Abstract
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 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 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.
Highlights
Recent developments in next-generation sequencing (NGS) technologies are revolutionizing various aspects of genomics
Because it relies on crosses to the parental line and eliminates the need for wide-crosses to genetically unrelated lines, MutMap is useful for identifying genes that determine quantitative minor effect phenotypes, which is a major challenge in crop improvement [7]
Phenotypes of dominant or semi-dominant mutations could be observed at this generation, M2 seeds obtained from selfing of M1 plants were advanced to M2 generation as our focus was on recessive mutations
Summary
Recent developments in next-generation sequencing (NGS) technologies are revolutionizing various aspects of genomics. Once whole genome draft sequences become available, resequencing of multiple individuals of the same species allows rapid identification of genomic variations, contributing to genetic analyses of medical conditions in human as well as important traits in crops, animals and microbes [328]. To exploit genome sequencing in crop breeding, we recently developed a method called MutMap and applied it to the identification of rice genes responsible for agronomically important traits [7]. The mutant lines show wide variations of phenotypes in agronomically important traits like plant height, grain number and disease tolerance. Because it relies on crosses to the parental line and eliminates the need for wide-crosses to genetically unrelated lines, MutMap is useful for identifying genes that determine quantitative minor effect phenotypes, which is a major challenge in crop improvement [7]
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