Targeted Identification of Rice Grain-Associated Gene Allelic Variation Through Mutation Induction, Targeted Sequencing, and Whole Genome Sequencing Combined with a Mixed-Samples Strategy

Abstract Background The mining of new allelic variation and the induction of new genetic variability are the basis for improving breeding efficiency. Results In this study, in total, 3872 heavy ion-irradiated M2 generation rice seeds and individual leaves were collected. The grain length was between 8 and 10.22 mm. The grain width was between 1.54 and 2.87 mm. The results showed that there was extensive variation in granulotype. The allelic variation in GS3 and GW5 was detected in 484 mixed samples (8:1) using targeted sequencing technology, and 12 mixed samples containing potential mutations and 15 SNPs were obtained; combined with Sanger sequencing and phenotype data, 13 key mutants and their corresponding SNPs were obtained; protein structural and functional analysis of key mutants screened out 6 allelic variants leading to altered grain shape, as well as the corresponding mutants, including long-grain mutants GS3-2 and GS3-7, short-grain mutants GS3-3 and GS3-5, wide-grain mutant GW5-1 and narrow-grain mutant GW5-4; whole genome sequencing identified new grain length gene allelic variants GS3-G1, GS3-G2 and GS3-G3. Conclusion Based on the above studies, we found 6 granulotype mutants and 9 granulotype-related allelic variants, which provided new functional gene loci and a material basis for molecular breeding and genotype mutation and phenotype analysis. We propose a method for targeted identification of allelic variation in rice grain type genes by combining targeted sequencing of mixed samples and whole genome sequencing. The method has the characteristics of low detection cost, short detection period, and flexible detection of traits and genes.