Mapping causal mutations by exome sequencing in a wheat TILLING population: a tall mutant case study

Abstract

Forward genetic screens of induced mutant plant populations are powerful tools to identify genes underlying phenotypes of interest. Using traditional techniques, mapping causative mutations from forward screens is a lengthy, multi-step process, requiring the identification of a broad genetic region followed by candidate gene sequencing to characterize the causal variant. Mapping by whole genome sequencing accelerates the identification of causal mutations by simultaneously defining a mapping region and providing information on the induced genetic variants. In wheat, although the availability of a high-quality draft genome assembly facilitates mapping and mutation calling, whole genome resequencing remains prohibitively expensive due to its large genome. In the current study, we used exome sequencing as a complexity reduction strategy to detect mutations associated with a target phenotype. In a segregating wheat EMS population, we identified a clear peak region on chromosome arm 4BS associated with increased plant height. Although none of the significant SNPs seemed causative for the mutant phenotype, they were sufficient to identify a linked ~ 1.9 Mb deletion encompassing nine genes. These genes included Rht-B1, which is known to have a strong effect on plant height and is a strong candidate for the observed phenotype. We performed simulation experiments to determine the impacts of sequencing depth and bulk size and discuss the importance of considering each factor when designing mapping-by-sequencing experiments in wheat. This approach can accelerate the identification of candidate causal point mutations or linked deletions underlying important phenotypes.Electronic supplementary materialThe online version of this article (10.1007/s00438-017-1401-6) contains supplementary material, which is available to authorized users.

Applying the latest advances in genomics and phenomics for trait discovery in polyploid wheat

“…Typically, for quantitative traits, phenotypes are clearer with successive backcross generations (Simmonds et al ., ). However, some qualitative traits can be studied directly in the initial mutant population (Mo et al ., ; Shorinola et al ., ). A caveat for the use of these populations is that not all genes will be present in the in silico database.…”

“…Instead, integrating known genetic mapping with new genomic approaches can accelerate trait discovery. Exome‐capture sequencing has been used to identify variation in the coding region of the wheat genome (Saintenac et al ., ) and can be used to identify candidate genes, often in combination with bulk‐segregant analysis (Gardiner et al ., ; Mo et al ., ). SNPs identified from the exome capture can be used to both develop new markers for genetic mapping and to identify mutations in putative candidate genes.…”

Mechanisms of powdery mildew resistance of wheat – a review of molecular breeding

“…To address this, different approaches have been developed to reduce such complexity in large‐genome species. One such approach is exome capture and sequencing, which greatly reduces sequencing volume and subsequently costs while giving good coverage of gene coding regions or sufficient mapping information of a genomic interval harbouring the causal gene (Mo et al , 2018). Exome capture assays yielded identification of gene candidates for plant height and resistance to leaf and yellow rust in wheat mutants (Hussain et al , 2018; Mo et al , 2018).…”

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

“…Genotyping-by-sequencing (GBS) entails restriction enzyme digests of genomic DNA to enrich for DNA fragments that begin with common restriction sites for high-throughput genome resequencing (Poland et al 2012;Truong et al 2012). Exome sequencing (exome-seq), on the other hand, enriches specifically for the genecontaining regions of the genome through affinity capture and purification of the predicted wheat coding regions for high throughput genome resequencing (Allen et al 2013;Henry et al 2014;Uauy et al 2017;Krasileva et al 2017;Mo et al 2017).…”

“…The RNA-seq approach has the drawback that relative gene expression levels vary in different tissues and times in development, leading to the possibility that the gene of interest might be underrepresented in the sampled tissue. 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).…”