Mapping and candidate gene identification defining BnChd1-1, a locus involved in chlorophyll biosynthesis in Brassica napus

The chlorophyll-deficient mutant (Bnchd1) is a spontaneous mutant of Brassica napus. Compared with the wild type, ‘Qingyou 10’, Bnchd1 exhibits distinct phenotypes, including interveinal yellowing leaves at the seedling stage and light-green leaves at the bolting stage, dwarfism throughout the lifespan, extremely low seed yields and abnormally shaped and early degradation of chloroplasts. Defective chloroplasts significantly reduce the levels of pigment in Bnchd1 at the seedling and bolting stages. Genetic analysis showed that two recessive genes, designated BnChd1-1 and BnChd1-2, are responsible for the light-green phenotype. BnChd1-1 was determined to be a single Mendelian factor in a BC2F1 population based on a phenotypic segregation ratio of 1:1. BnChd1-1 was mapped to a region of A01 using a BC3F1 population of 394 individuals with 198 green and 196 light-green plants. Within the collinear region in Brassica rapa, six genes that might be involved in chloroplast thylakoid development and NDH dehydrogenase activity were annotated. Among the six candidate genes, reverse transcription-polymerase chain reaction revealed that the mRNA levels of Bra021529 and Bra040517 were undetectable in the mutant and high in Qingyou10 and Westar plants at the seedling stage. Additionally, DNA sequence differences were identified across the gene and promoter region. Protein sequence differences were also observed in Bra040517, while no sequence differences in Bra021529 were observed between Bnchd1 and Qingyou10. Therefore, the homologue of Bra040517 is the most likely candidate gene for BnChd1-1.