Simultaneous Identification of Multiple Causal Mutations in Rice

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 elite indica rice cultivar in China. All mutant alleles were validated by phenotype association assay.Guangdong Innovative Research Team Program [201001S0104725509]; Ministry of Agriculture Transgenic Project [2012ZX08001001]; National Program on Key Basic Research Project of China [973 Program] [2011CB100101, 2013CBA01402]; National High Technology Research and Development Program of China [863 Program] [2014AA10A602]; Natural Science Foundation of China [31110103917]; Shenzhen Commission on Innovation and Technology [KQF201109160004A, CXZZ20140411140647863]SCI(E)ARTICLE