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Identification of EMS-Induced Causal Mutations in Arabidopsis thaliana by Next-Generation Sequencing

  • Naoyuki Uchida
  • Tomoaki Sakamoto
  • Masao Tasaka
  • Tetsuya Kurata
Part of the Methods in Molecular Biology book series (MIMB, volume 1062)

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.

Key words

Next-generation sequencing Whole-genome sequencing Ethyl methanesulfonate Bulked segregant analysis 

Notes

Acknowledgements

The authors thank Dr. Taku Ohshima and Mrs. Eiko Nakamoto (NAIST) for optimization of library preparation and GAIIx manipulation. We also thank Dr. Noriko Inada (NAIST) for the arrangement of the website to download our custom script.

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Naoyuki Uchida
    • 1
  • Tomoaki Sakamoto
    • 2
  • Masao Tasaka
    • 1
  • Tetsuya Kurata
    • 2
  1. 1.Graduate School of Biological SciencesNara Institute of Science and TechnologyIkomaJapan
  2. 2.Plant Global Education Project, Graduate School of Biological SciencesNara Institute of Science and TechnologyIkomaJapan

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