The introgression of BjMYB113 from Brassica juncea leads to purple leaf trait in Brassica napus

Abstract

The purple leaves of Brassica napus are abundant in anthocyanins, which are renowned for their role in conferring distinct colors, stress tolerance, and health benefits, however the genetic basis of this trait in B. napus remains largely unelucidated. Herein, the purple leaf B. napus (PL) exhibited purple pigments in the upper epidermis and a substantial increase in anthocyanin accumulation, particularly of cyanidin, compared to green leaf B. napus (GL). The genetic control of the purple leaf trait was attributed to a semi-dominant gene, pl, which was mapped to the end of chromosome A03. However, sequencing of the fragments amplified by the markers linked to pl indicated that they were all mapped to chromosome B05 from B. juncea. Within this B05 chromosomal segment, the BjMYB113 gene-specific marker showed perfect co-segregation with the purple leaf trait in the F₂ population, suggesting that the BjMYB113 introgression from B. juncea was the candidate gene for the purple leaf trait in B. napus. To further verify the function of candidate gene, CRISPR/Cas9 was performed to knock out the BjMYB113 gene in PL. The three myb113 mutants exhibited evident green leaf phenotype, absence of purple pigments in the adaxial epidermis, and a significantly reduced accumulation of anthocyanin compared to PL. Additionally, the genes involved in positive regulatory (TT8), late anthocyanin biosynthesis (DFR, ANS, UFGT), as well as transport genes (TT19) were significantly suppressed in the myb113 mutants, further confirming that BjMYB113 was response for the anthocyanin accumulation in purple leaf B. napus. This study contributes to an advanced understanding of the regulation mechanism of anthocyanin accumulation in B. napus.

Keywords: BjMYB113; Brassica; Anthocyanin accumulation; CRISPR/Cas9; Leaf color; Map-based cloning.

Fig. 1

Phenotypes of B. napus with purple and green leaves. a Phenotype and lower epidermis of B. napus with purple (PL), green leaves (GL), and F₁ progeny. b Content of total anthocyanin accumulation in PL and GL. The error bars represent the standard error of six biological replicates. c Accumulation of different types of cyanidin, flavonoid, and petunidin in the six biological replicates of PL and GL. Asterisks indicate significant differences in a t-test (**, P < 0.01). d Differentially accumulated metabolites in the leaves between PL and GL. The purple points indicate significantly higher accumulation of metabolites and the green points indicate significantly lower accumulation of metabolites in the PL as compared with GL

Fig. 2

Map-based cloning of the gene controls the purple leaf trait in the F2 population. a The distribution of the average value of ΔSNP-index plotted along the 19 chromosomes of B. napus. The red line indicates the threshold line. b Fine mapping of pl using 476 segregating individuals. The maker 598–1 was developed according to the BnAPR2 at the ends of chromosome A03 as described previously [36]. c Schematic diagram of predicted genes in the pl locus. The black broad arrows represent the predicted genes that present only in the PL. The white broad arrows represent the predicted genes that absent in both PL and GL by PCR method. The BjMYB113 (BjuVB05G51160) gene was in purple broad arrow, considered as the candidate gene. d Molecular markers of BjMYB113 were used to distinguish different genotypes in the parental line and the segregating population

Fig. 3

Creation and phenotype of BjMYB113 knockout lines. a Three target sites of exon 3 of the BjMYB113 and vector structure of CRISPR/Cas9 hosting sgRNA expression cassettes. The vertical arrow in the gene model indicates the target site. b Sequencing at the mutation sites of BjMYB113 in the T1 generation. Nucleotide indels or substitutions in three mutations are marked in purple, with details labeled at right. c Phenotype (scale bar = 2 cm) and transverse section of leaf from GL, PL, and related BjMYB113 knockout lines. d Content of total anthocyanin accumulation in PL, GL, and BjMYB113 knockout lines. The error bars represent the standard error of three biological replicates. Data were analyzed by one-way ANOVA, followed by comparisons of means using Tukey’s multiple comparisons test. Different letters indicate significant differences at p < 0.05

Fig. 4

The expression of BjMYB113 and anthocyanin biosynthetic genes in the parental and knockout lines. a The expression of BjMYB113 in the leaves of PL and GL. Asterisks indicate significant differences in a t-test (**, P < 0.01). b Expression of anthocyanin biosynthetic genes in the leaves of PL, GL, and knockout lines. The color scale at the left indicates Log2 transformed average FPKM values from three replicates after re-analyzing our previous RNA-Seq data. The color scale at the right indicates the relative expression of genes from three replicates using qRT-PCR methods. c The regulatory mechanism of BjMYB113 on the purple leaf trait in B. napus