Dynamics Anthocyanidin profiles of fleshy root in development of carmine radish
To demonstrate the dynamics Anthocyanidin profiles of fleshy root in development of carmine radish, Anthocyanidin profiles of fleshy root from seedling stage, initial expansion,full-expansion,Bolting stage, initial flowering stage,full-bloom stage and podding stage in five local cultivars of carmine radish (‘‘Hongxin 1’, ‘Guanguan’,’Longquan 1’,’ Yanzhi 1’ and ‘Yanzhi 2’) were investigated by HPLC analysis. The results showed that Anthocyanidin was significantly increased in ‘Hongxin 1’ differential fleshy roots types with the development of dynamic growing stages of fleshy roots than other local cultivars of carmine radish, from seedling stage to full-bloom stage, but decresed in podding stage (Fig. S1).
Illumina sequencing and De novo assembly
Based on results from HPLC analysis, young fleshy roots obtained from the development stage of carmine radish ‘Hongxin 1’ was used for RNA-Seq in this study. The cDNAs obtained from fleshy roots of seven growth phases (seedling stage, initial expansion, full-expansion, Bolting stage, initial flowering stage, full-bloom stage and podding stage) were sequenced using Illumina sequencing technology. After filtering out adaptor-only reads, trimming reads and low-quality reads (base quality ≤10), high-quality Reads were aligned to the SSU and LSU rRNA sequences to remove rRNA reads by a home-made perl script. And the percentage of clean reads counts almost 70% after removing rRNA sequences on average among raw tags in each library, respectively (Table 1). Moreover, 198, 342 assembled transcipts from the raw sequence reads were constructed with an average length of 411 bp, and 34,927 Unigenes were generated using paired-end reads with an average length of 768 bp through de novo assembly technology (Fig. S2)..
DEGs related to the dynamics growing stages of fleshy roots in carmine radish
To identify the putative candidate genes with great changes involved in the dynamics growing stages of fleshy roots in carmine radish, normalized expression levels for all global expressed genes were analyzed, indicating that high distinct gene expression profiles exists in the dynamics growing stages of fleshy roots (Fig. 1, Table S2).. More interestingly, we found that the putative candidate genes belong to Cluster 8 was consistently with dynamics anthocyanidin profiles of fleshy root in development of carmine radish, but the putative candidate genes categorized into Cluster 9 were found oppositely.
Furthermore, DEGs were identified the dynamics growing stages of fleshy roots among other different development periods (IE_root’, ‘FE_root’, ‘BS_root’, ‘IFS_root’, ‘FBS_root’ and ‘PS_root) between ‘SS_root’ group, the results indicated that 1,629, 1,037, 1,385, 1,521, 1,574 and 917 DEGs were generated in IE_root’, ‘FE_root’, ‘BS_root’, ‘IFS_root’, ‘FBS_root’ and ‘PS_root’, compared with ‘SS_root’, including up-regulated (878, 755, 718, 838, 852 and 555 transcripts) and down-regulated genes (751, 282, 667, 683, 722 and 362 transcripts) (Fig. 2A).. 126 Co-modulated DEGs (Common DEGs in the dynamic growing stages of fleshy roots in carmine radish) were identified based on venny graph (Fig. 2B),, and expression changes pattern of co-modulated DEGs were displayed with different colors using heatmap (Fig. 2C).. More importantly, we found some of co-modulated DEGs showed similar expression trends in the dynamics growing stages of fleshy roots, which was found consistently with anthocyanidin profiles of fleshy root in development of carmine radish, such as series functional enzymes acted as important regulators in anthocyanins biosynthesis, including dihydroflavonol 4-reductase (DFR: Cluster_13775), flavonoid 3’-monooxygenase (F3’H: Cluster_4431), leucoanthocyanidin dioxygenase (ANS: Cluster_3903) and Chalcone synthase (CHS: Cluster_39833), as well as some regulation enzymes comprised of anthocyanidin 3-O-glucoside 2’'‘-O-xylosyltransferase(F3GGT1: Cluster_9270), coumaroyl-CoA:anthocyanidin 3-O-glucoside–6’‘-O-coumaroyltransferase 1-like (3AT2: Cluster_46827), UDP-glycosyltransferase 75C1-like (UGT75C1: Cluster_2736) and UDP-glycosyltransferase 78D2-like (UGT78D2: Cluster_11854). In addition, sets of transport proteins and transcription factors, such as, glutathione S-transferase F12 (Cluster_24268), MYB transcription factor (Cluster_28373), as well as Zinc finger, RING-type protein (Cluster_7186) (Fig. 2C, Table S3). Moreover, these DEGs related to the dynamics growing stages of fleshy roots that involved in different biological processes were validated using qRT-PCR and the results showed higher consistent with expression profiles of RNASeq data (Fig. 3, Table S4)..
Functional annotation of DEGs related to the dynamics growing stages of fleshy roots in carmine radish
To explore the regulatory mechanisms of DEGs related to the dynamics growing stages of fleshy roots in carmine radish, GO annotation and KEGG pathway enrichment of those putative DEGs were conducted. The results illustrated that GO terms comprised of “anthocyanin-containing compound biosynthetic process” and “anthocyanin-containing compound metabolic process” were commonly overrepresented in the other dynamics growing stages of fleshy roots after initial expansion of fleshy roots (IE–40 days after planting), “flavoriod biosynthetic process” and “flavoriod metabolic process” were found overrepresented in fleshy roots of IFS, FBS and PS; but for “pigment biosynthetic process” and “pigment metabolic process”, which was only found overrepresented in fleshy roots of IFS and FBS; moreover, we found that GO terms comprised of “glucosinolate biosynthetic process” and “glucosinolate metabolic process” were only overrepresented in fleshy roots of IE. (Fig. 4A, Table S5).. By conducting pathway enrichment analysis, these results indicated that five significantly enrichment pathways DEG were identified for the dynamics growing stages of fleshy roots in carmine radish, including Flavonoid biosynthesis, Flavone and flavonol biosynthesis, Diterpenoid biosynthesis, Anthocyanin biosynthesis, as well as Benzoxazinoid biosynthesis (Fig. 4B, Table S6)..