Bulked Segregant - Genotyping-By-Sequencing: Cost-Effective and Background Independent Genetic Mapping of Mutants and QTL

Genetic mapping of new mutants, which allows us to map a mutant phenotype to a causal locus or loci in the genome, is a crucial step in forward genetics. Construction of a mapping population that consists of mutant and normal individuals is essential for genetic mapping. The mapping population can be used by different high-throughput methods for genetic mapping. Single Nucleotide Polymorphism (SNP) arrays and Sequenome-based methods detect presence and absence of pre-discovered SNPs, and therefore are not background independent. In contrast, high-throughput sequencing (HTS) based methods used for genetic mapping are generally background independent. Some HTS methods such as Genotyping-by-sequencing (GBS) and RAD-seq use DNA for mapping, while other methods such as BSR-seq and MMAPPR use RNA. Current DNA-based methods barcode DNA extracted from each individual in the mapping population to construct the sequencing library, and RNA-based methods construct a separate library from each of two pools, namely mutant and normal. Both approaches provide high resolution maps to identify causal loci, but are not cost-effective for screening a large number of mutant families such as may be recovered from an enhancer/suppressor screen. Here we present a low-resolution, but cost-effective, HTS-based method for genetic mapping. For each new mutant we pooled tissue from phenotyped individuals to create a mutant pool and a normal pool. We adapted the original GBS method to construct sequencing libraries, prepared libraries for several pairs of pools and determined rough map positions. Our method is cheaper than the current GBS protocol, easier than using RNA for library construction, and without sampling biases inherent in using RNA expressed in a certain tissue type(s). We are currently fine mapping the intervals identified by BS-GBS, and extending the method to map natural modifiers. Here we present the pipeline and results from these genetic mapping efforts in maize.