Sep 24, 2013

Identification of EMS-induced causal mutations in Arabidopsis thaliana by next-generation sequencing

Methods in Molecular Biology
Naoyuki UchidaTetsuya Kurata

Abstract

Emerging next-generation sequencing (NGS) technologies are powerful tools for the identification of causal mutations underlying phenotypes of interest in Arabidopsis thaliana. Based on a methodology termed bulked segregant analysis (BSA), whole-genome sequencing data are derived from pooled F2 segregants after crossing a mutant to a different polymorphic accession and are analyzed for single nucleotide polymorphisms (SNPs). Then, a genome region spanning the causal mutation site is narrowed down by linkage analysis of SNPs in the accessions used to produce the F1 generation. Next, candidate SNPs for the causative mutation are extracted by filtering the linked SNPs using multiple appropriate criteria. Effects of each candidate SNP on the function of the corresponding gene are evaluated to identify the causal mutation, and its validity is then confirmed by independent criteria. This chapter describes the identification by NGS analysis of causal recessive mutations derived from EMS mutagenesis.

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Mentioned in this Paper

Genes, Plant
Candidate Gene Identification
Massively-Parallel Sequencing
Arabidopsis thaliana extract
Mutant
High-Throughput Nucleotide Sequencing
Ethyl Methanesulfonate
Mutagenesis Procedure
Genome Sequencing
Single Nucleotide Polymorphism

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