Next-generation mapping of genetic mutations using bulk population sequencing.
Next-generation sequencing platforms have made it possible to very rapidly map genetic mutations in Arabidopsis using whole-genome resequencing against pooled members of an F2 mapping population. In the case of recessive mutations, all individuals expressing the phenotype will be homozygous for the mutant genome at the locus responsible for the phenotype, while all other loci segregate roughly equally for both parental lines due to recombination. Importantly, genomic regions flanking the recessive mutation will be in linkage disequilibrium and therefore also be homozygous due to genetic hitchhiking. This information can be exploited to quickly and effectively identify the causal mutation. To this end, sequence data generated from members of the pooled population exhibiting the mutant phenotype are first aligned to the reference genome. Polymorphisms between the mutant and mapping line are then identified and used to determine the homozygous, nonrecombinant region harboring the mutation. Polymorphisms in the identified region are filtered to provide a short list of markers potentially responsible for the phenotype of interest, which is followed by validation at the bench. Although the focus of recent studies has been on the mapping of point mutations exhibiting recessive phenotypes, the techniques employed can be extended to incorporate more complicated scenarios such as dominant mutations and those caused by insertions or deletions in genomic sequence. This chapter describes detailed procedures for performing next-generation mapping against an Arabidopsis mutant and discusses how different mutations might be approached.
Southern Crop Protection and Food Research Centre, Agriculture & Agri-Food Canada, London, ON, Canada.
Ryan S
RS
Austin
Steven P
SP
Chatfield
Darrell
D
Desveaux
David S
DS
Guttman
D017360:Arabidopsis / Q000235:genetics*; D002874:Chromosome Mapping / Q000379:methods*; D004252:DNA Mutational Analysis; D056726:Genetic Association Studies; D018745:Genome, Plant; D006239:Haplotypes; D059014:High-Throughput Nucleotide Sequencing; D010641:Phenotype; D016415:Sequence Alignment; D012984:Software