| High-throughput sequencing technology has become a powerful tool for genetic studies. Genes can be mapped quickly using the method of Bulked Segregation Analysis based on the high-throughput sequencing (abbreviated as BSA-seq), which has become more widely used. In plant, a mutant obtained from ethyl methane sulfonate (EMS) mutagenesis can be crossed with its wild-type to generate a F2 segregating population, or a heterozygous plant of the mutant locus of M2 generation can be selfed to produce a M3 segregating population. This is followed by BSA-seq, which helps in identifying and mapping the mutant gene quickly. This method is known as MutMap. Because BSA-seq requires sufficient single nucleotide polymorphism (SNP) loci in segregating populations, EMS mutagenesis efficiency (the number of SNPs generated by EMS mutagenesis) and the genetic diversity level (the number of SNPs remaining in the cultivar) are two key factors to determine whether the MutMap method can map the target genes. In this work, we used Nipponbare, a Japonica rice cultivar with available sequenced genome, and adopted high-throughput sequencing (Illumina HiSeq 2500) and bioinformatics analysis method to research EMS mutagenesis efficiency and genetic diversity level within the cultivar. The aim of this study was to provide a reference for the practical application of MutMap methods. The main results are as follows:1. Randomly taking a wild type Nipponbare plant for high-throughput sequencing(depth was about 50×), and comparing it with the Nipponbare genome reference sequence announced by International Rice Genome Sequencing Project (IRGSP), we found 19,209 single nucleotide differences genome-wide (the SNP differences between this plant and the IRGSP plant), most of which were located on chromosome 4,thesedifferences are mainly caused by the reference sequence of sequencing errors; besides, there are also 42,562 heterozygous SNPs in this plant, which were approximately evenly distributed in the genome.2. Experiments showed that the optimal conditions for Nipponbare EMS mutagenesis are as follows:at 23℃, water soaking 24 h,1% EMS Soaking 8 h, then washing seeds 2 h by rinsing water. After processing, the seed germination rate was 48%, close to the median lethal dose.3. Randomly selecting six M2 plants for high-throughput sequencing (depth was about 10×), we found that the number of SNPs generated by EMS mutagenesis in whole-genome varied from 360 to 2,705, with an average of 1071.4. Compared with the reference genome sequence, there are 17,566 single nucleotide differences between the 6 M2 plants and the IRGSP plant. The number was similar to that estimated in the wild-type plant. Furthermore, these SNPs were also mainly distributed in the 4th chromosome, these diffrences are mainly caused by the reference sequence of sequencing errors.5. The SNP number between the 6 M2 plants varied from 31,756 to 35,976, with an average of 34,488; the total number of SNPs among them was 37,182, close to the value estimated in the wild-type plant. These SNPs were also approximately evenly distributed in the genome.According to the results of the study, it can be seen that:there is about 1900 SNP between our laboratory Nipponbare material and the IRGSP Nipponbare material, but there are more than 35,000 SNPs within our lab Nipponbare material. The results indicate that SNPs within a rice cultivar is quite rich. In contrast, the number of SNPs generated by EMS mutagenesis is much smaller, with an average of only 1000 or so. Therefore, when apply the MutMap method to map genes, for better results, we should make full use of the SNPs exist within original cultivars. |
