Facile mutant identification via a single parental backcross method and application of whole genome sequencing based mapping pipelines
Allen, Robert S., Nakasugi, Kenlee, Doran, Rachel L., Millar, Anthony A., & Waterhouse, Peter M. (2013) Facile mutant identification via a single parental backcross method and application of whole genome sequencing based mapping pipelines. Frontiers in Plant Science, 4.
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.
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|Item Type:||Journal Article|
|Keywords:||Arabidopsis, mutant-mapping, NextGen-Sequencing, parental-backcross, EMS, mutant-screen|
|Subjects:||Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000)
Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000) > GENETICS (060400)
|Divisions:||Current > Schools > School of Earth, Environmental & Biological Sciences
Current > QUT Faculties and Divisions > Science & Engineering Faculty
|Copyright Owner:||Copyright © 2013 Allen, Nakasugi, Doran, Millar, and Waterhouse.|
|Copyright Statement:||This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.|
|Deposited On:||08 Jan 2014 02:16|
|Last Modified:||09 Jan 2014 03:08|
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