Gene Cloning via an RNA-seq Based Bulked Segregant Analysis: BSArSeq

Bulked segregant analysis (BSA) is an efficient method to rapidly and efficiently map genes responsible for mutant phenotypes.  This procedure, however, requires access to quantitative markers that are polymorphic in the mapping population. We have developed a modification of BSA (BSArSeq) that makes use of RNA-Seq reads to efficiently map genes from  populations for which no prior markers are available. Because of the digital nature of next-generation sequencing (NGS) data, it is possible to conduct de novo SNP discovery and quantitatively genotype BSA samples using the same RNA-seq data. In addition, analysis of the RNA-seq data provides information on the effects of the mutant on global patterns of gene expression at no extra cost.  In combination these results greatly simplify gene cloning experiments. To demonstrate the utility of this strategy we used BSArSeq to clone and characterize the glossy3 (gl3) gene of maize. Mutants of the glossy loci exhibit altered accumulation of epicuticular waxes on juvenile leaves.  We previously generated a large collection of glossy mutants using the Mu transposon system.  By subjected a Mu-induced allele to BSArSeq, we were able to map the gl3 locus to a ~2.3Mb interval that is consistent with results of prior mapping experiments. The single gene located in the 2.3Mb mapping interval that contained a Mu insertion and whose expression was down-regulated in the mutant pool was subsequently demonstrated to be the gl3 gene via the analysis of multiple independently induced mutant alleles. The gl3 gene encodes a putative myb transcription factor, which directly or indirectly affects the expression of a number of genes involved in the very-long-chain fatty acid pathway.