title: Simultaneous Identification of Multiple Causal Mutations in Rice.
abstract: Next-generation sequencing technologies (NGST) are being used to discover causal mutations in ethyl methanesulfonate (EMS)-mutagenized plant populations. However, the published protocols often deliver too many candidate sites and sometimes fail to find the mutant gene of interest. Accurate identification of the causal mutation from massive background polymorphisms and sequencing deficiencies remains challenging. Here we describe a NGST-based method, named SIMM, that can simultaneously identify the causal mutations in multiple independent mutants. Multiple rice mutants derived from the same parental line were back-crossed, and for each mutant, the derived F2 individuals of the recessive mutant phenotype were pooled and sequenced. The resulting sequences were aligned to the Nipponbare reference genome, and single nucleotide polymorphisms (SNPs) were subsequently compared among the mutants. Allele index (AI) and Euclidean distance (ED) were incorporated into the analysis to reduce noises caused by background polymorphisms and re-sequencing errors. Corrections of sequence bias against GC- and AT-rich sequences in the candidate region were conducted when necessary. Using this method, we successfully identified seven new mutant alleles from Huanghuazhan (HHZ), an eliteindicarice cultivar in China. All mutant alleles were validated by phenotype association assay. A pipeline based on Perl scripts for SIMM is publicly available at https://sourceforge.net/projects/simm/.
authors.affiliation: College of Life Sciences, Capital Normal University Beijing, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Yan
authors.affiliation: Shenzhen Institute of Molecular Crop Design Shenzhen, China.
authors.forename: Zhufeng
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Shenzhen Institute of Molecular Crop Design Shenzhen, China.
authors.forename: Jiawei
authors.initials: J
authors.lastname: Lu
authors.affiliation: Shenzhen Institute of Molecular Crop Design Shenzhen, China.
authors.forename: Chunjue
authors.initials: C
authors.lastname: Xu
authors.affiliation: Shenzhen Institute of Molecular Crop Design Shenzhen, China.
authors.forename: Gang
authors.initials: G
authors.lastname: Xie
authors.affiliation: Shenzhen Institute of Molecular Crop Design Shenzhen, China.
authors.forename: Yiqi
authors.initials: Y
authors.lastname: Li
authors.affiliation: Shenzhen Institute of Molecular Crop DesignShenzhen, China; College of Life Sciences, Peking UniversityBeijing, China.
authors.forename: Xing Wang
authors.initials: XW
authors.lastname: Deng
authors.affiliation: College of Life Sciences, Peking University Beijing, China.
authors.forename: Hang
authors.initials: H
authors.lastname: He
authors.affiliation: Shenzhen Institute of Molecular Crop DesignShenzhen, China; Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, College of Life Sciences, South China Normal UniversityGuangzhou, China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Tang
keywords: Euclidean distance; SIMM; SNP index; allele index; next-generation sequencing technology; sequence correction; single nucleotide polymorphism