Multiplex exome sequencing reveals genome-wide frequency and distribution of mutations in the 'Micro-Tom' Targeting Induced Local Lesions in Genomes (TILLING) mutant library.

While the 'Micro-Tom' TILLING mutant library is used for a wide range of purposes, including both basic research of gene function and breeding of commercial cultivars, genome-wide distribution and frequency of mutations have not yet been thoroughly elucidated on a population scale. In this study, we developed a 96-plex exome sequencing method to identify and analyze mutations within the TILLING mutants that were developed in the University of Tsukuba. First, an Illumina paired-end sequencing coupled with 96-plex exome capture resulted in the acquisition of an exome sequence dataset with an average read count of 5.6 million for the 95 mutants. Over 98% of the capture target region could be covered by the short reads with an averaged read depth of 12.8, which enabled us to identify single nucleotide polymorphisms and Indels in a genome-wide manner. By subtracting intra-cultivar DNA variations that are present between wild-type 'Micro-Tom' lines, we identified 241,391 mutation candidates in 95 mutant individuals. Of these, 64,319 and 6,480 mutations were expected to cause protein amino acid substitutions or premature stop codon, respectively. Based on the exome mutation dataset, a mutant line designated 'TOMJPW601' was found to carry a premature stop codon mutation (W261*) in a putative auxin influx carrier gene (), consistent with our previous report of its curly leaf phenotype. Our results suggested that a population-scale mutation database developed by multiplexed exome sequencing could be used for mutant screening, which in turn could contribute to both gene function research and breeding programs.