Advances in genome sequencing technologies have enabled researchers and breeders to rapidly associate phenotypic variation to genome sequence differences.We recently took advantage of next-generation sequencing technology to develop MutMap, a method that allows rapid identification of causal nucleotide changes of rice ankle-and-wrist-cuffs mutants by whole genome resequencing of pooled DNA of mutant F2 progeny derived from crosses made between candidate mutants and the parental line.Here we describe MutMap+, a versatile extension of MutMap, that identifies causal mutations by comparing Soap SNP frequencies of bulked DNA of mutant and wild-type progeny of M3 generation derived from selfing of an M2 heterozygous individual.Notably, MutMap+ does not necessitate artificial crossing between mutants and the wild-type parental line.This method is therefore suitable for identifying mutations that cause early development lethality, sterility, or generally hamper crossing.
Furthermore, MutMap+ is potentially useful for gene isolation in crops that are recalcitrant to artificial crosses.