Development of DNA Markers From Physically Mapped Loci in Aegilops comosa and Aegilops umbellulata Using Single-Gene FISH and Chromosome Sequences

Breeding of agricultural crops adapted to climate change and resistant to diseases and pests is hindered by a limited gene pool because of domestication and thousands of years of human selection. One way to increase genetic variation is chromosome-mediated gene transfer from wild relatives by cross hybridization. In the case of wheat (Triticum aestivum), the species of genus Aegilops are a particularly attractive source of new genes and alleles. However, during the evolution of the Aegilops and Triticum genera, diversification of the D-genome lineage resulted in the formation of diploid C, M, and U genomes of Aegilops. The extent of structural genome alterations, which accompanied their evolution and speciation, and the shortage of molecular tools to detect Aegilops chromatin hamper gene transfer into wheat. To investigate the chromosome structure and help develop molecular markers with a known physical position that could improve the efficiency of the selection of desired introgressions, we developed single-gene fluorescence in situ hybridization (FISH) maps for M- and U-genome progenitors, Aegilops comosa and Aegilops umbellulata, respectively. Forty-three ortholog genes were located on 47 loci in Ae. comosa and on 52 loci in Ae. umbellulata using wheat cDNA probes. The results obtained showed that M-genome chromosomes preserved collinearity with those of wheat, excluding 2 and 6M containing an intrachromosomal rearrangement and paracentric inversion of 6ML, respectively. While Ae. umbellulata chromosomes 1, 3, and 5U maintained collinearity with wheat, structural reorganizations in 2, 4, 6, and 7U suggested a similarity with the C genome of Aegilops markgrafii. To develop molecular markers with exact physical positions on chromosomes of Aegilops, the single-gene FISH data were validated in silico using DNA sequence assemblies from flow-sorted M- and U-genome chromosomes. The sequence similarity search of cDNA sequences confirmed 44 out of the 47 single-gene loci in Ae. comosa and 40 of the 52 map positions in Ae. umbellulata. Polymorphic regions, thus, identified enabled the development of molecular markers, which were PCR validated using wheat-Aegilops disomic chromosome addition lines. The single-gene FISH-based approach allowed the development of PCR markers specific for cytogenetically mapped positions on Aegilops chromosomes, substituting as yet unavailable segregating map. The new knowledge and resources will support the efforts for the introgression of Aegilops genes into wheat and their cloning.

Identification and Characterization of Wheat - Aegilops Comosa 7M/7A Disomic Substitution Lines with Stripe Rust Resistance

Aegilops comosa (MM, 2n = 2x = 14), an important diploid species belonging to wheat tertiary gene pools, contains many excellent genes/traits, including disease resistance for wheat breeding. In this study, three sister lines, NAL-32, NAL-33, and NAL-34, were identified from a wheat - Ae. comosa distant cross using fluorescence in situ hybridization (FISH) combined with single nucleotide polymorphism (SNP) microarray analysis. Genetically, NAL-32 contained neither an alien nor translocation chromosome, whereas NAL-33 and NAL-34 had disomic 7M/7A substitution chromosomes but differed in the absence (NAL-33) or presence (NAL-34) of 1BL/1RS translocation chromosomes. The substitution of 7M/7A in NAL-33 and NAL-34 was verified using wheat 55 K SNP arrays but 1BL/1RS translocation in NAL-34 was not. The two 7M/7A substitution lines, NAL-33 and NAL-34, had similar stripe rust resistance, and both showed higher stripe rust resistance than NAL-32 and their parents, suggesting that stripe rust resistance in NAL-33 and NAL-34 was derived from the 7M of Ae. comosa and that their resistance was likely irrelevant to 1BL/1RS translocation. Meanwhile, the three NAL lines also showed higher grain weights (grams per 50 grains) than one to three of their three wheat parents, and the two 7M/7A substitution lines, NAL-33 and NAL-34, had larger seed size-related traits than NAL-32, suggesting that both the 7M and 1BL/1RS chromosomes had positive effects on seed size-related traits. The results provide important bridge materials that can potentially be used for transferring stripe rust resistance, as well as seed size-related traits from Ae. comosa to wheat.

Development and characterization of Triticum turgidum – Aegilops comosa and T. turgidum – Ae. markgrafii amphidiploids

Aegilops comosa and Ae. markgrafii are diploid progenitors of polyploidy species of Aegilops sharing M and C genomes, respectively. Transferring valuable genes/traits from Aegilops into wheat is an alternative strategy for wheat genetic improvement. The amphidiploids between diploid species of Aegilops and tetraploid wheat can act as bridges to overcome obstacles from direct hybridization and can be developed by the union of unreduced gametes. In this study, we developed seven Triticum turgidum – Ae. comosa and two T. turgidum – Ae. markgrafii amphidiploids. The unreduced gametes mechanisms, including first-division restitution (FDR) and single-division meiosis (SDM), were observed in triploid F1 hybrids of T. turgidum – Ae. comosa (STM) and T. turgidum – Ae. markgrafii (STC). Only FDR was observed in STC hybrids, whereas FDR or both FDR and SDM were detected in the STM hybrids. All seven pairs of M chromosomes of Ae. comosa and C chromosomes of Ae. markgrafii were distinguished by fluorescent in situ hybridization (FISH) probes pSc119.2 and pTa71 combinations with pTa-535 and (CTT)12/(ACT)7, respectively. Meanwhile, the chromosomes of tetraploid wheat and diploid Aegilops parents were distinguished by the same FISH probes. The amphidiploids possessed specific valuable traits such as multiple tillers, large seed size related traits, and stripe rust resistance that could be utilized in the genetic improvement of wheat.

1Mx2.1, a novel high-molecular-weight glutenin subunit from Aegilops comosa and its relationship to high-quality dough

High-molecular-weight glutenin subunit (HMW-GS) of endosperm is mainly correlated with dough quality of bread wheat. In wheat cultivars, the HMW-GS genes with good processing quality are limited. However, there are an amount of excellent HMW-GS genes presenting in wheat-related species. In this work, two novel HMW-GS genes located on 1 M chromosome from Aegilops comosa have been cloned, designated as 1Mx2.1 and 1My12.1, respectively. The molecular structure of 1Mx2.1 and 1My12.1 showed high similarity with the published HMW-GS, but containing unique structures. 1Mx2.1 contained an extra cysteine residue in the repetitive domain, and 1My12.1 lost the conservative cysteine residue in the C-terminal domain. In vitro mixing test has indicated that 1Mx2.1 contributes excellent dough quality. The Ae. comosa can be used as an important genetic resource for wheat quality improvement.