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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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.
Uchida N et al (2011) Identification of EMS-induced causal mutations in a non-reference Arabidopsis thaliana accession by whole genome sequencing. Plant Cell Physiol 52:716–722PubMedCrossRefGoogle Scholar
Schneeberger K et al (2009) SHOREmap: simultaneous mapping and mutation identification by deep sequencing. Nat Methods 6:550–551PubMedCrossRefGoogle Scholar
Mokry M et al (2011) Identification of factors required for meristem function in Arabidopsis using a novel next generation sequencing fast forward genetics approach. BMC Genomics 12:256PubMedCrossRefGoogle Scholar
Marti L et al (2010) A missense mutation in the vacuolar protein GOLD36 causes organizational defects in the ER and aberrant protein trafficking in the plant secretory pathway. Plant J 63:901–913PubMedCrossRefGoogle Scholar
Laitinen RA et al (2010) Identification of a spontaneous frame shift mutation in a nonreference Arabidopsis accession using whole genome sequencing. Plant Physiol 153:652–654PubMedCrossRefGoogle Scholar
Cuperus JT et al (2010) Identification of MIR390a precursor processing-defective mutants in Arabidopsis by direct genome sequencing. Proc Natl Acad Sci USA 107:466–471PubMedCrossRefGoogle Scholar
Michelmore RW, Paran I, Kesseli RV (1991) Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proc Natl Acad Sci U S A 88:9828–9832PubMedCrossRefGoogle Scholar
Greene EA et al (2003) Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis. Genetics 164:731–740PubMedGoogle Scholar