Identification of a Pale Green Mutant pgm3 in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Chinese cabbage is one of the major vegetable crops in northern Asia. Its leaves are the major organ for photosynthesis and production, and leaf color directly influences its yield and quality. Here, we obtained a pale green mutant pgm3. This mutant line was derived from EMS mutagenesis of Chinese cabbage DH line FT. pgm3 exhibited chlorosis and etiolation, delayed growth, reduced photosynthetic pigment content and net photosynthetic rates, and impaired development of the chloroplast inner membrane system. Genetic analysis revealed that the pale green phenotype was controlled by a single recessive nuclear gene, Brpgm3. Mutmap analysis indicated that Brpgm3 is located on a 13.9 Mb region in A03. Within this region, a single SNP (A03: 7194530) with an SNP-index of 1, located in BraA03g015750.3C (BrClpC1), was identified from 40 differential SNPs. KASP genotyping demonstrated that the SNP co-segregated with the pale green phenotype in the F2 population. Sanger sequencing confirmed a G-to-A SNP in exon 4 of BrClpC1, which resulted in an amino acid substitution from S to G. Furthermore, multiple sequence alignment of homologs from 28 species demonstrated that this mutated residue is highly conserved. BrClpC1 was predominantly expressed in leaves and exhibited the highest transcript abundance among the nine members of the Class I Clp gene family in Brassica rapa. This is the first report identifying ClpC1 in Brassica crops. Our results not only confirmed BrClpC1 as a strong candidate gene for the pale green mutant of Chinese cabbage, but also highlighted BrClpC1 as a target for chloroplast biology research in Brassica crops.