Whole-Genome Identification and Comparative Expression Analysis of Anthocyanin Biosynthetic Genes in Brassica napus

Abstract

Anthocyanins contribute to most colors of plants and play protective roles in response to abiotic stresses. Brassica napus is widely cultivated worldwide as both an oilseed and a vegetable. However, only several high anthocyanin-containing cultivars have been reported, and the mechanisms of anthocyanin accumulation have not been well-elucidated in B. napus. Here, the phenotype, comparative whole-genome identification, and gene expression analysis were performed to investigate the dynamic change of the anthocyanin content and the gene expression patterns of anthocyanin biosynthetic genes (ABGs) in B. napus. A total of 152 ABGs were identified in the B. napus reference genome. To screen out the critical genes involved in anthocyanin biosynthesis and accumulation, the RNA-seq of young leaves of two B. napus lines with purple leaves (PL) or green leaves (GL), and their F₁ progeny at 41, 91, and 101 days were performed to identify the differentially expressed genes. The comparative expression analysis of these ABGs indicated that the upregulation of TT8 together with its target genes (such as DFR, ANS, UFGT, and TT19) might promote the anthocyanin accumulation in PL at the early developmental stage (41-91 days). While the downregulation of those ABGs and anthocyanin degradation at the late developmental stage (91-101 days) might result in the decrease in anthocyanin accumulation. Our results would enhance the understanding of the regulatory network of anthocyanin dynamic accumulation in B. napus.

Keywords: B. napus; anthocyanin biosynthetic genes; comparative genomic analysis; dynamic accumulation; transcriptome.

FIGURE 1

Phenotypes of B. napus with different leaf colors. The upper (A) and lower epidermis (B) of PL, F₁, and GL (scale bar = 2 cm). Transverse section of leaf from PL (C), F₁ (D), and GL (E) with different colors (scale bar = 2 μm). (F) The content of total anthocyanin accumulation at different stages. Values represent means ± SD from three biological replicates. Red dots in the x-axis indicate days for RNA-seq sampling.

FIGURE 2

Distribution and syntenic analysis of ABGs in Brassica napus. (A) The color on the chromosome indicates the gene density of all the genes. Redder color indicates the higher gene density, while the bluer color indicates lower gene density. The red boxes indicate tandem duplications. (B) The orange lines indicate the ABGs exhibited syntenic between A. thaliana, B. rapa, B. oleracea, and B. napus.

FIGURE 3

Summary of differentially expressed genes between GL and PL. (A) The number of DEGs among GL, F₁, and PL. (B) Venn diagrams of differentially expressed genes between PL and GL shared at different developmental stages. (C) The KEGG enrichment of DEGs between PL and GL at different developmental stages.

FIGURE 4

Anthocyanin biosynthetic pathway and expression patterns of ABGs in B. napus. (A) The expression level of ABGs which are differentially expressed between PL and GL is shown in the pathway. Purple and green colors are used to represent high to low expression levels, respectively. The color scale corresponds to the mean-centered log2-transformed average TPM values from three replicates. (B) The overview of ABG expression, which was differentially expressed at different stages. Asterisks indicate statistical differences between each other (two-tailed Student’s t-test, ** indicates p < 0.01; * indicates p < 0.05).

FIGURE 5

Model of anthocyanins accumulation in Brassica napus. The TT8, TT19, and LBGs are significantly expressed in PL compared with those in GL from 41 to 91 days, (p < 0.01). At these developmental stages, anthocyanin contents increase, and the purple pigments are accumulated in the adaxial epidermis and subepidermal cells. The expression levels of these genes are decreased in PL and exhibited no significant difference between that of GL from 91 to 101 days, (p = 0.1175). The anthocyanin degradation at this developmental stage might reduce the total anthocyanin accumulation and result in the leaves to gradually turn to green. Circles filled with purple color indicate upregulated genes expressed in PL at 41 and 91 days.