Identification of a Dominant Chlorosis Phenotype Through a Forward Screen of the Triticum turgidum cv. Kronos TILLING Population

Abstract

Durum wheat ( Triticum turgidum ) derives from a hybridization event approximately 400,000 years ago which led to the creation of an allotetraploid genome. The evolutionary recent origin of durum wheat means that its genome has not yet been fully diploidised. As a result, many of the genes present in the durum genome act in a redundant fashion, where loss-of-function mutations must be present in both gene copies to observe a phenotypic effect. Here, we use a novel set of induced variation within the cv. Kronos TILLING population to identify a locus controlling a dominant, environmentally dependent chlorosis phenotype. We carried out a forward screen of the sequenced cv. Kronos TILLING lines for senescence phenotypes and identified a line with a dominant early senescence and chlorosis phenotype. Mutant plants contained less chlorophyll throughout their development and displayed premature flag leaf senescence. A segregating population was classified into discrete phenotypic groups and subjected to bulked-segregant analysis using exome capture followed by next-generation sequencing. This allowed the identification of a single region on chromosome 3A, Yellow Early Senescence 1 ( YES-1 ), which was associated with the mutant phenotype. While this phenotype was consistent across 4 years of field trials in the United Kingdom, the mutant phenotype was not observed when grown in Davis, CA (United States). To obtain further SNPs for fine-mapping, we isolated chromosome 3A using flow sorting and sequenced the entire chromosome. By mapping these reads against both the cv. Chinese Spring reference sequence and the cv. Kronos assembly, we could identify high-quality, novel EMS-induced SNPs in non-coding regions within YES-1 that were previously missed in the exome capture data. This allowed us to fine-map YES-1 to 4.3 Mb, containing 59 genes. Our study shows that populations containing induced variation can be sources of novel dominant variation in polyploid crop species, highlighting their importance in future genetic screens. We also demonstrate the value of using cultivar-specific genome assemblies alongside the gold-standard reference genomes particularly when working with non-coding regions of the genome. Further fine-mapping of the YES-1 locus will be pursued to identify the causal SNP underpinning this dominant, environmentally dependent phenotype.

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

“…We combined these two approaches to map a mutation using bulked segregant analysis of exome sequence from pools of genomic DNA from WT-like and ERA8-like individuals from a BC 3 F 2:3 population. A similar approach was used in the tetraploid Kronos TILLING population to map tall versus short EMS-induced alleles from a segregating M 4 population and a leaf chlorosis phenotype in an F 2 population (Mo et al 2017;Harrington et al 2019). In both of these studies, the phenotypic differences between wild-type and mutant individuals were relatively large compared to the more difficult-tophenotype ERA8 mutation.…”

“…Exome capture was used to restrict genomic DNA sequencing to those regions encoding predicted genes (Krasileva et al 2017). Library preparation and sequencing was carried out at the Earlham Institute, UK as described by Harrington et al (2019). Separate barcoded libraries were prepared from the ERA8 parent (ERA8/ERA8), wild-type Zak parent (+/+), the bulked ERA8-like, and bulked Zak-like DNA with average insert sizes of 327, 345, 348, and 340 bp, respectively.…”

“…But such a population has the drawback that there may be multiple sequence polymorphisms within a candidate gene(s), making it more challenging to identify the mutation causing the phenotype of interest (Shorinola et al 2017). Future efforts to determine if the ERA8 phenotype is caused by the TaMKK3-A-G1093A allele could exploit additional polymorphisms identified by complete chromosome sequencing of the ZakERA8 mutant chromosome (as in Harrington et al 2019), or make use of a population where both parents carry the PHS susceptible TaMKK3-A-A660 S allele, such as a population derived from Otis/ZakERA8.…”

“…Exome capture has enabled affinity-purification of the predicted wheat coding regions before performing whole genome DNA resequencing (Allen et al 2013;Henry et al 2014;Uauy et al 2017;Krasileva et al 2017;Mo et al 2017). This approach has been used to characterize the genome-sequence of EMS-mutagenized wheat TILLING populations (Harrington et al 2019). Since this method was effective in identifying EMS-induced mutations responsible for a phenotype, this study used exome capture in a BSA-exome sequencing approach to map EMS-induced mutations linked to the ERA8 allele.…”

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

“…Exome capture has enabled affinity-purification of the predicted wheat coding regions before performing whole genome DNA resequencing (Allen et al 2013;Henry et al 2014;Uauy et al 2017;Krasileva et al 2017;Mo et al 2017). This approach has been used to characterize the genome sequence of EMSmutagenized tetraploid wheat TILLING populations (Harrington et al 2019). Since this method was effective in identifying EMS-induced mutations responsible for a phenotype, this study used exome capture in a BSA-exome sequencing approach to map EMS-induced mutations linked to the ERA8 allele.…”