Genetic and Transcription Profile Analysis of Tissue-Specific Anthocyanin Pigmentation in Carrot Root Phloem

Abstract

In purple carrots, anthocyanin pigmentation can be expressed in the entire root, or it can display tissue specific-patterns. Within the phloem, purple pigmentation can be found in the outer phloem (OP) (also called the cortex) and inner phloem (IP), or it can be confined exclusively to the OP. In this work, the genetic control underlying tissue-specific anthocyanin pigmentation in the carrot root OP and IP tissues was investigated by means of linkage mapping and transcriptome (RNA-seq) and phylogenetic analyses; followed by gene expression (RT-qPCR) evaluations in two genetic backgrounds, an F₂ population (3242) and the inbred B7262. Genetic mapping of 'root outer phloem anthocyanin pigmentation' (ROPAP) and inner phloem pigmentation (RIPAP) revealed colocalization of ROPAP with the P₁ and P₃ genomic regions previously known to condition pigmentation in different genetic stocks, whereas RIPAP co-localized with P₃ only. Transcriptome analysis of purple OP (POP) vs. non-purple IP (NPIP) tissues, along with linkage and phylogenetic data, allowed an initial identification of 28 candidate genes, 19 of which were further evaluated by RT-qPCR in independent root samples of 3242 and B7262, revealing 15 genes consistently upregulated in the POP in both genetic backgrounds, and two genes upregulated in the POP in specific backgrounds. These include seven transcription factors, seven anthocyanin structural genes, and two genes involved in cellular transport. Altogether, our results point at DcMYB7, DcMYB113, and a MADS-box (DCAR_010757) as the main candidate genes conditioning ROPAP in 3242, whereas DcMYB7 and MADS-box condition RIPAP in this background. In 7262, DcMYB113 conditions ROPAP.

Keywords: Daucus carota; RNA-Seq; RT-qPCR; anthocyanin; candidate genes; genetic mapping; phloem pigmentation.

Figure 1

Genetic mapping of the phenotypic traits ‘root outer phloem anthocyanin pigmentation’ (ROPAP) and ‘root inner phloem anthocyanin pigmentation’ (RIPAP) in the 3242-F₂ population (A). Bars indicate the 1.5 LOD support interval and the position of the maximum LOD value for each binary trait. The P₁ and P₃ regions in chromosome 3, as delimited by Bannoud et al. [12], are highlighted in purple. Asterisks indicate markers that were significantly distorted from the expected segregation ratio for codominant markers in an F₂ population, at p < 0.05 (*) and p < 0.01 (**). Magnified images correspond to the physical map (in nucleotide distances) of the P₁ (B) and P₃ (C) regions associated with ROPAP and RIPAP, and include candidate regulatory (in red letters) and structural genes involved in anthocyanin biosynthesis (green) and transport (blue), along with their performance in different gene-expression experiments and genetic backgrounds (D,E). Red and green arrows indicate up and downregulation in the outer phloem relative to the inner phloem, respectively, whereas black dashes indicate not differentially expressed. nt: No transcripts detected. Root section phenotypes of the different genetic backgrounds analyzed are illustrated for each gene expression experiment.

Figure 2

Comparative analysis of transcript levels by RT-qPCR for 19 selected genes in purple outer phloem (OP) (gray bars) vs. non-purple inner phloem (IP) (white bars) tissues from roots of 3242 and B7262 genetic backgrounds. For 3242, roots from a derivative F₃ family of 3242-F₂ (used for mapping and transcriptomic analyses) were used. Root phenotypes for both genetic backgrounds are shown in the upper-left images. The selected anthocyanin-related genes were consistently differentially expressed in all of the POP vs. NPIP transcriptome comparisons (16 genes), and/or they colocalized with ROPAP and RIPAP in the linkage map, and/or they were previously reported to be involved in the biosynthesis, modification or transport of anthocyanins. Bars indicate mean values for the fold change in gene expression relative to the expression of the internal control gene (actin), as calculated from the ratio ∆Cq-target gene/∆Cq-actin [34] and their standard errors. Asterisks indicate significantly higher gene expression at p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

Figure 3

Gene expression analysis (RT-qPCR) of DcMYB7, DcMYB113 and MADS-box (DCAR_010757) in the root purple outer phloem (OP) (gray bars) and purple inner phloem (IP) (white bars) of the carrot inbred line P9547 (A), which is genetically related to 3242, and the Indian cultivar Pusa asita (B). Root phenotypes with cross-sections indicating the OP and IP tissues, and their gene expression profiles are presented. Bars indicate mean values for the fold change in gene expression relative to the expression of the internal control gene (actin), as calculated from the ratio ∆Cq-target gene/∆Cq-actin [34], and their standard errors. Asterisks (***) indicate significantly higher gene expression at p < 0.001, whereas OP vs. IP comparisons without asterisks is not statistically different at p ≤ 0.05. The absence of bars for DcMYB113 indicates that no transcripts were detected.