Mapping A Mutant Gene In Arabidopsis Using Deep Sequencing

Gene mapping is an important content of genetic research. The technology of next generation sequencing (or named deep sequencing) has the advantage of high throughput and low cost, and has become an efficient tool for genetic and genomic studies. In this study, according to the principle of bulked segregant analysis (BS A), the method of combining deep sequencing and BSA for gene mapping was investigated, and the feasibility and efficiency of the method was validated with a practical example in Arabidopsis.Data source:An F2population was created from a cross between a single-gene mutant and wild type of Arabidopsis. A mutant and a wild-type DNA pools were established, respectively. The two pools were sequenced using the Solexa system, generating a set of sequence data consisting of reads of76bp in length with an average depth of13x.Methods for data analysis:With the known genome sequence of Arabidopsis as reference, the sequence data filtering and matching and SNP detection were performed using the software SOAPaligner and SOAPsnp. Using the SNPs as markers, the allelic frequency difference (d) between the two pools within a certain window (genome segment) was estimated, and the window moved with a given step length along the genome to obtain a profile of d value changing along the genome. The threshold corresponding to an overall significance level of0.05was estimated using the method of permutation tests. The d value peaks higher than the threshold are likely to be the position of the mutation site.Main results:(1) A total of29,264,012reads were generated from the two DNA pools, and27,215,532reads were left after data filtering.83.35%of these filtered reads could be matched to the reference genome, of which84.55%and15.45%were unique matching and multiple matching, respectively.(2) A total of292,725SNP sites were identified. After further removing the SNP sites with depth≤2or≥50,285,671SNP sites were finally obtained, with an average density of1.6/kb in the genome.(3) By scanning the whole genome with a10-kb step length and four window sizes (100kb,200kb,300kb and400kb, the mutation site (target gene) was mapped in an interval of2853000-2898000on the end of chromosome1.This study demonstrated the feasibility and efficiency of combining deep sequencing and BSA for gene mapping. An obvious advantage of this approach is that it can directly map the target gene onto the physical map. With the further development of deep sequencing technology and decrease of its cost, the approach will be extensively utilized in gene mapping.