Gene Mapping via Bulked Segregant RNA-Seq (BSR-Seq)

• Published in: PloS one Vol. 7; no. 5; p. e36406
• Main Authors: Liu, Sanzhen, Yeh, Cheng-Ting, Tang, Ho Man, Nettleton, Dan, Schnable, Patrick S.
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 07.05.2012; Public Library of Science (PLoS)
• Subjects: Alleles; Analysis; Apoptosis; Arabidopsis; Arabidopsis thaliana; Bayes Theorem; Bayesian analysis; Binding sites; Bioinformatics; Biology; Biosynthesis; Chromosome Mapping; Cloning; Cloning, Molecular; Corn; Deoxyribonucleic acid; DNA; DNA methylation; Empirical analysis; Epigenetics; Fatty acids; Fatty Acids - biosynthesis; Fatty Acids - genetics; Gene expression; Gene Expression Profiling; Gene mapping; Gene mutation; Genes; Genetic aspects; Genetic markers; Genomes; Genomics; Genotype & phenotype; GL3 gene; High-Throughput Nucleotide Sequencing; Identification; Loci; Mapping; Markers; Mutants; Mutation; Oryza; Plant Proteins - genetics; Polymorphism, Single Nucleotide; Ribonucleic acid; RNA; RNA sequencing; Seeds - genetics; Single-nucleotide polymorphism; Transcription (Genetics); Transcription factors; Transcription Factors - genetics; Transposons; Trends; Zea mays - genetics; United States > US; Iowa
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0036406

Bulked segregant analysis (BSA) is an efficient method to rapidly and efficiently map genes responsible for mutant phenotypes. BSA requires access to quantitative genetic markers that are polymorphic in the mapping population. We have developed a modification of BSA (BSR-Seq) that makes use of RNA-Seq reads to efficiently map genes even in populations for which no polymorphic markers have been previously identified. 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 by analyzing the same RNA-Seq data using an empirical Bayesian approach. 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 BSR-Seq was used to clone the glossy3 (gl3) gene of maize. Mutants of the glossy loci exhibit altered accumulation of epicuticular waxes on juvenile leaves. By subjecting the reference allele of gl3 to BSR-Seq, we were able to map the gl3 locus to an ≈ 2 Mb interval. The single gene located in the ≈ 2 Mb mapping interval whose expression was down-regulated in the mutant pool was subsequently demonstrated to be the gl3 gene via the analysis of multiple independent transposon 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 biosynthesis of very-long-chain fatty acids.