Genome-Wide Identification and Evolution Analysis of R2R3-MYB Gene Family Reveals S6 Subfamily R2R3-MYB Transcription Factors Involved in Anthocyanin Biosynthesis in Carrot

Abstract

The taproot of purple carrot accumulated rich anthocyanin, but non-purple carrot did not. MYB transcription factors (TFs) condition anthocyanin biosynthesis in many plants. Currently, genome-wide identification and evolution analysis of R2R3-MYB gene family and their roles involved in conditioning anthocyanin biosynthesis in carrot is still limited. In this study, a total of 146 carrot R2R3-MYB TFs were identified based on the carrot transcriptome and genome database and were classified into 19 subfamilies on the basis of R2R3-MYB domain. These R2R3-MYB genes were unevenly distributed among nine chromosomes, and Ka/Ks analysis suggested that they evolved under a purified selection. The anthocyanin-related S6 subfamily, which contains 7 MYB TFs, was isolated from R2R3-MYB TFs. The anthocyanin content of rhizodermis, cortex, and secondary phloem in 'Black nebula' cultivar reached the highest among the 3 solid purple carrot cultivars at 110 days after sowing, which was approximately 4.20- and 3.72-fold higher than that in the 'Deep purple' and 'Ziwei' cultivars, respectively. The expression level of 7 MYB genes in purple carrot was higher than that in non-purple carrot. Among them, DcMYB113 (DCAR_008994) was specifically expressed in rhizodermis, cortex, and secondary phloem tissues of 'Purple haze' cultivar, with the highest expression level of 10,223.77 compared with the control 'DPP' cultivar at 70 days after sowing. DcMYB7 (DCAR_010745) was detected in purple root tissue of 'DPP' cultivar and its expression level in rhizodermis, cortex, and secondary phloem was 3.23-fold higher than that of secondary xylem at 110 days after sowing. Our results should be useful for determining the precise role of S6 subfamily R2R3-MYB TFs participating in anthocyanin biosynthesis in carrot.

Keywords: R2R3-MYB family; anthocyanin; carrot; evolution; expression profile; transcription factor.

Figure 1

Summary of MYB TFs among plants. Daucus carota was highlighted with purple box.

Figure 2

Phylogenetic tree of R2R3-MYB TFs of Arabidopsis and carrot and chromosomal determination and collinearity analysis of R2R3-MYB genes in carrot. Phylogenetic trees were constructed from the R2R3-MYB protein sequence of carrot and Arabidopsis (A). The phylogenetic trees were established by the neighbor-joining (NJ) method. (B,C) represent the carrot chromosomes. The black lines represent collinear gene pairs. The genes were mapped onto different chromosomes and the numbers along the chromosome boxes represent sequence lengths in megabases.

Figure 3

Distribution of carrot R2R3-MYB TFs subfamilies. R2R3-MYB TFs subfamilies represented on the Abscissa. Ordinate represents the number of each subfamily member.

Figure 4

Sequence logos of conserved domains of R2R3-MYB proteins in carrot.

Figure 5

Ka/Ks density distribution in carrot.

Figure 6

The color of cross-sections and the taproots of nine purple and non-purple carrots cultivars at two different stages. (A) represents the purple and nonpurple carrots grown for 70 days. (B) represents the purple and nonpurple carrots grown for 110 days. Cultivar abbreviations: DPP, Deep purple; BN, Black nebula; ZW, Ziwei; PPHZ, Purple haze; CPP, Cosmic purple; ZL, Zilong; KRD, Kurodagosun; SHBC, Sanhongbacun; MGH, Meiguihong. White bar represents 5 cm.

Figure 7

Total anthocyanin contents in the taproot of nine carrot cultivars at two. different stages. Error bars represent standard deviation (SD) of three replicates and are computed as cyanidin 3-O-galactoside equivalents.

Figure 8

Site of MYB TFs in S6 subfamily on chromosome and transcript abundances of DcMYB genes in four different tissue parts of D. carota cvs. DPP, PPHZ, CPP and KRD. (A) represents the site of MYB TFs. Heat map was constructed based on the log2 (RPKM + 1) transformation values (B), and bar notes represent different expression levels. Yellow represents high expression level, and blue represents little expression level.

Figure 9

Expression profiles of S6 subfamily DcMYB genes (DcMYB6 (DCAR_000385), DcMYB7 (DCAR_010745), DcMYB8 (DCAR_010746), DcMYB9 (DCAR_010747), DcMYB11 (DCAR_010751), DcMYB016 (DCAR_016451) and DcMYB113 (DCAR_008994)) in different root tissues of carrot at two growth stages. (A) represents expression profiles of DcMYB genes in purple and nonpurple carrots at 70 days after sowing. (B) represents expression profiles of DcMYB genes in purple and nonpurple carrots at 110 days after sowing. The relative gene expression was calculated with the 2^−ΔΔCT method. Error bars represent standard deviation (SD) of three replicates.