lines have stable homozygous introgressions from Ae. caudata and will be used for future trait analysis. Using cytological techniques and molecular marker analysis of the recombinant lines, 182 KASP markers were physically mapped 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 are useful resources that can be used in breeding programs worldwide for wheat improvement. Additionally, the genome-specific KASP markers could prove to be a valuable tool for the rapid detection and marker-assisted selection of other Aegilops species in a wheat background.

The wild relatives of wheat provide an important source of genetic variation for wheat improvement. Much of the work in the past aimed at transferring genetic variation from wild relatives into wheat has relied on the exploitation of the ph1b mutant, located on the long arm of chromosome 5B. This mutation allows homologous recombination to occur between chromosomes from related but different genomes, e.g. between the chromosomes of wheat and related chromosomes from a wild relative resulting in the generation of interspecific recombinant chromosomes. However, the ph1b mutant also enables recombination to occur between the homologous genomes of wheat, e.g. A/B, A/D, B/D, resulting in the generation of wheat intergenomic recombinant chromosomes. In this work we report on the presence of wheat intergenomic recombinants in the genomic background of hexaploid wheat/Amblyopyrum muticum introgression lines. The transfer of genomic rearrangements involving the D-genome through pentaploid crosses provides a strategy by which the D-genome of wheat can be introgressed into durum wheat. Hence, a pentaploid crossing strategy was used to transfer D-genome segments, introgressed with either the A- and/or the B-genome, into the tetraploid background of two durum wheat genotypes Karim and Om Rabi 5 in either the presence or absence of different Am. muticum (2n = 2x = 14, TT) introgressions. Introgressions were monitored in backcross generations to the durum wheat parents via multi-color genomic in situ hybridization (mc-GISH). Tetraploid lines carrying homozygous D-genome introgressions, as well as simultaneous homozygous D- and T-genome introgressions, were developed. Introgression lines were characterized via Kompetitive Allele-Specific PCR (KASP) markers and multi-color fluorescence in situ hybridization (FISH). Results showed that new wheat sub-genomic translocations were generated at each generation in progeny that carried any Am. muticum chromosome introgression irrespective of the linkage group that the segment was derived from. The highest frequencies of homologous recombination were observed between the A- and the D-genomes. Results indicated that the genotype Karim had a higher tolerance to genomic rearrangements and T-genome introgressions compared to Om Rabi 5. This indicates the importance of the selection of the parental genotype when attempting to transfer/develop introgressions into durum wheat from pentaploid crosses.

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 ...

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.

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.