Exome sequencing of bulked segregants identified a novel TaMKK3-A allele linked to the wheat ERA8 ABA-hypersensitive germination phenotype

Abstract

Author contribution statement: SAM, OS, CMS, and CU designed the experiments. SAM and CMS developed the populations. SAM and SC performed the Louise/ZakERA8 QTL analysis. DZS and DS conducted GBS and analyzed the raw sequence data. SAM performed germination assays for the bulked segregant analysis, fine mapped ERA8, and introgressed ERA8 into breeding lines. OS performed the exome capture, BSA, and designed KASP markers. SAM and OS conducted the RNA sequencing and TaMKK3-A alignment. CMS and CU provided resources. SAM and CMS wrote the manuscript. All authors read and approved the final submission.Abstract: Preharvest sprouting (PHS) is the germination of mature grain on the mother plant when it rains before harvest. The ENHANCED RESPONSE TO ABA8 (ERA8) mutant increases seed dormancy and, consequently, PHS tolerance in soft white wheat 'Zak'. ERA8 was mapped to chromosome 4A in a Zak/'ZakERA8' backcross population using bulked segregant analysis of exome sequenced DNA (BSA-exome-seq). ERA8 was fine-mapped relative to mutagen-induced SNPs to a 4.6 Mb region containing 70 genes. In the backcross population, the ERA8 ABA hypersensitive phenotype was strongly linked to a missense mutation TaMKK3-A-G1093A (LOD 16.5), a gene associated with natural PHS tolerance in barley and wheat. The map position of ERA8 was confirmed in an 'Otis'/ZakERA8 but not in a 'Louise'/ZakERA8 mapping population. This is likely because Otis carries the same natural PHS susceptible MKK3-A-A660 S allele as Zak, whereas Louise carries the PHS tolerant MKK3-A-C660 R allele. Thus, the variation for grain dormancy and PHS tolerance in the Louise/ZakERA8 population likely resulted from segregation of other loci rather than segregation for PHS tolerance at the MKK3 locus. This inadvertent complementation test suggests that the MKK3-A-G1093A mutation causes the ERA8 phenotype. Moreover, MKK3 was a known ABA signaling gene in the 70-gene 4.6 Mb ERA8 interval. None of these 70 genes showed the differential regulation in wild-type Zak versus ERA8 expected of a promoter mutation. Thus, the working model is that the ERA8 phenotype results from the MKK3-A-G1093A mutation. Key MessageUsing bulked segregant analysis of exome sequence, we fine-mapped the ABA hypersensitive mutant ERA8 in a wheat backcross population to the TaMKK3-A locus of chromosome 4A.

Development of sorghum mutant genotypes and analysis of their agro-morphological diversity

“…Finally, our research allowed the identi cation of new promising mutant genotypes that could be tested in multi-local trials to assess their agronomic e ciency. recent developments in high-thrsoughput next-generation sequencing (Irshad et al, 2020;Martinez et al, 2020).Ethiopian sorghum has been used in a variety of traditional and molecular studies for various purposes (Belay and Wale, 2021;Tirfessa et al, 2020). The majority of this research, however, concentrated on grain sorghums or their wild relatives, and there is an extensive study of sweet sorghum germplasm selection and genotypes from Ethiopia.…”

“…Finally, our research allowed the identi cation of new promising mutant genotypes that could be tested in multi-local trials to assess their agronomic e ciency. recent developments in high-thrsoughput next-generation sequencing (Irshad et al, 2020;Martinez et al, 2020).…”