Abstract
Map-based cloning (MBC) is the conventional approach for linking phenotypes to genotypes, and has been successfully used to identify causal mutations in diverse organisms. Next-generation sequencing (NGS) technologies offer unprecedented possibilities to sequence the entire genomes of organisms, thereby in principle enabling direct identification of causal mutations without mapping. However, although mapping-by-sequencing has proven to be a cost effective alternative to classical MBC in particular situations, methods based solely on NGS still have limitations and need to be refined. Aiming to identify the causal mutations in suppressors of Arabidopsis thaliana superroot2 phenotype, generated by ethyl methane sulfonate (EMS) treatment, we combined NGS and classical mapping, to rapidly identify the point mutations and restrict the number of testable candidates by defining the chromosomal intervals containing the causal mutations, respectively. The NGS-assisted mapping approach we describe here facilitates unbiased identification of virtually any causal EMS-generated mutation by overlapping the identification (deep sequencing) and validation (mapping) steps. To exemplify the useful marriage of the two approaches we discuss the strategy used to identify a new viable recessive allele of the Arabidopsis CULLIN1 gene in the non-reference Wassilewskija (Ws-4) accession.
Highlights
Map-based cloning (MBC) has been, and still is, widely used to identify genetic changes underlying mutant phenotypes in diverse organisms
A reference genome is needed as a scaffold in both approaches, a method based on comparing k-mers in whole-genome sequencing (WGS) datasets that eliminates the need for segregating populations and reference sequences has been recently described [16]
Mutant Isolation and Mapping Genetic and physiological studies have shown that adventitious root (AR) formation is a heritable quantitative genetic trait controlled by multiple endogenous and environmental factors
Summary
Map-based cloning (MBC) has been, and still is, widely used to identify genetic changes underlying mutant phenotypes in diverse organisms. Several methods for identifying ethyl methane sulfonate (EMS)-induced point mutations in Arabidopsis and various other organisms have been developed using whole genome (re) sequencing following advances and reductions in cost of generation sequencing (NGS) technologies. The included methods all combine bulk segregant analysis (pooling recombinant genomes) with whole-genome sequencing (WGS) [1,6,7,8,9,10] Their main advantage over classical MBC is that they allow simultaneous mapping and mutant identification, by analyzing NGS-generated data from a pool of recombinant F2 individuals and subtracting the putative causal mutations after comparing the sequences to a reference genome. A reference genome is needed as a scaffold in both approaches, a method based on comparing k-mers in WGS datasets that eliminates the need for segregating populations and reference sequences has been recently described [16]
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.
