Fast mapping of a chlorophyll b synthesis-deficiency gene in barley (Hordeum vulgare L.) via bulked-segregant analysis with reduced-representation sequencing

Bulked-segregant analysis coupled with next-generation sequencing (BSA-seq) has emerged as an efficient tool for genetic mapping of single genes or major quantitative trait loci controlling (agronomic) traits of interest. However, such a mapping-by-sequencing approach usually relies on deep sequencing and advanced statistical methods. Application of BSA-Seq based on construction of reduced-representation libraries and allele frequency analysis permitted anchoring the barley pale-green (pg) gene on chromosome 3HL. With further marker-assisted validation, pg was mapped to a 3.9 Mb physical-map interval. In the pg mutant a complete deletion of chlorophyllide a oxygenase (HvCAO) gene was identified. Because the product of this gene converts Chl a to Chl b, the pg mutant is deficient in Chl b. An independent Chl b-less mutant line M4437_2 carried a nonsynonymous substitution (F263L) in the C domain of HvCAO. The study demonstrates an optimized pooling strategy for fast mapping of agronomically important genes using a segregating population.