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Article

Analysis of Transcriptome and Expression of C4H and FLS Genes on Four Flower Colors of Impatiens uliginosa

by
Xiaoli Zhang
†,
Yi Tan
†,
Xinyi Li
,
Zengdong Liu
,
Fan Li
,
Haiquan Huang
* and
Meijuan Huang
*
College of Landscape Architecture and Horticulture Sciences, Southwest Research Center for Engineering Technology of Landscape Architecture (State Forestry and Grassland Administration), Yunnan Engineering Research Center for Functional Flower Resources and Industrialization, Research and Development Center of Landscape Plants and Horticulture Flowers, Southwest Forestry University, Kunming 650224, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2024, 10(4), 415; https://doi.org/10.3390/horticulturae10040415
Submission received: 25 March 2024 / Revised: 13 April 2024 / Accepted: 18 April 2024 / Published: 19 April 2024
(This article belongs to the Special Issue Physiological and Molecular Biology of Ornamental Plants—2nd Edition)
Browse Figures
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Abstract

:
Flower color is a major feature of ornamental plants, and the rich flower color of plants is an important factor in determining their ornamental and economic values, so flower color is an important research target for gardening and horticulture breeders at home and abroad. Our research group collected four colors of Impatiens uliginosa (white, pink, red, and deep red) during the collection of germplasm resources in the field. In this study, we analyzed the transcriptomes of the four flower colors of I. uliginosa by using RNA-Seq technology. The transcriptomes were screened to identify candidate genes related to flower color, and the coloring mechanisms of four flower colors were revealed at the molecular level. The main findings were as follows: (1) The number of the four different transcripts ranged from 64,723 to 93,522 and contained a total of 100,705 unigenes. (2) The analysis of differentially expressed genes revealed structural genes including C4H, FLS, PAL, and ANS and transcription factors including MYB, MYB-related, AP2-EREBP, and bHLH. (3) Among the four flower colors of I. uliginosa, the C4H1 gene had the highest expression in pink flowers, and the C4H2 gene had the highest expression in red flowers. This indicated that C4H genes positively regulated the red flower color of I. uliginosa. However, FLS expression was the highest in white flowers, and with deepening flower color, FLS gene expression gradually weakened, acting as a negative regulator. The results of this study could lay the theoretical foundation for investigating the mechanism of coloration and flower color variation in I. uliginosa.

1. Introduction

Flower color is an important ornamental trait of garden plants, which determines the economic and ornamental value of plants and has important biological significance such as attracting insect pollinators, reducing stress, and perpetuating the race [1]. The main coloring pigments of plant flowers and fruits are carotenoids, flavonoids, and betalains [2]. The formation of flower color is the result of a combination of many factors. pH, pigment type, auxin effect, metal ions, petal epidermal cell structure, and pigment number affect the coloration and variation mechanism of plant flower color [3,4].
There are large differences in the coloration mechanism of anemones originating from different regions and with different flower colors, including pigment types, mineral elements, and developmental stages. Researchers have isolated kaempferol, quercetin, geranium pigments, cornflower pigments, delphinium pigments, and their derivatives from the petals of red Impatiens balsamina [5,6]. Clevenger found that cornflower pigments were the most widely distributed, followed by mallow pigments, by determining the anthocyanins of 19 species of Impatiens [7]. Hasan identified nine flavonoids, three of which were new, by determining the leaves of the Impatiens bicolor [8]. Tatsuzawa et al. isolated mallow pigments and their derivatives from purple Impatiens textori Miqs [9]. Isolation of two new quercetin glycoside compounds from Impatiens balsamina was achieved by Lei et al. [10]. Mariana found dihydromyricetin, quercetin, kaempferol, and their derivatives by determining the flavonoids in the petals of Impatiens glandulifera [11].
Genes play an important role in the biosynthesis of anthocyanin glycosides, and the expression of these genes is closely related to anthocyanin content, thus affecting the flower color of plants [12]. Cinnamate-4-hydroxylase (C4H) alias trans-cinnamate-4-monooxygenase is an important enzyme for step 2 in the synthesis of anthocyanin. It can form a complex with PAL and 4CL and then immobilize it through the N-terminal hydrophobic helix region, which plays an important role in the chemical transfer and subcellular localization of the electron chain [13], catalyzing the conversion of trans-cinnamic acid to 4-coumaric acid, which then efficiently modulates carbon metabolism pathways [14,15]. C4H is a member of the cytochrome P450 oxidase (Cytochrome P450, CYP450) family and belongs to the CYP73 subfamily [16]. The first P450 enzyme in plants to be clonally characterized and functionally determined is C4H, which maintains high activity in plant tissues compared to other P450s [17]. It has been shown that the protein activity of C4H directly affects the synthesis of flavonoid compounds, lignin, aromatic compounds, etc., in plants [18] and is considered to be an important regulatory point. Baek found that the changes in C4H gene expression during the development of blackberries (Korean black raspberry) were the same as the changes in flavonoid accumulation [19]. Studies on the expression pattern of the C4H gene in plants inferred that its expression level is also closely related to lignin biosynthesis [20].
In addition, the flavonol synthase gene (FLS) has an effect on the accumulation of anthocyanin glycosides and the synthesis of flavonols as well as the color presentation of plants [21], and the FLS gene determines the production of plant flavonoids. The FLS gene dihydroflavonols was catalyzed by FLS to produce flavonols. FLS is a member of the 2-ketoglutarate-dependent dioxygenase (2-ODD) family of enzymes, to which anthocyanidinsynthase (ANS) and flavonol synthase (FLS) in the flavonoid metabolic pathway belong [22]. Tanaka isolated and cloned the FLS gene from Petunia hybrida and also enabled its successful expression in yeast [23]. Subsequently, isolation and cloning of FLS genes have been reported in a large number of plants such as Solanum tuberosum, Arabidopsis thaliana, Ginkgo biloba, strawberry (Fragaria × ananassa), etc. [24,25,26,27,28]. It has also been explored that the reaction substrates of the ANS gene and the FLS gene are identical, i.e., there was competition and the amino acid sequence similarity between the two was high [29], which further confirms that the FLS gene has an effect on the synthesis of both phytoflavonols and anthocyanins.
The group collected I. uliginosa, which belongs to the genus Impatiens of the family Balsaminaceae of the herbaceous plants, mainly distributed in Yunnan, Guizhou, Guangxi, and other places in China in the early stage of the collection of germplasm resources in the field [30]. There are four different flower colors of I. uliginosa, deep red, red, pink, and white, in different habitats or in the same habitat, and the color is bright. The study of the coloring mechanism of this mutant strain is of great significance to the molecular breeding of flower color in Impatiens. Currently, the study of flower color in Impatiens is mainly focused on the determination of flavonoids in petals and the cloning and expression of structural genes in the synthesis pathway of anthocyanin, while the transcriptional regulation of the molecular mechanism of flower color presentation in the same species with multiple colors has not yet been resolved. Therefore, in this study, we mined the transcriptome data of four different flower colors of I. uliginosa to screen the genes related to the regulation of flower color.

2. Materials and Methods

2.1. Plant Materials

The test material used in this experiment was I. uliginosa, a wild species of Impatiens, which has four different flower colors: white, pink, red, and deep red. They were collected from Aziying, Laoyuhe Wetland Park, Kunming City, Yunnan Province, and Dadieshui, Shilin Yi Autonomous County, Yunnan Province, respectively. Five plants of each of the four flower colors of I. uliginosa were selected to be robust and free of pests and diseases, and the whole flower organ of one flower in the state of full bloom was extracted from each plant, which was separated from the plant and quickly put into liquid nitrogen. The four flower colors were mixed separately, labeled, and then stored at −70 °C (Figure 1).

2.2. Transcriptome Sequencing and Analysis

2.2.1. RNA Extraction and Detection

Total RNA was extracted from 1 mg of the combined samples by a grinder using the OMEGA Quick RNA Isolation Kit according to the instructions. Contaminating genomic DNA was removed using RNAase-free DNase I. RNA purity was determined using a spectrophotometer, and RNA integrity was verified using an agarose gel. Library and RNA-Seq construction were performed by UW Genetics; the constructed libraries were sequenced using the Illumina sequencing platform after passing quality control.

2.2.2. Second-Generation Sequencing and Assembly

The cDNA library was constructed by Illumina’s NEB Next®Ultra™ RNA Library Prep to obtain raw data, followed by QC to obtain clean data to assemble a non-redundant unigene from scratch. In this study, we sequenced four different flower colors and assembled the I. uliginosa flower color transcriptomes named IUDR (deep red), IUR (red), IUP (pink), and IUW (white), respectively.

2.2.3. Sequence Analysis and Annotation

After the sequencing data were offloaded, it was necessary to assemble the nucleotide sequences of unigenes of I. uliginosa through quality control, filtering, and assembly steps. In order to fully obtain the functional annotation of unigene, this study used the comparison software BLASTX v2.2.23 (BasicLocal Alignment Search Tool) (E-value ≤ 1 × 10−5) to compare the unigene sequences with the open databases, so that they can be functionally annotated and categorized. The public alignment databases include GO (Gene Ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes, http://www.genome.jp/kegg, accessed on 3 December 2023), Swiss-Prot (A manually annotated and reviewed protein sequence database, http://www.genome.jp/kegg, accessed on 4 December 2023), and the Unigene Sequence Search Tool (E-value ≤ 1 × 10−5). And the reviewed protein sequence database (http://www.ebi.ac.uk/uniprot/, accessed on 6 December 2023), COG/KOG (EuKaryotic Orthologous Group, http://ftp.ncbi.nih.gov/pub/COG/KOG, accessed on 7 December 2023), eggNOG (evolutionary genealogy of genes: Non-supervised Orthologous Groups, http://eggnogdb.embl.de/, accessed on 10 December 2023), Pfam (a database of conserved Protein families or domains), and NR (NCBI non-redundant proteins, https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 11 December 2023) databases were compared (E-value ≤ 1.0 × 10−5) [30].

2.2.4. Screening and Enrichment Analysis of Differentially Expressed Genes (DEGs)

The assembled sequences were compared with the protein database for Blastx (http://www.ncbi.nlm.nih.gov/BLAST/, accessed on 15 December 2023), and based on the comparison results, the expression levels of the genes were quantified using the software RSEM v1.2.12, after which the expression levels of the genes were normalized using the FPKM method, and the differential genes were screened on the basis of the expression amount.

2.2.5. Related Gene Expression and Analysis

Primers were designed according to the sequences of IuC4H1, IuC4H2, and IuFLS, and IuActin was used as the internal reference gene (Table S1). The expression differences of IuC4H1, IuC4H2, and IuFLS were analyzed by qRT-PCR on the bloom samples of four flower colors of I. uliginosa, and three biological replicates were set for each sample. qRT-PCR was carried out on a Roche: LightCycler® 480 II Fluorescent Quantitative PCR Instrument. The qRT-PCR reaction system (20 µL) included the following: 10 µL of qPCR SYBR Green Master Mix, 7 µL of double-distilled water, 1 µL each of forward and reverse primers, and 1 µL of template cDNA. The specific program was as follows: predenaturation at 95 °C for 15 s, denaturation at 60 °C for 30 s, and annealing at 72 °C for 1 min, for 40 cycles. The relative expression of genes was analyzed by using the 2−ΔΔCt calculation method. Data were analyzed with the help of SPSS software 27.0 and plotted by Origin software 64.

3. Results

3.1. RNA Sequencing and Transcriptome De Novo Assembly

In order to understand the molecular mechanism of flower color variation in I. uliginosa, the transcripts were assembled from scratch using a reference-free genome approach to construct four high-throughput sequencing libraries of four different flower colors of I. uliginosa, namely, IUDR (deep red), IUR (red), IUP (pink), and IUW (white). After removing the articulated subsequence, ambiguous reads, and low-quality reads, the sequencing depths from the transcriptome data of IUDR, IUR, IUP, and IUW, respectively, ranged from 6.65 to 6.77 GB with an average depth of 6.5 GB. The Q30 values ranged from 95.94 to 96.09% with an average of 96.00%, and the net number of reads ranged from 44.35 to 45.11 M, with a net reads ratio of 83.02~87.71% (Table 1).
A count of the length and number of transcripts revealed that the number of the four different transcripts ranged from 64,723 to 93,522, with an average length of around 900 bp and an N50 of around 1500 bp (Table 2). The total sample of the four different transcriptomes of unigenes contained 100,705 full-length cDNA sequences with 115,842,379 bases, of which the average length of transcript unigene was 1150 bp (Figure 2), the N50 was 1890 bp, and the highest number of fragments distributed at 300 bp was 22,613, which was 22,613, which accounted for a total unigene percentage of 22.45%. The number of unigenes larger than 3000 bp accounted for 6.54% (6592 fragments) of the total number (Table 3).

3.2. Functional Annotation of Transcriptome Genes of I. uliginosa

The results of sequence match analysis with other species showed that Vitis vinifera had 10,213 homologous genes, accounting for 14.32% of the total, followed by Sesamum indicum with 4318 homologous genes, accounting for 6.06% of the total, Coffea canephora with 3458 homologous genes, accounting for 4.85% of the total, and Solanum tuberosum with 2983 homologous genes, accounting for 4.18% of the total (Figure 3). A total of 100,705 unigenes were successfully annotated by searching five public databases based on BLASTx (E-value < 1 × 10−5), among which the NCBI-NR database contained 71,307 unigenes, Swiss-Prot contained 50,638 unigenes, the KEGG database contained 55,276 unigenes, the InterPro database contained 59,058 unigenes, and the KOG database contained 32,641 unigenes (Figure 4, Table 4).

3.3. GO (Gene Ontology) and COG (Clusters of Orthologous Group of Proteins) Annotation Analysis of I. uliginosa Transcriptome Data

The GO gene pool contained three gene attribute sections: molecular function, biological process, and cellular component. The transcriptome of I. uliginosa contains 71,307 GO-annotated genes, accounting for 70.81% of the whole annotation, and consists of 58 different annotated gene functional regions. Among them, the biological process section contained 14,490 genes, the metabolic process contained 14,948 genes, and the single-organism process contained 9082 genes; the molecular function section contained 13,079 genes and the catalytic activity process contained 14,757 genes; the cellular component section contained 9905 genes, the cell cellular component contained 9905 genes, the cell part contained 11,934 genes, and the cell contained 12,094 genes. In the three categories of “translation regulator activity”, “locomotion”, and “cell killing”, the unigenes were annotated to 1, 2, and 4 in each category (Figure 5).
The COG database had the ability to infer unknown gene functions and compare homologous protein sequences, which accounted for 32.41% of the total annotated genes in the I. uliginosa transcriptome COG database, of which a total of 32,641 genes were annotated. The annotated genes were categorized into 25 classes according to their functions. General function prediction only contained a total of 9925 genes; replication, recombination, and repair contained 4816 genes; transcription contained 5406 genes. Post-translational modification, protein turnover, and chaperones contained 4029 genes (Figure 6).

3.4. Identification and Functional Annotation of Differential Genes (DEGs) in I. uliginosa

Based on the results of the gene expression levels of each sample, the number of genes obtained for each different flower color was analyzed using a Venn diagram, and the results showed that there was a large gap in the expression of genes in the four samples of different flower colors, with a total of 1402 genes between IUDR and IUP; a total of 1639 genes between IUDR and IUR; and a total of 1842 genes between IUDR and IUW; a total of 992 genes between IUP and IUR. The number of genes between IUP and IUP totaled 9929; the number of genes between IUP and IUW totaled 1157; and the number of genes between IUR and IUW totaled 1087. There were a total of 38,191 genes between the four samples, namely, IUDR, IUP, IUW, and IUR. Among them, the C4H and FLS genes were better expressed (Figure 7A).
Differentially expressed genes (DEGs) were detected using the DEseq2 algorithm, and the results of DEG detection are shown in Figure 7B. Subsequently, gene expression in the IUDR, IUP, IUW, and IUR samples was compared two-by-two, and the results showed that downregulated expression of differentially expressed genes (DEGs) predominated in the comparison of the four different flower colors (Figure 7B). A total of 6197 DEGs were expressed in IUDR vs. S IUW, of which 6098 were upregulated and 14,650 were downregulated. A total of 6197 DEGs were expressed in IUDR vs. IUP, of which 15,519 were upregulated and 13,894 were downregulated. A total of 6197 DEGs were expressed in IUW vs. IUP, of which 6234 were upregulated and 13,790 were downregulated. There were a total of 6197 DEGs in IUDR vs. IUR, with 14,353 upgrades and 15,166 downgrades; a total of 6197 DEGs in IUR vs. IUP, of which 5844 were upgraded and 7216 were downgraded; and a total of 6197 DEGs in IUR vs. IUW, with 6496 upgrades and 16,618 downgrades. In the IUDR vs. IUP, IUDR vs. IUR, and IUR vs. IUP two-by-two comparisons, there was little difference between the number of upward and downward adjustments in DEGs, while the number of downward adjustments was greater than the number of upward adjustments in the other three comparisons.
The distribution of DEGs was also demonstrated using a volcano plot. In the IUDR vs. IUR comparison, the number of DEGs had a statistically significant change in the expression level in the volcano plot over the other comparisons (Figure 7C).

3.5. Identification and Expression Analysis of Transcription Factors in I. uliginosa

Transcription factors in plants are involved in the regulation of various metabolic pathways in plants, which enable plants to have a wider range of adaptations, and transcription factors were thought to have a wider regulation in anthocyanin biosynthesis than structural genes. Therefore, we predicted the unigenes encoding transcription factors from the transcriptome data of four different flower colors of I. uliginosa, and a total of 3493 unigenes were identified, which could be classified into 59 families (Figure 8), most of which were from the “MYB” family (429 unigenes), which accounted for 12.47% of the total number of unigenes, and the “MYB-unigenes” family (12.47% of the total number of unigenes). The “MYB-related” family (355 unigenes) accounted for 10.32% of the total, the “AP2-EREBP” family (227 unigenes) accounted for 6.60% of the total, and the “bHLH” family (227 unigenes) accounted for 6.60% of the total. The “bHLH” family (221 unigenes) accounted for 6.42% of the total. MYB and MYB-related transcription factors are essential for the plant flavonoid metabolic pathway.

3.6. qRT-PCR Validation of the Candidate Genes

Two candidate genes were selected for qRT-PCR to verify the accuracy of the transcriptome data (Figure 9). There were significant differences in the expression of IuC4H1, IuC4H2, and IuFLS genes in different flower colors of I. uliginosa. The results showed that the C4H1 gene was most expressed in pink flowers, slightly more in white flowers than in deep red flowers, and least in red flowers, with a significant difference in expression between pink and red. Expression of the C4H2 gene was highest in red flowers, slightly higher in pink flowers than in deep red flowers, and weaker in white flowers, significantly different from the other three flower colors. FLS expression was highest in white flowers and lowest in deep red flowers and was significantly different from the other two flower colors. As the flower color deepened, the expression of FLS gradually weakened. Thus, among the four flower colors of I. uliginosa, the C4H1 gene was expressed most frequently in pink flowers, the C4H2 gene was expressed most frequently in red flowers, and the FLS was expressed most frequently in white flowers, suggesting that both the C4H and FLS genes are involved in the formation of I. uliginosa flower color.

4. Discussion

The origin and evolution of flowers is a key issue in exploring the origin and evolution of angiosperm species. Meanwhile, flower color is an epigenetic and evolutionarily adapted phenotypic trait in ornamental plants, and the quality of flower color determines the ornamental and economic value of plants. Flowering plants will show a wide range of flower colors, with multiple colors of the same species and multiple species of the same color [2,31]. From previous studies, the genus Rhododendron (Ericaceae) has more than 1000 flower colors with unique ornamental value and a model system for flower color studies. Nie et al. investigated the differences in color-presenting mechanisms of two rhododendron species, yellow-flowered R. molle and red-flowered R. simsii, through a multilocus study [32]. For plants of the same genus, differences in key enzymes or gene regulatory mechanisms for pigment synthesis can also result in differences in flower color [33]. Researchers also explored genetically related genes for flower color and pigmentation by studying small-flowered chrysanthemums with different flower colors [34]. Studies on the flower color of homozygous ornamental plants are still scarce.
In this study, four flower colors of I. uliginosa were used as research materials, and transcriptome sequencing was performed to lay the foundation for investigating the molecular mechanism of flower color synthesis in I. uliginosa. The length and number of transcripts were counted, and it was found that the number of different transcripts of the four flower colors of I. uliginosa ranged from 64,723 to 93,522, with an average length of about 900 bp and an N50 of about 1500 bp. The total sample of the four different transcriptomes of unigenes contained 100,705 full-length cDNA sequences with 115,842,379 bases, of which the average length of transcript unigene was 1150 bp, the N50 was 1890 bp, and the highest number of fragments distributed in the 300 bp was 22,613 fragments, which accounted for 22.45% of the total number of unigenes. The number of unigenes larger than 3000 bp was 6592, accounting for 6.54% of the total number of unigenes.
The C4H gene is a key enzyme gene in the second step of the anthocyanin biosynthesis pathway [35], and also, the activity of this enzyme can affect the synthesis of compounds such as lignin and aromatic species, which has an important role in plant secondary metabolism [36]. In the four flower colors of I. uliginosa at full bloom, C4H1 was expressed highest in pink and lowest in red, whereas C4H2 was expressed highest in red and lowest in white, and thus it was hypothesized that the C4H2 gene had a stronger positive effect than C4H1 on flower color formation in I. uliginosa, in the same way that only one of the two copies of osmanthus is highly expressed in petals [37] and may separately fulfill their respective functions.
Flavonols are one of the major co-pigments that enhance the stability of anthocyanin glycosides and influence flower color formation. In the metabolic pathway of flavonoid compounds, flavonols share a presynthesis pathway with anthocyanin glycoside biosynthesis. Flavonol synthase (FLS) catalyzes the conversion of dihydroflavonols to flavonols [38]. The relative expression of the FLS gene in the four flower colors differed more significantly, and the highest expression was found in white flowers of I. uliginosa. This inference was consistent with the results of Ping in his functional validation of the FLS gene in roses, where they found that transgenic tobacco had increased flavonol content and flower differentiation to white [39]. Suppression of the FLS gene in Chinese bellflower was followed by substantial red coloration at the bud stage of the transgenic strain and pigment accumulation in the pistil, a phenotype that was evident in the progeny after crosses [40]. It can be hypothesized that the FLS gene may play an important role in the formation of white flowers of I. uliginosa.

5. Conclusions

In this study, four flower colors (white, pink, red, and deep red) of I. uliginosa were used as materials, and transcriptome sequencing was performed on the petals of I. uliginosa at the full bloom stage to investigate the intrinsic mechanism of four flower colors in I. uliginosa from the point of view of molecular biology. At present, there are still few studies on the specific color formation mechanism of the four flower colors of I. uliginosa, and the previous studies mainly focused on physiological and biochemical experiments, such as the determination of anthocyanin and pH. This study is the first to perform transcriptome sequencing of the four flower colors of I. uliginosa, and it can lay a theoretical foundation for further study on the molecular mechanism of different flower colors of I. uliginosa and other ornamental plants.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae10040415/s1, Table S1: Sequences of primers related to qRT-PCR amplification.

Author Contributions

Software, F.L.; Investigation, Z.L.; Data curation, X.L.; Writing—original draft, X.Z.; Writing—review & editing, Y.T.; Supervision, H.H. and M.H. All authors have read and agreed to the published version of the manuscript.

Funding

This work was carried out with the support of the National Natural Science Foundation of China [32060364, 32060366, 31860230], Joint Special Key Project for Agricultural Basic Research in Yunnan Province(202101BD070001-018), Major scientific and technological projects in Yunnan Province [202102AE090052], and Program for Doctoral Supervisors Team in Genetic Improvement and High-efficient Propagation of Landscape Plants in Yunnan Province.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Acknowledgments

Thanks to Southwest Forestry University for providing experimental instruments and equipment for this experiment, and also thanks to teachers Haiquan Huang and Meijun Huang for their patient guidance during the experiment.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Blooming period of four different color flowers of I. uliginosa. Bars = 1 cm.
Figure 1. Blooming period of four different color flowers of I. uliginosa. Bars = 1 cm.
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Figure 2. Length distribution of All-Unigene.
Figure 2. Length distribution of All-Unigene.
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Figure 3. Percentages of unigenes that align with sequences from top five species in the NR database.
Figure 3. Percentages of unigenes that align with sequences from top five species in the NR database.
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Figure 4. Annotation from five databases. Note: KEGG: Kyoto Encyclopedia of Genes and Genomes; COG: Clusters of Orthologous Groups of proteins; NR: NCBI non-redundant protein sequences.
Figure 4. Annotation from five databases. Note: KEGG: Kyoto Encyclopedia of Genes and Genomes; COG: Clusters of Orthologous Groups of proteins; NR: NCBI non-redundant protein sequences.
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Figure 5. Annotation of unigenes searched against GO databases.
Figure 5. Annotation of unigenes searched against GO databases.
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Figure 6. Annotation of unigenes searched against KOG databases.
Figure 6. Annotation of unigenes searched against KOG databases.
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Figure 7. The expressed genes analysis between petals at four different stages: (A) Venn diagram shows the expression of genes in IUDR, IUP, and IUR samples. (B) Venn diagram showing the DEGs between IUDR vs. IUP, IUDR vs. IUR, and IUP vs. IUR. (C) The numbers of up- and downregulated genes between IUDR vs. IUP, IUDR vs. IUR, and IUP vs. IUR. Volcano plots of transcriptome between IUDR vs. IUP, IUDR vs. IUR, and IUP vs. IUR. Red dots represent upregulated DEGs. Blue dots represent downregulated DEGs. Black points represent non-DEGs.
Figure 7. The expressed genes analysis between petals at four different stages: (A) Venn diagram shows the expression of genes in IUDR, IUP, and IUR samples. (B) Venn diagram showing the DEGs between IUDR vs. IUP, IUDR vs. IUR, and IUP vs. IUR. (C) The numbers of up- and downregulated genes between IUDR vs. IUP, IUDR vs. IUR, and IUP vs. IUR. Volcano plots of transcriptome between IUDR vs. IUP, IUDR vs. IUR, and IUP vs. IUR. Red dots represent upregulated DEGs. Blue dots represent downregulated DEGs. Black points represent non-DEGs.
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Figure 8. Transcription factor family classification of unigenes.
Figure 8. Transcription factor family classification of unigenes.
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Figure 9. The relative expression of C4H1, C4H2, and FLS genes in four flower colors of I. uliginosa. Data are shown as the mean ± standard error of the mean, based on three replicates. Different letters indicate significant differences at p < 0.05 according to Duncan’s multiple range test.
Figure 9. The relative expression of C4H1, C4H2, and FLS genes in four flower colors of I. uliginosa. Data are shown as the mean ± standard error of the mean, based on three replicates. Different letters indicate significant differences at p < 0.05 according to Duncan’s multiple range test.
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Table 1. Sequencing quality of transcripts of four different colors.
Table 1. Sequencing quality of transcripts of four different colors.
SampleTotal Raw Reads (Mb)Total Clean Reads (Mb)Total Clean Bases (Gb)Clean Reads Q20 (%)Clean Reads Q30 (%)GC (%)Clean Reads Ratio (%)
IUDR52.6944.626.6998.7896.0941.4684.68
IUP54.3345.116.7798.759642.7783.02
IUR52.6944.356.6598.7896.1142.284.17
IUW51.0444.776.7198.7295.9441.7187.71
Table 2. Quality indicators for transcripts of four different colors.
Table 2. Quality indicators for transcripts of four different colors.
SampleTotal NumberTotal LengthMean LengthN50N70N90GC (%)
IUDR79,27769,699,393879154594633641.46
IUP91,10976,919,883844152186831842.77
IUR93,52280,820,572864153090332942.2
IUW64,72359,663,736921160098236141.71
Table 3. Unigene data summary of four different colors.
Table 3. Unigene data summary of four different colors.
SampleTotal NumberTotal LengthMean LengthN50N70N90GC (%)
IUDR55,00358,568,93010641689113546241.44
IUP67,73566,343,5599791646101639042.76
IUR70,68570,639,2989991640104640542.2
IUW48,34551,364,23410621702112145341.68
All-Unigene100,705115,842,37911501890126749541.98
Table 4. Annotation of unigenes searched against NR, Nt, Swiss-Prot, KEGG, KOG, InterPro, and GO databases.
Table 4. Annotation of unigenes searched against NR, Nt, Swiss-Prot, KEGG, KOG, InterPro, and GO databases.
ValuesNumberPercentage
NR-Annotated71,30770.81%
Nt-Annotated59,00158.59%
Swissprot-Annotated50,63850.28%
KEGG-Annotated55,27654.89%
COG-Annotated32,64132.41%
Interpro-Annotated59,05858.64%
GO-Annotated29,47329.27%
Overall77,87777.33%
Total100,705100%
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MDPI and ACS Style

Zhang, X.; Tan, Y.; Li, X.; Liu, Z.; Li, F.; Huang, H.; Huang, M. Analysis of Transcriptome and Expression of C4H and FLS Genes on Four Flower Colors of Impatiens uliginosa. Horticulturae 2024, 10, 415. https://doi.org/10.3390/horticulturae10040415

AMA Style

Zhang X, Tan Y, Li X, Liu Z, Li F, Huang H, Huang M. Analysis of Transcriptome and Expression of C4H and FLS Genes on Four Flower Colors of Impatiens uliginosa. Horticulturae. 2024; 10(4):415. https://doi.org/10.3390/horticulturae10040415

Chicago/Turabian Style

Zhang, Xiaoli, Yi Tan, Xinyi Li, Zengdong Liu, Fan Li, Haiquan Huang, and Meijuan Huang. 2024. "Analysis of Transcriptome and Expression of C4H and FLS Genes on Four Flower Colors of Impatiens uliginosa" Horticulturae 10, no. 4: 415. https://doi.org/10.3390/horticulturae10040415

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