Abstract
BackgroundAnalysis of mutants isolated from forward-genetic screens has revealed key components of several plant signalling pathways. Mapping mutations by position, either using classical methods or whole genome high-throughput sequencing (HTS), largely relies on the analysis of genome-wide polymorphisms in F2 recombinant populations. Combining bulk segregant analysis with HTS has accelerated the identification of causative mutations and has been widely adopted in many research programmes. A major advantage of HTS is the ability to perform bulk segregant analysis after back-crossing to the parental line rather than out-crossing to a polymorphic ecotype, which reduces genetic complexity and avoids issues with phenotype penetrance in different ecotypes. Plotting the positions of homozygous polymorphisms in a mutant genome identifies areas of low recombination and is an effective way to detect molecular linkage to a phenotype of interest.ResultsWe describe the use of single nucleotide polymorphism (SNP) density plots as a mapping strategy to identify and refine chromosomal positions of causative mutations from screened plant populations. We developed a web application called CandiSNP that generates density plots from user-provided SNP data obtained from HTS. Candidate causative mutations, defined as SNPs causing non-synonymous changes in annotated coding regions are highlighted on the plots and listed in a table. We use data generated from a recent mutant screen in the model plant Arabidopsis thaliana as proof-of-concept for the validity of our tool.ConclusionsCandiSNP is a user-friendly application that will aid in novel discoveries from forward-genetic mutant screens. It is particularly useful for analysing HTS data from bulked back-crossed mutants, which contain fewer polymorphisms than data generated from out-crosses. The web-application is freely available online at http://candisnp.tsl.ac.uk.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-014-0041-7) contains supplementary material, which is available to authorized users.
Highlights
Analysis of mutants isolated from forward-genetic screens has revealed key components of several plant signalling pathways
We demonstrate the utility of CandiSNP by analysing sequence data generated from two allelic mob mutants, bak1-5 mob1 and bak1-5 mob2, which are caused by mutations in the gene encoding the calcium-dependent protein kinase CALCIUM-DEPENDENT PROTEIN KINASE 28 (CPK28) [13]
Case study Bulk segregant analysis of two mob mutants using Illumina sequencing identifies thousands of polymorphisms As a case study for CandiSNP we examined high-throughput sequencing (HTS) data obtained for two allelic recessive mutants, bak1-5 mob1 and bak1-5 mob2, that were isolated from the modifier of bak1-5 screen [13]
Summary
Analysis of mutants isolated from forward-genetic screens has revealed key components of several plant signalling pathways. Either using classical methods or whole genome high-throughput sequencing (HTS), largely relies on the analysis of genome-wide polymorphisms in F2 recombinant populations. Combining bulk segregant analysis with HTS has accelerated the identification of causative mutations and has been widely adopted in many research programmes. A major advantage of HTS is the ability to perform bulk segregant analysis after back-crossing to the parental line rather than out-crossing to a polymorphic ecotype, which reduces genetic complexity and avoids issues with phenotype penetrance in different ecotypes. Plotting the positions of homozygous polymorphisms in a mutant genome identifies areas of low recombination and is an effective way to detect molecular linkage to a phenotype of interest. Mutations are identified by sequence analysis and the causative mutation is usually confirmed by complementation with a non-mutated (wild-type) copy of the gene
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
