Abstract
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.
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Acknowledgments
The authors thank Peter McCourt, Nicholas J. Provart, Pauline W. Wang, Danielle Vidaurre, George Stamatiou, Robert Breit, Dario Bonetta, Jianfeng Zhang, Pauline Fung, and Yunchen Gong for their help in the development of NGM. We would also like to express our gratitude to the McCourt and Desveaux Labs (University of Toronto), Haughan Lab (University of British Columbia), and Bonnetta Lab (University of Ontario Institute of Technology) for their provision of sequence data. This work was funded through grants by the Natural Sciences and Engineering Research Council of Canada to D.S. Guttman and D. Desveaux.
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Austin, R.S., Chatfield, S.P., Desveaux, D., Guttman, D.S. (2014). Next-Generation Mapping of Genetic Mutations Using Bulk Population Sequencing. In: Sanchez-Serrano, J., Salinas, J. (eds) Arabidopsis Protocols. Methods in Molecular Biology, vol 1062. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-580-4_17
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DOI: https://doi.org/10.1007/978-1-62703-580-4_17
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