Mapping Maize Mutants Using Bulked-Segregant Analysis and Next-Generation Sequencing

Forward genetics is used to identify the genetic basis for a phenotype. The approach involves identifying a mutant organism exhibiting a phenotype of interest and then mapping the causative locus or gene. Bulked-segregant analysis (BSA) is a quick and effective approach to map mutants using pools of mutants and wild-type plants from a segregating population to identify linkage of the mutant phenotype, and this approach has been successfully used in plants. Traditional linkage mapping approaches are outdated and time intensive, and can be very difficult. With the highly evolved development and reduction in cost of high-throughput sequencing, this new approach combined with BSA has become extremely effective in multiple plant species, including Zea mays (maize). While the approach is incredibly powerful, careful experimental design, bioinformatic mapping techniques, and interpretation of results are important to obtain the desired results in an effective and timely manner. Poor design of a mapping population, limitations in bioinformatic experience, and inadequate understanding of sequence data are limitations of these approaches for the researcher. Here, we describe a straightforward protocol for mapping mutations responsible for a phenotype of interest in maize, using high-throughput sequencing and BSA. Specifically, we discuss relevant aspects of developing a mutant mapping population. This is followed by a detailed protocol for DNA preparation and analysis of short-read sequences to map and identify candidate causative mutations responsible for the mutant phenotype of interest. We provide command-line and perl scripts to complete the bioinformatic analysis of the mutant sequence data. This protocol lays out the design of the BSA, bioinformatic approaches, and interpreting the sequencing data. These methods are very adaptable to any forward genetics experiment and provide a step-by-step approach to identifying the genetic basis of a maize mutant phenotype. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Bulked-segregant analysis and high-throughput sequencing to map maize mutants.