Identification of a biomass unaffected pale green mutant gene in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

Abstract

Chlorophyll (Chl) is an essential component of the photosynthetic apparatus and pigments in plant greening. Leaf color is an important agronomic and commercial trait of Chinese cabbage. In this study, we identified a pale green mutant pgm created by ethyl methane sulfonate (EMS) mutagenesis in Chinese cabbage. Compared with wild-type (FT), pgm had a lower Chl content with a higher Chl a/b ratio, imperfect chloroplast structure, and lower non-photochemical quenching. However, its net photosynthetic rate and biomass showed no significant differences. Genetic analysis revealed that the pale green phenotype of pgm was controlled by a recessive nuclear gene, designated as Brpgm. We applied BSR-Seq, linkage analysis, and whole-genome resequencing to map Brpgm and predicted that the target gene was BraA10g007770.3C (BrCAO), which encodes chlorophyllide a oxygenase (CAO). Brcao sequencing results showed that the last nucleotide of its first intron changed from G to A, causing the deletion of the first nucleotide in its second CDS and termination of the protein translation. The expression of BrCAO in pgm was upregulated, and the enzyme activity of CAO in pgm was significantly decreased. These results provide an approach to explore the function of BrCAO and create a pale green variation in Chinese cabbage.

Figure 1

Phenotypic characterization and ultrastructure of chloroplasts of pale green mutant (pgm) and wild-type (FT). (a) Phenotypic characterization of pgm (right) and FT (left) at 6 weeks. (b) The fresh weight and dry weight in pgm and FT at 6 weeks. (c, d) Ultrastructure of chloroplasts in FT (c) and pgm (d). Scale is shown at the bottom.

Figure 2

The distribution of ED⁵ values on chromosomes.

Figure 3

Genetic and physical maps of the Brpgm gene locus in Brassica rapa. (a) The mutant gene Brpgm was initially located between the molecular markers SSR10-17 and SSR5-1 in the A10 linkage group, with genetic distances of 2.20 and 0.43 cM, respectively. (b) The mutation gene Brpgm was located between SS9-27 and SSR 7–18 with genetic distances of 0.02 and 0.11 cM, respectively. (c) The physical size of the candidate region was 4499.6 Kb, containing 336 candidate genes.

Figure 4

Sequence alignment and gene structure. (a) DNA sequence alignment of wild-type (FT), pale green mutant (pgm), and F₂ recombinant individuals: 204, 332, 354, 919, 1153, and 1542. (b) cDNA sequence alignment of FT, pgm, and F₂ recombinant individuals. (c) Gene structure of BrCAO. (d) BrCAO protein sequence of FT and pgm. The red box indicates the mutation position.

Figure 5

Expression analysis of BrCAO and enzyme assay of BrCAO. (a) The expression pattern of BrCAO in leaves of four stages (cotyledon, seedling, rosette, and heading stage) in pale green mutant (pgm) and wild-type (FT). (b) The CAO activity in pgm and FT. U/L = activity unit. The ‘*’ represents significant differences at 5% levels.