The whole-genome sequencing-based bulked segregant analysis (WGS-BSA) has facilitated the mapping candidate causal variations for cloning target plant genes. Here, we report an improved WGS-BSA method termed as M2-seq to expedite the mapping candidate mutant loci by studying just M-2 generation. It is an efficient mutant gene mapping tool, rapid, and comparable to the previously reported approaches, such as Mutmap and Mutmap+ that require studying M-3 or advanced selfed generations. In M2-seq, background variations among the M-2 populations can be removed efficiently without knowledge of the variations of the wild-type progenitor plant. Furthermore, the use of absolute delta single-nucleotide polymorphism (SNP) index values can effectively remove the background variation caused by repulsion phase linkages of adjacent mutant alleles; and thereby facilitating the identification of the causal mutation in target genes. Here, we demonstrated the application of M2-seq in successfully mapping the genomic regions harboring causal mutations for mutant phenotypes among 10 independent M-2 populations of soybean. The mapping candidate mutant genes just in M-2 generation with the aid of the M2-seq method should be particularly useful in expediting gene cloning especially among the plant species with long generation time.

The whole-genome sequencing-based bulked segregant analysis (WGS-BSA) has facilitated the mapping candidate causal variations for cloning target plant genes. Here, we report an improved WGS-BSA method termed as M2-seq to expedite the mapping candidate mutant loci by studying just M-2 generation. It is an efficient mutant gene mapping tool, rapid, and comparable to the previously reported approaches, such as Mutmap and Mutmap+ that require studying M-3 or advanced selfed generations. In M2-seq, background variations among the M-2 populations can be removed efficiently without knowledge of the variations of the wild-type progenitor plant. Furthermore, the use of absolute delta single-nucleotide polymorphism (SNP) index values can effectively remove the background variation caused by repulsion phase linkages of adjacent mutant alleles; and thereby facilitating the identification of the causal mutation in target genes. Here, we demonstrated the application of M2-seq in successfully mapping the genomic regions harboring causal mutations for mutant phenotypes among 10 independent M-2 populations of soybean. The mapping candidate mutant genes just in M-2 generation with the aid of the M2-seq method should be particularly useful in expediting gene cloning especially among the plant species with long generation time.