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Research Project: Genetic and Physiological Mechanisms Underlying Complex Agronomic Traits in Grain Crops

Location: Plant Genetics Research

Title: Mapping maize mutants using bulked-segregant analysis and next-generation sequencing

item Best, Norman
item MCSTEEN, PAULA - University Of Missouri

Submitted to: Current Protocols in Plant Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/3/2022
Publication Date: 11/9/2022
Citation: Best, N.B., McSteen, P. 2022. Mapping maize mutants using bulked-segregant analysis and next-generation sequencing. Current Protocols in Plant Biology. 2(11). Article e591.

Interpretive Summary: Forward genetics is a molecular biology technique to associate gene function with a trait of interest. Mutant screens are highly effective to identify novel molecular mechanisms that control plant growth and development. To associate the trait of interest with a genetic locus, linkage mapping is necessary. Classical approaches, especially in maize, are very time intensive, cost a lot of money for reagents, and require large populations to associate a genomic region with a particular trait. The development of new next-generation sequencing technologies, and reductions in price, has opened the door to develop new mapping approaches that are highly efficient and cost-effective. This manuscript describes a method that combines bulked-segregant analysis, pooling mutant plants together, with next-generation sequencing analysis. The mutant pool is sequenced, and mutations are identified that associate with the trait of interest to identify the molecular mechanisms responsible for the phenotype. This manuscript provides a step-by-step approach, including experimental design, DNA prep, bioinformatic analysis, and interpretation of expected results. Furthermore, this manuscript is also the first presentation of a new novel allele of the barren stalk1 gene. This allele is a weak mutation that will allow for better genetic interaction studies in the future, as the phenotype can be both enhanced and suppressed.

Technical Abstract: 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.