MutMap+: Genetic Mapping and Mutant Identification without Crossing in Rice

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.

Development and utilization of a new chemically‐induced soybean library with a high mutation density

“…These approaches include building reduced representation libraries (RRL) (Hyten et al ; Varala et al ; Sun et al ), utilizing restriction site‐associated DNA (RAD) (Baird et al ) sequencing, genotyping by sequencing (GBS) (Elshire et al ; Sonah et al ; Liu et al ), exome sequencing (Mascher et al , ) or reducing sequence depth (Huang et al ; Xie et al ). With the decreased cost of DNA sequencing technologies, whole‐genome deep re‐sequencing based on NGS, including sequencing pooled DNA (Schneeberger et al ; Austin et al ; Abe et al ; Leshchiner et al ; Fekih et al ), resequencing different individuals or accessions (Lam et al ; Xu et al ; Nordström et al ; Zhou et al ) and transcriptome sequencing (Trick et al ; Islam et al ) have been used to greatly accelerate the identification of mutagen‐ induced or naturally occurring mutations. An increasing number of successful examples have demonstrated the feasibility of the method by identification of EMS‐induced, causal mutations in Arabidopsis (Ashelford et al ; Hartwig et al ; Leshchiner et al ), rice (Abe et al ), soybean (Zhou et al ), barley (Mascher et al ) and other species.…”

Mutant Resources and Mutagenomics in crop plants

“…TILLING ensures detection of mutations within genes of interest and to associate such mutations to a definite phenotype, for which gene(s) linked to the phenotype and the gene sequence is known (Sikora et al, 2011). MutMap is a recent development for cloning EMS-induced alleles in rice using a bulked segregation strategy, and the method is further extended to enable alleles to be cloned without outcrossing (Abe et al, 2012;Fekih et al, 2013). Wilde et al (2012) presented an overview of screening in horticultural crops for natural or induced allelic diversity in over 100 candidate genes for traits of commercial interest, such as longer shelf-life (tomato, melon), improved starch quality (potato), and virus-resistance (peppers, tomato).…”

Mutagenesis for Crop Breeding and Functional Genomics

“…With smaller genome plants, it is possible to sequence whole genomes and clone genes by associating co-segregation of genotype to phenotype (Schneeberger et al 2009;Cuperus et al 2010). An approach known as MutMap has been described for cloning EMS-induced alleles in rice using a bulked segregant strategy, and the method further adapted so that alleles can be cloned without outcrossing (Abe et al 2012;Fekih et al 2013). This is much more challenging in larger genome crops due to throughput and cost limitations of whole-genome sequencing.…”