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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.

SUBMITTER: Mo Y

PROVIDER: S-EPMC5854723 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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

Mo Youngjun Y Howell Tyson T Vasquez-Gross Hans H de Haro Luis Alejandro LA Dubcovsky Jorge J Pearce Stephen S

Molecular genetics and genomics : MGG 20171129 2

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 reg ...[more]

PMID: 29188438

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Project description:Wheat (Triticum ssp.) is an important food source for humans in many regions around the world. However, the ability to understand and modify gene function for crop improvement is hindered by the lack of available genomic resources. TILLING is a powerful reverse genetics approach that combines chemical mutagenesis with a high-throughput screen for mutations. Wheat is specially well-suited for TILLING due to the high mutation densities tolerated by polyploids, which allow for very efficient screens. Despite this, few TILLING populations are currently available. In addition, current TILLING screening protocols require high-throughput genotyping platforms, limiting their use.We developed mutant populations of pasta and common wheat and organized them for TILLING. To simplify and decrease costs, we developed a non-denaturing polyacrylamide gel set-up that uses ethidium bromide to detect fragments generated by crude celery juice extract digestion of heteroduplexes. This detection method had similar sensitivity as traditional LI-COR screens, suggesting that it represents a valid alternative. We developed genome-specific primers to circumvent the presence of multiple homoeologous copies of our target genes. Each mutant library was characterized by TILLING multiple genes, revealing high mutation densities in both the hexaploid (~1/38 kb) and tetraploid (~1/51 kb) populations for 50% GC targets. These mutation frequencies predict that screening 1,536 lines for an effective target region of 1.3 kb with 50% GC content will result in ~52 hexaploid and ~39 tetraploid mutant alleles. This implies a high probability of obtaining knock-out alleles (P = 0.91 for hexaploid, P = 0.84 for tetraploid), in addition to multiple missense mutations. In total, we identified over 275 novel alleles in eleven targeted gene/genome combinations in hexaploid and tetraploid wheat and have validated the presence of a subset of them in our seed stock.We have generated reverse genetics TILLING resources for pasta and bread wheat and achieved a high mutation density in both populations. We also developed a modified screening method that will lower barriers to adopt this promising technology. We hope that the use of this reverse genetics resource will enable more researchers to pursue wheat functional genomics and provide novel allelic diversity for wheat improvement.

Project description:Durum wheat (Triticum turgidum L.) is a cereal crop widely grown in the Mediterranean regions; the amber grain is mainly used for the production of pasta, couscous and typical breads. Single nucleotide polymorphism (SNP) detection technologies and high-throughput mutation induction represent a new challenge in wheat breeding to identify allelic variation in large populations. The TILLING strategy makes use of traditional chemical mutagenesis followed by screening for single base mismatches to identify novel mutant loci. Although TILLING has been combined to several sensitive pre-screening methods for SNP analysis, most rely on expensive equipment. Recently, a new low cost and time saving DHPLC protocol has been used in molecular human diagnostic to detect unknown mutations.In this work, we developed a new durum wheat TILLING population (cv. Marco Aurelio) using 0.70-0.85% ethyl methane sulfonate (EMS). To investigate the efficiency of the mutagenic treatments, a pilot screening was carried out on 1,140 mutant lines focusing on two target genes (Lycopene epsilon-cyclase, ?-LCY, and Lycopene beta-cyclase, ?-LCY) involved in carotenoid metabolism in wheat grains. We simplify the heteroduplex detection by two low cost methods: the enzymatic cleavage (CelI)/agarose gel technique and the denaturing high-performance liquid chromatography (DHPLC). The CelI/agarose gel approach allowed us to identify 31 mutations, whereas the DHPLC procedure detected a total of 46 mutations for both genes. All detected mutations were confirmed by direct sequencing. The estimated overall mutation frequency for the pilot assay by the DHPLC methodology resulted to be of 1/77 kb, representing a high probability to detect interesting mutations in the target genes.We demonstrated the applicability and efficiency of a new strategy for the detection of induced variability. We produced and characterized a new durum wheat TILLING population useful for a better understanding of key gene functions. The availability of this tool together with TILLING technique will expand the polymorphisms in candidate genes of agronomically important traits in wheat.

Dataset Information

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

Publications

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

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