Abstract

   Panax japonicus C. A. Mey. is a rare and endangered Class II important
   protected wild medicinal plant in China with a wide range of
   pharmacological activities. The development of the fruit is special,
   only the apical capitulum can develop into fruit normally, and the
   color trait changes during the maturation process showed a pattern from
   green to red and from red to purple-black. In order to reveal the
   phenomenon of color trait changes during fruit development, this paper
   analyzed the correlation between color trait changes and pigment
   contents (chlorophyll, carotenoids) and secondary metabolite contents
   (anthocyanin, flavonoids, and total phenols) of the fruits, and used
   transcriptome sequencing to explore the correlation between color trait
   changes and synthetic genes, and then verified the fluorescence
   quantification with qRT-PCR. The results showed that chlorophyll was
   gradually converted and carotenoids began to appear in the pericarp
   when the fruit entered the color change stage from the green fruit
   stage; the content of anthocyanin, flavonoids and total phenolic
   substances gradually increased during fruit ripening. Transcriptome
   sequencing obtained transcriptome data of 15 samples from five periods
   of color trait changes in Panax japonicus fruits 93.94 Gbp, with
   462,117 functionally annotated Total Unigenes; the total number of DETs
   generated by intercomparison of the five periods (G, Y, R, B, P) was
   17,895 (up-regulated 9435 and down-regulated 8460). Weighted gene
   co-expression network analysis (WGCNA) indicated that 448 were hub
   differential genes (DETs) associated with common involvement in the
   color changes (carotenoids, anthocyanins, flavonoids, total phenols) in
   the fruits of Duchess. GO and KEGG were enriched to participate in the
   photosynthesis—antenna protein pathway 19; involved in flavonoid
   biosynthesis a total of 205. The results of qRT-PCR fluorescence
   quantitative validation showed that chlorophyll degradation related 6
   candidate genes PjCAB and fruit color change flavonoid biosynthesis 6
   candidate genes PjF3’H, PjCHI, PjCHS, PjDFR, PjANS, and PjC3’H at five
   periods were consistent with the transcriptome sequencing results.

   Keywords: Panax japonicus, Fruit color traits, Pigment content,
   Transcriptome, Gene discovery, qRT-PCR validation

   Subject terms: Molecular biology, Physiology

Introduction

   Fruit color in plants is an important signal for fruit ripening and
   plays an important role in the ecosystem. The study of fruit phenotypic
   traits and transcriptome to reveal the mechanism of fruit coloration is
   an important issue of special interest in the field of botany. In the
   study of fruit color in plants, many scholars have found that
   chlorophyll, carotenoids, flavonoids, anthocyanin, etc. are the main
   physiological indicators of fruit color, and this system of indicators
   has been confirmed in the color of fruits of a variety of plants, such
   as chilli peppers^[32]1, kiwifruit^[33]2–[34]4, begonias^[35]5,
   tomatoes^[36]6,[37]7, mandarins^[38]8, peaches^[39]9, and so on.
   Transcriptome analysis of the molecular mechanism of plant fruit
   coloring elucidated the molecular mechanism of fruit discoloration
   through flavonoid pathway and gene co-expression network analysis
   (WGCNA)^[40]10. Key enzyme genes involved in fruit coloration were
   identified in passion fruit^[41]11, jujube^[42]12, red mango^[43]13,
   blood orange^[44]14, apricot^[45]15, grapefruit^[46]16 and
   cranberry^[47]17.Panax japonicus (Panax japonicus C. A. Mey.) is a wild
   endangered valuable medicinal plant under Grade II protection in China,
   and is a perennial herb of the genus Ginseng in the family Wugazhidae;
   it has the effect of supplementing blood with Panax ginseng and
   supplementing qi with ginseng, so it is also called bamboo Panax
   ginseng and Panaxnotoginseng^[48]18. The special requirements of its
   living environment limit the distribution range and migration ability
   of this species to a certain extent, resulting in a narrow ecological
   range, which is mainly distributed in the southwestern region of China
   (Yunnan, Guizhou, Sichuan) and Enshi, Hubei^[49]19. The fruit is a
   spherical berry, which is usually green when the flower fades for half
   a month, and then starts to change color from light green to red when
   the fruit matures, with the upper part purple-black and the lower part
   red^[50]20,[51]21. In this study, we measured the physiological indexes
   and pigment content changes in the fruits of Salvia miltiorrhiza, and
   based on RNA-Seq sequencing analysis, we obtained the candidate
   transcripts and key functional genes of metabolic pathway biosynthesis
   related to the change of color traits in Panax japonicus fruits. We
   also revealed the physiological and molecular aspects of the metabolic
   pathways related to color changes in Panax japonicus fruits, and the
   key functional genes of the metabolic pathway biosynthesis related to
   color changes in Panax japonicus fruits. This study reveals the causes
   of the changes in fruit color traits and its molecular regulation
   mechanism, and provides reference for further exploration of the key
   genes for the changes in fruit color traits in ginseng plants of the
   family Ginseng, Araliaceae (Fig. [52]1).

Fig. 1.

   [53]Fig. 1
   [54]Open in a new tab

   Changes of fruit color traits of Panax japonicus in 5 periods.

Results and analysis

Pigment and physiological index content of Panax japonicus fruit

   To determine the dynamic changes of chlorophyll, carotene, anthocyanin,
   flavonoids and total phenols in the pericarp of Panax japonicus fruits
   during five different color trait change periods by enzymatic assay,
   and to reveal the causes of color trait changes in Drosera fruits from
   physiological indexes. The results showed Chlorophyll content showed a
   decreasing trend in fruit G to P. The chlorophyll content ranged from
   0.164 to 0.743 mg.g^−1 over the five color change periods, with G being
   the highest at 0.743 mg.g^−1, Y chlorophyll content being the next
   highest, and P chlorophyll content being the lowest (Fig. [55]2a). The
   carotenoid content showed an increasing trend from G to P. However, the
   carotenoid content of the pericarp was synthesised slowly from G to B,
   and the highest value of carotenoid content was reached at P pericarp.
   The carotenoid content ranged from 0.042 to 0.234 mg.g^−1 during the
   five color change periods, with P having the highest content of
   0.234 mg.g^−1, followed by B carotenoids, and G carotenoids being the
   lowest (Fig. [56]2b). The anthocyanin content showed a gradual increase
   from G to P, with a slow rate of synthesis from G to B. The highest
   anthocyanin content was reached in the P pericarp. The anthocyanin
   content ranged from 0.200 to 3.393 mg.g^−1 during the five color change
   periods, with the highest content of 3.393 mg.g^−1 at the ripening
   stage, followed by the average anthocyanin content of B, and the lowest
   average anthocyanin content of G (Fig. [57]2c). The synthesis of
   flavonoid content showed an exponential increase from G to P. Flavonoid
   content ranged from 1.713 to 7.256 mg.g^−1 during the course of the
   five color change periods, with the highest content of 7.256 mg.g^−1
   during the ripening period, followed by the average flavonoid content
   of B, and the lowest average flavonoid content of G. The flavonoid
   content of G was the highest during the ripening period (Fig. [58]2d).
   The total phenolic content showed a gradual increase from G to P. The
   trend was the same as that of the anthocyanin content, with total
   phenolic content ranging from 0.940 to 2.790 mg.g^−1 during the five
   color-presentation phases, with the highest content at the maturity
   phase of 2.790 mg g^−1, followed by the average total phenolic content
   of B, and the lowest average total phenolic content of G. The total
   phenolic content of G was the highest at the maturity phase
   (Fig. [59]2e). In summary, the results showed that the chlorophyll in
   the fruit was gradually destroyed, carotenoids began to appear in the
   pericarp, and anthocyanin, flavonoids and total phenolics began to be
   synthesized and played a decisive role in the color change during the
   maturation process of the fruit of Panax japonicus.

Fig. 2.

   [60]Fig. 2
   [61]Open in a new tab

   The physiological index content of Panax japonicus fruit in 5 different
   periods.

Sequencing data quality control and transcript splicing clustering

   The transcriptome data of 15 samples for the color trait variation of
   Panax japonicus fruits were obtained by sequencing on the Illumina
   platform 93.94 Gbp, the values of Q30 were all greater than 99%, the GC
   contents were all greater than 42%, and the sequence error rate was
   0.03% < 0.1%, which indicated that the quality of sequencing of these
   values was qualified (Supplementary Table 1). Trinity [[62]6] was used
   to splice the clean reads and the longest Cluster sequences were
   obtained by Corset hierarchical clustering for analysis. The lengths of
   transcript and unigene sequences were counted separately (Supplementary
   Fig. 1). The most lengths were 200–400 bp, 311,390 and 177,932,
   respectively; followed by lengths of 400–600 bp, with the numbers of
   93,869 and 89,119; the number of transcripts and unigenes with sequence
   lengths longer than 2Kbp was 51,614, and the least number was in the
   interval of 1.8–2Kbp. The number of transcripts and Unigenes in the
   1.8–2Kbp interval was 9773.

Functional annotation of identified transcripts

   The 462,117 unigenes were annotated, and the sequence information in
   the NR database annotated the most 239,867 unigenes, accounting for
   51.91% of the total number; the GO database annotated the least 116,105
   unigenes, accounting for 25.12% of the total number (Supplementary
   Table 2). Plotting venn diagram through five databases (Fig. [63]3A),
   there were 133,297 unigene sequences co-annotated to five databases,
   20,986 unigene sequences annotated to the Nr database, 16,063 unigene
   sequences annotated to the Nt database, 1,135 sequences annotated to
   the KOG database, 26 sequences annotated to the Swiss-prot database, 26
   sequences annotated to the Uniprot database, and 1,135 sequences
   annotated to the Nt database (KOG database). 26 entries, and 2 entries
   to the Uniprot database. Nr Homlogous Species Distribution annotations
   were performed to obtain information on the gene sequences of bamboo
   ginseng fruits and gene functions, and the analysis revealed that
   Daucus carota subsp. sativus (37.43%), Actinidia chinensis var.
   chinensis (6.63%), Camellia sinensis var. sinensis (3.07%), and Vitis
   vinifera (5.11%) (Fig. [64]3B). Functionally enriched GOs were
   classified one level below the three major categories (BP Biological
   process, CC Cellular component, MF Molecular Function) (Fig. [65]3C;
   Supplementary Table 3) while all Unigenes in the KOG database were
   annotated in the KOG database (Fig. [66]3D), and were classified
   according to the KEGG metabolic pathways they participated in
   (Fig. [67]3E; Supplementary Table 4), It was divided into five broad
   categories, cellular processes, environmental information processing,
   genetic information processing, metabolism and organic systems, with
   the highest percentage of genes annotated in the metabolism category at
   9.88%, followed by secondary metabolite biosynthesis at 5.04%, and
   microbial metabolism in different environments at 2.26%.To further
   investigate the transcription factors that may be involved in the color
   trait changes in Panax japonicus fruits, all the assembled transcripts
   were predicted using iTAK [[68]30] software to screen the transcription
   factors that may be involved in the color trait changes in Panax
   japonicus fruits. As a result, 10,139 transcripts encoding TFs were
   identified and classified into 93 different TF families including
   subfamilies (Supplementary Table 5). Due to the large number of
   predicted transcript families, the top 20 predicted more abundant
   families were selected for histogram display, among which the most
   abundant TFs were MYB-related, AP2/ERF-ERF, C2H2, bHLH, C3H, and WRKY
   families (Supplementary Fig. 2).

Fig. 3.

   [69]Fig. 3
   [70]Open in a new tab

   Functional annotation of fruit transcripts of Panax japonicus.

Differential gene expression analysis

   In order to identify the genes involved in the variation of color
   traits in the fruits of Panax japonicus, transcriptome differential
   gene expression (DEG) analysis was performed. The results indicated
   that the total number of DEGs generated by comparing each of the five
   periods (G, Y, R, B, P) with each other was 17,895, of which
   groupGvsgroupB had the highest number of 3,115 DEGs, including 1,794
   up-regulated and 1,321 down-regulated genes (Fig. [71]4). The
   distribution of DEGs with different color change periods was further
   screened, and the results of 10 subgroups were compared and plotted in
   Venn diagrams. Comparing the Y, R, B, and P color change periods using
   the G period as a control, 129 shared differential genes were obtained
   (Fig. [72]4A); 29 shared differential genes were obtained using the Y
   period as a control (Fig. [73]4B); 18 shared differential genes were
   obtained using the R period as a control (Figure 4C); and 25 shared
   differential genes were obtained using the B period as a control
   (Fig. [74]4D); 220 shared differential genes were obtained using the P
   period as a control (Fig. [75]4E). It is worth noting that 129
   differential genes were differentially expressed from the G to P stage
   of fruit in the comparison of fruit ripening color trait changes in
   Panax japonicus, which can be used as a candidate gene pool for fruit
   color trait changes in the next step of research.

Fig. 4.

   [76]Fig. 4
   [77]Open in a new tab

   Statistical histogram and Venn diagram of differential gene number.

WGCNA analysis of differential genes (DETs)

   A clustering tree was constructed based on the correlation of
   expression between genes and divided into modules, genes have similar
   expression changes in a physiological process, then these genes may be
   functionally related, defining them as a module (module), and the
   longitudinal distance represents the distance between two nodes
   (between genes) (Fig. [78]5A). Heat map of module-trait relationship,
   when the correlation between module and trait is significant, the
   redder the color of the square, the stronger the correlation.The
   correlation between MElightcyan module and G-phase and chlorophyll
   trait is significant at 0.98 and 0.91 respectively, the correlation
   between Mecyan module and Y-phase is significant at 0.96, the
   correlation between Mered module and R-phase is significant at 0.90,
   and the correlation between Meblue module and B-phase is significant at
   0.90. correlation was significant at 0.90. It is noteworthy that the
   MEblack module was significantly correlated with P phase, carotenoids,
   anthocyanins and flavonoids traits of 0.80, 0.84, 0.81, 0.77, 0.84,
   respectively (Fig. [79]5B), which may be correlated with the changes in
   the color traits of the Panax japonicus fruits. The 448 relevant
   sequences from the MEblack module were subjected to KEGG Pathway
   analysis, which revealed seven enrichments for the biosynthesis of
   secondary metabolites and four additional enrichments for the
   metabolism of terpenoids and polyketides (Fig. [80]5C).

Fig. 5.

   [81]Fig. 5
   [82]Open in a new tab

   WGCNA analysis of differentially expressed genes in fruit color traits
   of Panax japonicus.

GO enrichment analysis of differential genes

   The total number of DETs obtained during the process of fruit color
   trait changes in Panax japonicus, was 17,895, of which 9435 were
   up-regulated and 8460 were down-regulated. In order to obtain the
   functional annotation of the differential genes, GO enrichment
   histogram analysis was carried out using DETs, and because of the large
   number of GO terms annotated, 15 of the three categories were selected
   for the graph (Supplementary Table 6), and in the four subgroups
   groupGvsgroupY, groupGvsgroupR, and groupGvsgroupB, groupGvsgroupP,
   groupGvsgroupR, groupGvsgroupB, and groupGvsgroupP DETs enriched the
   highest number of biological_processes in the Term was flavonoid
   metabolic process; among them, groupGvsgroupB enriched the most 45
   genes (Fig. [83]6A), followed by groupGvsgroupB enriched 45 genes.
   GroupGvsgroupB was the most enriched group with 45 genes (Fig. [84]6A),
   followed by groupGvsgroupR with 31 genes (Fig. [85]6B), groupGvsgroupY
   with 20 genes (Fig. [86]6C), and groupGvsgroupP with the least enriched
   group with 12 genes (Fig. [87]6D). A Wayne diagram using the DETs of
   the enriched flavonoid metabolism process in each subgroup revealed
   five common DETs in four subgroups, and nine common DETs in three other
   subgroups, except for the groupGvsgroupP subgroup (Fig. [88]6E). Heat
   map analysis of these shared differential genes indicated that there
   were significant differences in flavonoid metabolism processes in the
   color traits of Panax japonicus fruits, and that these genes went from
   non-expression or low expression at the very beginning to high
   expression at the time of fruit ripening. These genes were
   preliminarily predicted to be candidate genes for the variation of
   fruit color traits in Panax japonicus (Fig. [89]6F).

Fig. 6.

   [90]Fig. 6

   [91]Fig. 6
   [92]Open in a new tab

   Differential gene GO enrichment histogram.

KEGG pathway enrichment analysis of differential genes

   KEGG Pathway enrichment bubble map analysis was performed with 17,895
   DETs for the color trait of Panax japonicus fruits to further elucidate
   their potential biological functions in the variation of color traits
   in Panax japonicus fruits. The results showed that the metabolic
   pathways enriched in DEGs compared in group GvsgroupY were
   photosynthesis, carbon metabolism, flavonoid biosynthesis,
   photosynthesis-haptoglobin, glyoxylate and dicarboxylic acid metabolism
   (Fig. [93]7A); The metabolic pathways enriched in DEGs compared in
   groupGvsgroupR are, Flavonoid biosynthesis, Carbon metabolism, Carbon
   fixation in photosynthetic organisms, Photosynthesis, Glycine, serine
   and threonine metabolism, Photosynthesis—antenna proteins
   (Fig. [94]7B). The metabolic pathways enriched in DEGs compared in
   groupGvsgroupB are Carbon metabolism, Carbon fixation in photosynthetic
   organisms, Glycine, serine and threonine metabolism, Photosynthesis,
   Flavonoid biosynthesis, (Fig. [95]7C); The metabolic pathways enriched
   in groupGvsgroupP are Carbon metabolism, Carbon fixation in
   photosynthetic organisms, Protein processing in endoplasmic reticulum,
   Protein processing in endoplasmic reticulum, Propanoate metabolism,
   Glycolysis/Gluconeogenesis, Citrate cycle (TCA cycle). Glyoxylate and
   dicarboxylate metabolism (Fig. [96]7D). It is noteworthy that the
   metabolic pathway for color trait changes in Drosera fruits, Flavonoid
   biosynthesis, was enriched in all three subgroups (groupGvsgroupY,
   groupGvsgroupR, groupGvsgroupB), while the metabolic pathway for
   chlorophyll degradation, Photosynthesis—antenna proteins, was only
   enriched in groupGvsgroupG and groupGvsgroupR. photosynthesis—antenna
   proteins) were only enriched in groupGvsgroupY and groupGvsgroupR.

Fig. 7.

   [97]Fig. 7
   [98]Open in a new tab

   KEGG enrichment bubble diagram analysis.

Candidate genes for photosynthesis-antenna proteins and qRT-PCR validation

   Screening of differential candidate genes for chlorophyll degradation
   in fruit through the KEGG-enriched photosynthesis—antenna proteins
   metabolic pathway. The total number of DETs enriched for the five color
   trait change periods was 30 (Supplementary Table 7), of which
   groupGvsgroupY was enriched by up to 11 (Fig. [99]8A); in addition, the
   30 genes enriched were collated for gene expression, and the top 10
   genes with higher expression were selected for heatmap clustering
   analysis. The clustered heat map of the expression of the selected
   genes showed that the expression of the first seven genes was highest
   at the green fruit stage (G) and decreased as the fruit color began to
   change, the last three genes were not expressed in the fruit (G), were
   expressed during the (Y, R, B) period, and were not expressed up to the
   fruit ripening stage (P) (Fig. [100]8B). To validate the RNA-Seq
   results, among the 10 candidate genes clustered in the heat map, six
   highly expressed candidate genes of the photosynthesis-antenna protein
   metabolism pathway were selected for expression analysis by qRT-PCR.
   The results showed that the expression of all six candidate genes was
   significantly higher in the (G) period than in its (Y, R, B, P) period,
   which was highly consistent with the transcript expression
   (Fig. [101]8a–f), and these photosynthesis-antenna protein metabolism
   pathway candidate genes conformed to the pattern of the gradual
   decrease of chlorophyll in the color trait of the fruits of Panax
   japonicus. Sequence comparison was performed using NCBI database, and
   the information of six candidate genes were obtained, all of which were
   highly similar to chlorophyll a/b binding (CAB), and they were named as
   PjCABI, PjCABII, PjCABIII, PjCAB1, PjCAB2 and PjCAB3 key candidate
   genes, respectively.

Fig. 8.

   [102]Fig. 8
   [103]Open in a new tab

   KEGG enriched the candidate differential genes of
   photosynthesis-antenna protein metabolic pathway.

Candidate genes for flavonoid biosynthesis and qRT-PCR validation

   The Flavonoid biosynthesis pathway of KEGG-enriched DETs was used to
   screen candidate genes associated with changes in fruit color traits of
   Panax japonicus, mainly analysing the Flavonoid biosynthesis pathway,
   which starts with phenylpropane biosynthesis and ends with anthocyanin
   biosynthesis. The results showed that 90 genes were obtained from
   groupG vs groupY, 154 from groupG vs groupR, and 145 from groupG vs
   groupB, and a total of 290 flavonoid biosynthesis genes were obtained
   from each group (Supplementary Table 8), which were analysed by using a
   Wayne diagram, and 81 common candidate genes were obtained that were
   associated with the changes in the color traits of the fruits of Panax
   japonicus, (Supplementary Fig. 3). Based on the flavonoid metabolic
   pathway map, 81 candidate genes were further examined for differences
   at the gene level during the five stages of color change in Drosera
   fruits, and five pathways of flavonoid biosynthesis genes were obtained
   (Supplementary Table 9); The top 6 highly expressed candidate genes of
   these 5 pathways were selected for heat map clustering analysis and
   these highly expressed candidate genes were subjected to NCBI database
   sequence comparison to obtain the gene information and were named as
   PjF3’H, PjCHI, PjCHS, PjDFR, PjANS, respectively. Mapping of flavonoid
   biosynthesis pathway was analysed to identify gene products in the
   flavonoid biosynthesis pathway; whether it is the PjCHS (Fig. [104]9A)
   and PjCHI (Fig. [105]9B) candidate genes upstream of PjF3’H candidate
   genes on the synthesis of coumaroyl CoA, naringenin chalcones,
   naringenin metabolites enriched in PjDFR (Fig. [106]9D) and PjANS
   (Fig. [107]9E) candidate genes downstream of PjF3’H (Fig. [108]9C) on
   the synthesis enrichment of dihydrosorbinol, dihydroquercetin,
   dihydromyricetin, white cornflowerin, cornflowerin, and white geranium
   glycoside ligand metabolite were all significantly upregulated
   (Fig. [109]9). Therefore, more substances were synthesized in the
   flavonoid biosynthesis pathway regarding the color trait changes in
   Panax japonicus fruits, and thus the whole flavonoid biosynthesis
   metabolism pathway was critically oriented in the promotion of color
   trait changes in the ripening of Panax japonicus fruits. In order to
   validate the RNA-Seq results, nine flavonoid biosynthesis-related
   candidate genes were randomly selected among the five categories of
   candidate genes, and expression analysis was carried out by qRT-PCR.
   The results pointed out that the relative expression levels of these
   candidate genes were highly consistent with their corresponding RNA-Seq
   expressions during the five periods of changes in the color traits in
   the fruits of Panax japonicus (Fig. [110]10a–i), thus confirming the
   validity of the RNA-Seq data. These candidate genes had the highest
   expression of PjDFR (Fig. [111]10i) and the lowest expression of PjC3’H
   (Fig. [112]10c) during the flavonoid biosynthesis of Panax japonicus
   fruits.

Fig. 9.

   [113]Fig. 9
   [114]Open in a new tab

   KEGG enrichment of candidate differential genes in flavonoid
   biosynthesis pathway.

Fig. 10.

   [115]Fig. 10
   [116]Open in a new tab

   qRT-PCR of flavonoid biosynthesis pathway candidate genes enriched by
   KEGG.

Conclusion and discussion

   In this study, we used five different periods of color trait changes in
   the fruit of Panax japonicus as test material, and determined the
   effects of chlorophyll, carotenoids, anthocyanin, flavonoids and total
   phenols on the changes of fruit color traits during the growth and
   development of Panax japonicus by using enzyme labeling assay. The
   results of the physiological indexes of Panax japonicus fruits
   indicated that the pattern of chlorophyll content in the pericarp
   decreased with the color change of the fruits, which was verified in
   the process of color trait changes of fruits of chilli peppers^[117]22,
   yellow-fleshed kiwifruit^[118]23, and cherry tomatoes^[119]24,
   suggesting that in the maturation of the fruits of the plants, their
   chlorophyll content would gradually decrease and be converted into
   other pigments to show different colors; and the effect of chlorophyll
   content on the color trait changes of Drosera fruits during growth and
   development of Drosera fruits was determined by carotenoids and total
   phenols. This conclusion is the same as that of Guo Yong mei^[120]22,
   Huang Chunhui^[121]23 and Ruan Meiying^[122]24 in the study of chilli,
   kiwifruit and tomato; the changes in the content of anthocyanin in the
   fruits of Drosera showed a trend of gradual increase, while the
   synthesis of flavonoids and total phenolics showed a trend of
   increasing, then decreasing, and then increasing again. This phenomenon
   was also confirmed in the changes of fruit color traits of
   Viburnum^[123]25, Capsicum annuum^[124]26, blueberry^[125]27–[126]29,
   apple^[127]30 and mulberry^[128]31. The above analysis pointed out that
   the gradual degradation of chlorophyll and the gradual accumulation of
   carotenoids in the ripening process of Drosera fruits, and the
   simultaneous synthesis of anthocyanin, flavonoids and total phenolic
   substances are conducive to the change of fruit color traits.

   High-throughput sequencing technology was used to sequence the
   transcriptome of Panax japonicus fruit for the first time, and a total
   of 93.94 Gbp of transcriptome data was obtained from 15 samples of
   Panax japonicus fruit color change, and 462,117 functional annotations
   were obtained after clustering by Corset hierarchy. Transcriptome
   sequencing analyses of Pentaphyllaceae plants revealed that Total
   Unigenes 135,235 single genes were obtained by transcriptome sequencing
   of flowers, leaves, roots and rhizomes of Panax japonicus^[129]32; and
   Panax notoginseng roots, leaves and flowers were transcribed and
   sequenced. Total Unigenes 107,340 single genes^[130]33; Panax
   quinquefolius root transcript sequencing annotated a total of
   21,684^[131]34, which shows that the same genus of Pentaphyllum is
   differently organised and the results of transcript sequencing are
   different. The total number of DETs generated by comparing the five
   periods of G, Y, R, B, and P in the fruit of Duchess with each other
   was 17,895 (9435 up-regulated and 8460 down-regulated) differentially
   expressed Unigenes (DETs), respectively. Transcriptome sequencing of
   ’Ningqi No.1’ and ‘Ningqi No. 7’ fruits at the green fruit stage, color
   change stage and ripening stage obtained 2827, 2552 and 2311
   differentially expressed genes at the green fruit stage, color change
   stage and ripening stage^[132]35; Sequencing of black fruit Lycium
   barbarum and white fruit Lycium barbarum obtained 25,279 differentially
   expressed genes of black fruit Lycium barbarum and white fruit Lycium
   barbarum^[133]36. It can be seen that the total number of DETs obtained
   by sequencing the transcriptome of the fruits of Panax japonicus is
   larger than that of ‘Ningqi No. 1’ and ‘Ningqi No.7’, but smaller than
   that of the black-fruit goji berry and white-fruit goji berry.

   Weighted co-expression network analysis of color trait variations
   (DETs) in Panax japonicus fruits was carried out by WGCNA method to
   reveal candidate hub gene modules regulating color trait variations in
   Panax japonicus fruits. The results indicated that a total of 10
   co-expression modules were obtained in the co-expression network
   analysis, and 448 hub genes related to carotenoids, anthocyanins,
   flavonoids and total phenols in DETs were identified in the MEblack
   module. In the study of Capsicum annuum fruit color traits, 17 modules
   were obtained for co-expression network analysis, of which 397 genes
   were involved in the accumulation of carotenoids in the ‘Darkgreen’
   module^[134]37, and 14 co-expression modules were identified in nine
   developmental stages of Luffa aegyptiaca fruit, of which the Turquoise
   module was associated with fruit length and length. The Turquoise
   module was significantly correlated with fruit length and fruit
   diameter, and 10 candidate genes for the potential control of fruit
   length in lucerne were obtained^[135]38. Four of the 23 modules in the
   analysis of the gene coexpression network related to quality traits in
   okra were involved in regulation^[136]39, 15 modules in the gene
   coexpression network related to drought tolerance in the root system of
   potato, and a one-guinea gene coexpression module for root drought
   tolerance was obtained, and 15 modules were obtained from the Genetic
   Basis of Biological Functions of Maize Plant Height and Spike
   Height^[137]40. co-expression modules^[138]41. Six modules of gene
   co-expression were obtained in transcriptional regulators of banana
   fruit ripening^[139]42. It can be seen that different plant fruit
   traits (DETs) can be obtained as candidate genes for trait-related
   modules by the WGCNA method.

   Anthocyanins are one of the important parts of pigments produced by the
   flavonoid biosynthetic pathway, and the key enzymes and structural
   genes involved in the flavonoid biosynthetic pathway have been
   confirmed accordingly in fruits^[140]43. GO and KEGG Pathway functional
   enrichment analyses of DETs for color trait changes in Panax japonicus
   fruits yielded 56 genes for flavonoid metabolism in the GO database and
   149 genes for flavonoid biosynthesis in the KEGG Pathway database,
   which may be involved in the regulation of color trait changes in D.
   japonicus fruits during ripening;A total of 29 differentially expressed
   genes were significantly enriched in the isoflavonoid biosynthesis
   pathway by KEGG Pathway analysis of DETs at three stages of Capsicum
   annuum green ripening fruit color development^[141]37; Ficus carica
   pericarp transcriptome KEGG Pathway flavonoid biosynthesis has 74
   differentially expressed genes enriched^[142]44, 4 different varieties
   of chilli pepper fruit color metabolome and transcriptome comprehensive
   analysis, KEGG Pathway based on the WGCNA method to identify 7
   flavonoid synthesis candidate genes^[143]45. Studying five aubergines
   with different fruit color traits in KEGG Pathway obtained 144 DETs
   enriched in aubergines, including flavonoid metabolites^[144]46; the
   flavonoid pathway enriched DETs in the fruits of Panax japonicus were
   all more than that of chilli pepper green ripening^[145]37,[146]45,
   Figs.^[147]44 and aubergines^[148]46, which is also the process color
   trait variability. Secondly, KEGG Pathway enrichment of DETs for fruit
   color trait changes in Panax japonicus yielded 19 genes in the
   photosynthesis-antenna protein pathway, which may be involved in the
   chlorophyll metabolism pathway; the green color of aubergine pericarp
   is mainly affected by chlorophyll KEGG Pathway enrichment yielded 27
   DETs related to chlorophyll metabolism, and wheat breeders indicated
   that the up-regulation of photosynthesis-antenna protein genes could
   maintain the green color of leaves for a long time^[149]46, wheat
   breeders indicated that the up-regulation of photosynthesis-antenna
   proteins genes can maintain the green color of leaves for a long time,
   KEGG Pathway enrichment found 46 differentially expressed genes
   involved in photosynthesis-antenna eggs^[150]47, in grape (Vitis
   vinifera) fruit study, 122 DETs were found in KEGG Pathway enriched
   photosynthetic antenna proteins^[151]48. The candidate genes enriched
   in the KEGG Pathway for photosynthesis-antenna proteins for color trait
   changes in the fruits of DETs were less than those of
   aubergine^[152]46, wheat^[153]47, and grapes^[154]48, suggesting that
   DETs’ unique growth environments of high altitude, high humidity, and
   high shade are inherently caused by the unique growth environment of
   DETs.

   In order to further prove the reliability of the transcriptome data for
   the functional enrichment of key candidate genes for the color trait
   changes in the fruits of Panax japonicus, six highly expressed
   candidate genes of the photosynthesis-antenna protein pathway were
   selected by real-time fluorescence quantitative PCR (qRT-PCR) for
   qRT-PCR validation. The results pointed out that with the same pattern
   of change of transcriptome data, the expression of green fruit stage
   (G) was the highest; the gene expression decreased gradually with the
   maturity of fruit. The information of the candidate genes was obtained
   and named as PjCABI, PjCABII, PjCABIII, PjCAB1, PjCAB2, PjCAB3 through
   NCBI database comparison. 9 highly expressed key candidate genes for
   color trait changes were selected in KEGG Pathway flavonoid
   biosynthesis metabolism pathway for qRT-PCR verification. The results
   pointed out the same results with the transcriptome data, and the
   information of key candidate genes were obtained and named as PjF3’H,
   PjF3’H1, PjCHI, PjCHI1, PjCHS, PjCHS1, PjDFR, PjANS, and PjC3’H through
   the comparison of NCBI database, and it is noteworthy that the PjDFR
   gene had the highest expression among the nine candidate genes. It was
   found that the chlorophyll content during kiwifruit fruit development
   was highly correlated with chlorophyll degradation-related genes,
   especially three chlorophyll a/b binding proteins (CABs) genes play an
   important role in chlorophyll degradation^[155]49. Chlorophyll a/b
   binding protein (CAB) genes were also obtained during peach fruit
   (Prunus persica ’ Hujingmilu ’) development as key degradation proteins
   for chlorophyll metabolism^[156]50. These studies are highly consistent
   with the decrease in chlorophyll content and expression of six CAB
   genes of the related photosynthesis—antenna protein pathway during
   fruit development in Panax japonicus. Flavonoid metabolites are
   important pigmentation factors that play important roles in leaf,
   flower, fruit and seed tissues^[157]51,[158]52. The key enzyme genes
   CHI, CHS and F3′H were found to be the main factors affecting pericarp
   color in the color formation mechanism of passion fruit^[159]11. The
   key flavonoid biosynthesis genes F3’H and MiANS genes in the
   post-ripening process of green ripe fruits of the red mango variety
   Zill were also expressed in the variation of color traits^[160]13. All
   of these findings indicated the same as the nine candidate genes
   screened in the flavonoid biosynthesis pathway in Zill fruits, further
   suggesting that these candidate genes are the key enzyme genes for
   color trait changes in Panax japonicus fruits.

   The changes of color traits in the fruit of Panax japonicus showed an
   opposite trend in the dynamic pattern of chlorophyll content and
   carotenoids, anthocyanins, flavonoids and total phenolics in
   physiological and biochemical aspects. 19 candidate genes in the
   photosynthesis-antenna protein pathway related to chlorophyll
   degradation were also obtained in the transcriptome analysis, and 6 key
   candidate genes for high-expression were verified by qRT-PCR, and 81
   candidate genes were obtained in the flavonoid biosynthesis metabolic
   pathway, and 9 key candidate genes for high-expression were selected.
   81 candidate genes were obtained in flavonoid biosynthesis metabolism
   pathway, and 9 highly expressed key candidate genes were selected for
   qRT-PCR validation. Physiological, biochemical and transcriptomic
   analyses revealed the mechanism of fruit color trait changes in S.
   dubiae, and that the degradation of chlorophyll and the synthesis of
   carotenoids, anthocyanin and flavonoids were synchronized in the fruit.

Materials and methods

Plant material

   Panax japonicus fruits were collected from the Conservation Base of
   Bamboo Ginseng Germplasm Resources of ’Guizhou Innovation Centre of
   Mountain Specialty and Efficient Agriculture’ in Anshun university,
   Anshun City, Guizhou Province, China. Five different color change
   periods (G, Y, R, B, P) of the fruit were selected as shown in
   Fig. [161]1, and the fruit was washed with pure water, the excess water
   on the pericarp was sucked up by filter paper, and the seeds and
   pericarp were removed with a scalpel; the pericarp was frozen in liquid
   nitrogen for 15 min, and then the pericarp was grinded in a mortar and
   pestle, and then stored in a refrigerator at -80℃ for later use.

Determination of physiological index items

   Using the enzyme standard detection method, the powder samples of Panax
   japonicus pericarp stored in the ultra-low-temperature refrigerator at
   – 80 ℃ for five periods were taken out, and each sample was weighed
   into three portions of 0.1 g, and the extraction method was carried out
   in accordance with the (solarbio) kit for the determination of the
   contents of chlorophylls (BC0995), carotenoids (BC4335), anthocyanin
   (BC1385), flavonoids (BC1335), and total phenols (BC1345). BC1345)
   content determination.

RNA extraction and detection

   Total RNA was extracted from 15 samples of Panax japonicus pericarp (5
   different fruit color traits, 3 biological replicates) using Tiangen
   Kit, RNAprep Pure. The integrity and quality of RNA was assessed by
   agarose gel (Agarose horizontal electrophoresis machine model DYCP-32C)
   and Agilent Agilent 2100 Bioanalyzer.

Library construction and sequencing

   The first strand of cDNA was synthesised in M-MuLV reverse
   transcriptase system using fragmented mRNA as template and random
   oligonucleotides as primers, and the second strand of cDNA was
   synthesised using dNTPs as raw material. The double-stranded cDNA was
   purified and subjected to PCR amplification to finally obtain the
   library. Qubit2.0 Fluorometer was used for preliminary quantification,
   and the library was diluted to 1.5 ng/ul. Agilent2100 bioanalyzer was
   used to insertsize the library to ensure the quality of the library.
   Quality control of sequencing data (Trimmomatic) and de-hosting
   sequences (Bowtie2)^[162]53 were performed to obtain valid analysed
   sequences (clean data).

Transcript splicing and clustering

   After obtaining clean reads, the clean reads were spliced to obtain
   reference sequences for subsequent analyses. Trinity^[163]54 was used
   to splice the clean reads, and Corset^[164]55 aggregated the Shared
   Reads transcripts into many clusters, combining the transcript
   expression levels and the H-Cluster algorithm to create new clusters
   defined as ’Gene ’. Single-copy direct homologous gene pools were
   evaluated using BUSCO^[165]56 to ensure the integrity of the assembly.

Functional annotation and gene expression level analysis

   The Trinity spliced Unigene sequence was used as the reference sequence
   (Ref), and the clean reads of each sample were compared towards the Ref
   to obtain the spliced transcripts using (RSEM^[166]57 software bowtie2
   as the default parameter), and the transcripts were subjected to Nr,
   Nt, KOG, Swiss-prot, Uniprot, KEGG, and Nr, Nt, KOG, Swiss-prot,
   Uniprot, KEGG, GO functional annotations were performed on the
   transcripts to obtain functional information of the gene.

Differential gene expression analysis (DETs)

   Normalisation (normalization) of the raw read count, which is mainly a
   correction for sequencing depth, was performed by statistical modeling
   for probability of hypothesis testing (P-value), and correction for
   multiple hypothesis testing (BH) was performed to obtain FDR values.
   Genes were considered to have expression differences if they differed
   in expression by more than twofold. Padj was introduced to correct the
   hypothesis test P-value, thus controlling the proportion of false
   positives.

WGCNA of differential genes (DETs)

   The BioMorker Cloud Platform Weighted Gene Co-Expression Network
   Analysis (WGCNA) tool was used to mine gene module (module) information
   in the expression data. We uploaded genes.TMM.fpkm gene expression
   summary table and Trait file (analysis parameters: Fold 0.5, min
   ModuleSize 30, ntop 150) to explore the correlation between the modules
   and the phenotypes of color change traits and physiological indexes of
   the fruits of Panax japonicus for the identification of target network
   genes.

GO and KEGG pathway enrichment analysis of DETs

   The GO enrichment analysis was performed by the enricher function of
   clusterProfiler, using padj < 0.05 as the significant enrichment,
   mapping all the differentially expressed genes to the Gene Ontology
   database term, and counting the number of genes significantly enriched
   for each GO term. KEGG Pathway integrates genome chemistry and systemic
   function. KEGG Pathway integrates genome chemistry and system function
   information content, and Pathway significant enrichment analysis was
   performed with absolute log2 (Foldc hange) > 1& padj < 0.05.

Quantitative RT-PCR (qRT-PCR) validation

   The cDNA of Panax japonicus fruit was used as template for PCR
   reaction, and Primer3web version 4.1.0 was used to design gene-specific
   primers for the selected candidate genes (Supplementary Table 10).
   qRT-PCR was performed using a real-time fluorescence quantification
   instrument (LightCycler480II, 384). Actin (nei) was used as an internal
   reference gene to normalise the expression level^[167]58, and the
   relative expression level of each candidate gene was quantified using
   the 2^−ΔΔCt method.

Supplementary Information

   [168]Supplementary Information 1.^ (13.7KB, xlsx)
   [169]Supplementary Information 2.^ (11.3KB, xlsx)
   [170]Supplementary Information 3.^ (88.7KB, docx)
   [171]Supplementary Information 4.^ (11.9KB, xlsx)
   [172]Supplementary Information 5.^ (10.9KB, xlsx)
   [173]Supplementary Information 6.^ (12.5KB, xlsx)
   [174]Supplementary Information 7.^ (13.1KB, xlsx)
   [175]Supplementary Information 8.^ (12.9KB, xlsx)
   [176]Supplementary Information 9.^ (1.2MB, xlsx)
   [177]Supplementary Information 10.^ (57.1KB, xlsx)
   [178]Supplementary Information 11.^ (11.4KB, xlsx)

Acknowledgements