Genetic and transcription profile analysis of tissue-specific anthocyanin pigmentation in carrot root phloem

Purple carrots can accumulate large quantities of anthocyanins in their roots. Depending on the genetic background, anthocyanin pigmentation can be expressed in the entire root, or it can display tissue specific-patterns, confined to the root phloem or xylem tissues. Within the phloem, the tissue usually contributing most of the overall anthocyanin concentration in the carrot root, purple pigmentation can be found in the outer phloem (OP) (also called cortex) and inner phloem (IP) tissues, or it can be confined exclusively to the OP. The latter is a fairly-common phenotype in many purple carrot cultivars. 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, transcriptome (RNA-seq), phylogenetic, and gene expression (RT-qPCR) analyses in two genetic backgrounds; an F2 mapping population (3242) and the inbred line B7262. Genetic mapping of the 'root outer phloem anthocyanin pigmentation' (ROPAP) and inner phloem pigmentation (RIPAP) revealed co-localization 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 (4 MYBs, 1 bHLH, 1 MADS-box, 1 ERF), 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, which roots present purple pigmentation only in the outer phloem, DcMYB113 conditions ROPAP Overall design: Outer purple phloem and inner non-purple phloem

紫胡萝卜可在其根部积累大量花青素（anthocyanins）。根据遗传背景的不同，花青素着色可在整个根部表现，或呈现组织特异性模式，仅局限于胡萝卜根的韧皮部（phloem）或木质部（xylem）组织。在通常占胡萝卜根部总花青素含量比例最高的韧皮部中，紫色着色可在外韧皮部（outer phloem, OP，又称皮层（cortex））和内韧皮部（inner phloem, IP）组织中出现，或仅局限于外韧皮部。该表型在诸多紫胡萝卜栽培品种中较为常见。本研究通过连锁作图、转录组测序（RNA-seq）、系统发育分析以及实时定量荧光PCR（RT-qPCR）基因表达分析，在两种遗传背景下探究了胡萝卜根外韧皮部与内韧皮部组织中组织特异性花青素着色的遗传调控机制：一种为F2作图群体（3242），另一种为近交系B7262。对“根外韧皮部花青素着色”（ROPAP）与“根内韧皮部着色”（RIPAP）的遗传作图分析显示，ROPAP与此前在不同遗传材料中被报道调控花青素着色的P1和P3基因组区域共定位，而RIPAP仅与P3区域共定位。通过对紫色外韧皮部（POP）与非紫色内韧皮部（NPIP）组织的转录组测序分析，结合连锁作图与系统发育数据，研究团队初步鉴定出28个候选基因；其中19个通过RT-qPCR在3242与B7262的独立根部样本中进行了验证，结果显示在两种遗传背景的POP中，共有15个基因持续上调表达，另有2个基因仅在特定遗传背景的POP中上调表达。这些基因包括7个转录因子（4个MYB类、1个bHLH类、1个MADS-box类、1个ERF类）、7个花青素生物合成结构基因，以及2个参与细胞转运过程的基因。综上，本研究结果表明，在3242背景中，DcMYB7、DcMYB113以及MADS-box基因DCAR_010757是调控ROPAP的核心候选基因，而DcMYB7与该MADS-box基因则调控该背景下的RIPAP；在仅在外韧皮部呈现紫色着色的7262背景中，DcMYB113负责调控ROPAP。实验设计：外紫色韧皮部与内非紫色韧皮部