Abstract
Forward genetic screens have identified numerous genes involved in development and metabolism, and remain a cornerstone of biological research. However, to locate a causal mutation, the practice of crossing to a polymorphic background to generate a mapping population can be problematic if the mutant phenotype is difficult to recognize in the hybrid F2 progeny, or dependent on parental specific traits. Here in a screen for leaf hyponasty mutants, we have performed a single backcross of an Ethane Methyl Sulphonate (EMS) generated hyponastic mutant to its parent. Whole genome deep sequencing of a bulked homozygous F2 population and analysis via the Next Generation EMS mutation mapping pipeline (NGM) unambiguously determined the causal mutation to be a single nucleotide polymorphisim (SNP) residing in HASTY, a previously characterized gene involved in microRNA biogenesis. We have evaluated the feasibility of this backcross approach using three additional SNP mapping pipelines; SHOREmap, the GATK pipeline, and the samtools pipeline. Although there was variance in the identification of EMS SNPs, all returned the same outcome in clearly identifying the causal mutation in HASTY. The simplicity of performing a single parental backcross and genome sequencing a small pool of segregating mutants has great promise for identifying mutations that may be difficult to map using conventional approaches.
Highlights
A forward genetic approach has been the foundation of determining gene function for many decades
We demonstrate that the parental backcross method, combined with the Next Generation EMS mutation mapping pipeline (NGM) pipeline that was originally designed for the outcross method, can be utilized to unambiguously map an Arabidopsis mutant from a small pool of F2s with relatively low sequence coverage and without resorting to successive rounds of deep sequencing
We have identified an Ethane Methyl Sulphonate (EMS) generated mutant using solely the position and frequency of EMS single nucleotide polymorphisim (SNP) determined by whole genome sequencing
Summary
A forward genetic approach has been the foundation of determining gene function for many decades. Map-based cloning has historically been a labor intensive and cumbersome process, often involving outcrossing of the mutant to a polymorphic line followed by fine mapping using a pool of potentially thousands of individual F2 plants carrying the mutation (Jander et al, 2002). Recent advances in deep sequencing have revolutionised the identification of causal mutations underlying a particular mutant phenotype. An underlying tenet relies on evaluating the frequency and position of mutation induced single nucleotide polymorphisms (SNPs) in the pool of mutant F2 individuals. The recessive causal mutation will be always homozygous, whereas the homozygosity of linked SNPs will decrease with distance from the causal mutation. The position of these linked SNPs, and the measure of their homozygosity/allelic frequency can be used as markers to identify the causal mutation with strong likelihood
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