Mapping of a Pale Green Mutant Gene and Its Functional Verification by Allelic Mutations in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Abstract

Leaves are the main organ for photosynthesis, and variations in leaf color affect photosynthesis and plant biomass formation. We created two similar whole-plant pale green mutants (<i>pem1</i> and <i>pem2</i>) from the double haploid (DH) Chinese cabbage line "FT" through ethyl methanesulfonate (EMS) mutagenesis of seeds. Photosynthetic pigment contents and net photosynthetic rates were significantly lower in the mutants than in the wild-type "FT," and the chloroplast thylakoid endomembrane system was poor. Genetic analysis showed that the mutated phenotypes of <i>pem1</i> and <i>pem2</i> were caused by a single nuclear gene. Allelism tests showed that <i>pem1</i> and <i>pem2</i> were alleles. We mapped <i>Brpem1</i> to a 64.25 kb region on chromosome A10, using BSR-Seq and map-based cloning of 979 F₂ recessive individuals. Whole-genome re-sequencing revealed a single nucleotide polymorphism (SNP) transition from guanine to adenosine on <i>BraA10g021490.3C</i> in <i>pem1</i>, causing an amino acid shift from glycine to glutamic acid (G to E); in addition, <i>BraA10g021490.3C</i> in <i>pem2</i> was found to have a single nucleotide substitution from guanine to adenosine, causing an amino acid change from E to lysine (K). <i>BraA10g021490.3C</i> is a homolog of the <i>Arabidopsisdivinyl chlorophyllide a 8-vinyl-reductase</i> (<i>DVR</i>) gene that encodes 3,8-divinyl protochlorophyllide a 8-vinyl reductase, which is a key enzyme in chlorophyll biosynthesis. Enzyme activity assay and chlorophyll composition analysis demonstrated that impaired DVR had partial loss of function. These results provide a basis to understand chlorophyll metabolism and explore the mechanism of a pale green phenotype in Chinese cabbage.