Transcriptome and flavonoid metabolome analysis reveals the molecular basis of floral color divergence between the hybrid and their parental species in Paphiopedilum

Transcriptome and flavonoid metabolome analysis reveals the molecular basis of floral color divergence between the hybrid and their parental species in Paphiopedilum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Transcriptome and flavonoid metabolome analysis reveals the molecular basis of floral color divergence between the hybrid and their parental species in Paphiopedilum Quanshu Luo, Junming Chen, Cheng Wan, Leijia Li, Sheng Chen, Jihong Wang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6878590/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The artificial hybridization of Paphiopedilum began in 1869. With the breakthrough in aseptic sowing techniques, the number of hybrids surged. Over 30,000 hybrids have been registered by the Royal Horticultural Society (RHS). Hybrids derived from Paphiopedilum delenatii typically retain its prominent characteristics: large, symmetrical petals and a magenta-purple labellum. In this study, the hybrid of P. delenatii and P. primulinum exhibited an overall creamy-white coloration. This study focuses on the divergence in floral coloration between the hybrid and their parental lines. Through comparative transcriptome analysis and flavonoid metabolic profiling across three distinct Paphiopedilum , we aim to elucidate the molecular mechanisms underlying how hybrids deviate from the red and yellow-green phenotypes of their parents to develop a cream-colored pigmentation. Results The labellum and dorsal sepal morphology closely resembled P. primulinum , while the petal traits combined the wavy margins of P. primulinum and smooth surface of P. delenatii . Flavonoid metabolome analysis of the floral tissues, a total of 29 flavanones, 93 flavones and flavonols, 13 anthocyanins, 23 isoflavonoids, six flavonoids, and four chalcones and dihydrochalcones were detected. Remarkably, anthocyanin content is significantly higher in P. delenatii compared to P. primulinum and the hybrid. Transcriptome analysis highlighted significant up-regulation of genes in the flavonoid biosynthesis pathway and phenylpropanoid biosynthesis pathway when comparing hybrids to P. primulinum . Conversely, early-stage biosynthetic genes were down-regulated in hybrid relative to P. delenatii , likely contributing to metabolite content differences. Conclusion This study employed multi-omics analysis to compare the floral tissues of P. delenatii and P. primulinum , along with their hybrid progeny. It identified differences in flavonoid metabolite content among them. By analyzing differentially expressed genes (DEGs) within the KEGG flavonoid biosynthesis pathway, the molecular mechanism underlying the divergent floral pigmentation pattern of the hybrid compared to both parental species was explored. Analysis of transcriptomic data, through comparison with the non-redundant (NR) protein database, revealed a closer phylogenetic relationship between Paphiopedilum and the genus Dendrobium , whereas morphologically Paphiopedilum is classified under Cypripedium -related taxa. These findings provide novel insights and molecular evidence for understanding Paphiopedilum floral coloration and phylogenetic relationships. Paphiopedilum floral color Transcriptome flavonoid metabolome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Orchidaceae are the most diverse family of angiosperms, comprising 28000 species spread across 736 genera [1,2]. The Paphiopedilum genus (commonly known as " Lady's Slipper Orchid ") comprises 96-100 species globally and is primarily distributed in Southeast Asia, the Himalayan lowlands, and Southwest China [3]. Renowned for its slipper-shaped floral morphology and vibrant color variations, this genus holds significant horticultural value and is a prized ornamental plant in global markets [4]. China is the country with the richest diversity of Paphiopedilum species in the world. There are 27 native species indigenous to China, accounting for one-third of the globally recorded wild Paphiopedilum species, and most are distributed in Yunnan or Guangxi Province [3]. All Paphiopedilum species are included in Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). The natural distribution range of Paphiopedilum delenatii Guill. ( Orchidaceae ) spans northern Guangxi Province and southeastern Yunnan Province in China, as well as southern Vietnam [5]. Its floral morphology exhibits a diameter of 6-8 cm, with the dorsal sepal and synsepal presenting a pure white coloration, while the labellum displays a distinct pink pigmentation. This species displays notable adaptability to cultivation under controlled horticultural conditions [3]. On the other hand, Paphiopedilum primulinum M. W. Wood & P. Taylor is native to Sumatra and Java, Indonesia [6]. The flower has a diameter of 6.5-7 cm, with the dorsal sepal and synsepal displaying greenish-yellow coloration, while the petals and labellum are yellow[7]. Because of their uniqueness in flower color and morphology, they are widely sought after by orchid lovers. Therefore, the hybrid varieties of P. delenatii or P. primulinum as their parents are also welcomed by Paphiopedilum lovers, and have won a large number of prizes in the orchid exhibition [3]. Flowering plants typically rely on multi-sensory media, such as color, scent, and morphology, to attract pollinators, thereby ensuring successful reproduction [8]. Although some plants increase their attractiveness to pollinators through a combination of olfactory and visual signals [9], visual signals are still decisive for attracting pollinators [10]. The color of flowers is determined by secondary metabolites, including flavonoids, carotenoids, and betalains, which collectively regulate pigmentation based on their chemical structures and cellular environment [11]. Numerous studies on floral pigmentation have shown that flowers displaying red, purple, or blue colors exhibit high anthocyanin content, whereas pale or white flowers primarily contain flavonols or chalcones [12, 13]. Generally, within the same species, darker flowers colors are typically associated with higher flavonoid content, particularly anthocyanins [14], but the effect of total flavonoid content on flower color did not show obvious correlation [15]. The flavonoid biosynthesis pathway constitutes a key branch of the phenylpropanoid pathway extended via chalcone synthase (CHS), through which a multitude of flavonoid secondary metabolites are generated. These metabolites are broadly categorized into six classes: flavones, flavonols, flavanones, flavanols, anthocyanins, and isoflavones [16]. The flavonoid biosynthesis pathway is governed by a series of critical enzymes and genes, including phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL) [17]. The enzymatic pathway for flavonoid biosynthesis as follows: 4CL generates flavanones, such as naringenin, through the catalytic actions of chalcone synthase (CHS) and chalcone isomerase (CHI). When catalyzed by dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS), flavanone can be converted into anthocyanins; under the catalysis of flavonol synthase (FLS) along with flavonoid 3'-hydroxylase (F3'H) or flavonoid 3',5'-hydroxylase (F3'5'H), it generates flavonols; additionally, isoflavone synthase (IFS) and flavone synthase (FNS) mediate the production of isoflavones and flavones respectively[17]. These enzymes are regulated by their corresponding genes [18]. With the development of science and technology, transcriptomics has been gradually applied to botanical research. Through RNA sequencing technology, the gene expression pattern of plants can be comprehensively analyzed, and the regulatory mechanism of genes can be explained [19], and the complex gene expression network can be revealed [20]. In research of orchid plants, using metabolomics-transcriptomics combined analysis, reveal Dendrobium nobile flavonoid metabolite content and metabolic pathway different genes on flower color diversity impact, and obtain regulatory gene network [21]. The three Paphiopedilum plants used in this study exhibited yellow-green, purplish-red, and cream-white flower colors, respectively. Through integrated analysis of flavonoid metabolomics and transcriptomics, we performed a comparative analysis of the differences in flavonoid metabolism and gene expression between the hybrid and its parental lines. This approach revealed the molecular mechanisms underlying the segregation phenomenon of flower coloration between the hybrid plant and its parents. Results Characteristics of flavonoid metabolizing substances in Paphiopedilum L. The floral structure of the native species P. delenatii exhibit two broad, smooth, and glabrous white petals. The dorsal sepal is nearly white, while both the synsepal and the labellum display a uniform purple coloration (Fig. 1a). P. primulinum features petals with undulate margins and ciliate edges. The labellum is yellow-green, while the dorsal sepal and synsepal are green (Fig. 1b). The hybrid combines the broad petal morphology inherited from P. delenatii and the undulate petal margins from P. primulinum . The synsepal is yellow-green with sparse reddish pigmentation, while the dorsal sepal, petals, and labellum are uniformly cream-colored (Fig. 1c). Results of our determination of flavonoid metabolic content in these three Paphiopedilum plants，a total of 29 flavanones were detected, among which Hesperetin 5-O-glucoside, Eriocitrin, Poncirin, Naringenin and Butin were much higher in P. delenatii than in the other two. The contents of Naringenin and Butin in the cream-colored hybrid were higher than P. primulinum (Fig. 2A, Table S1). 93 flavones and flavonols were detected. There was no significant difference in the content of most compounds between P. primulinum and hybrid, while some metabolites, such as Kaempferol-3-O-rutinoside, Chrysoeriol 7-O-rutinoside, were found to be higher in P. delenatii than in the others (Fig. 2B). Among the 13 anthocyanins detected, the content of Cyanidin in P. delenatii was consistently higher than that in P. primulinum and the hybrid. In contrast, the concentration of Procyanidin A2 in the hybrid significantly exceeded those in its parental species (Fig. 2C). Additionally, 23 isoflavonoids, 6 flavonoids, and 4 chalcones and dihydrochalcones were identified. Among 168 flavonoid metabolites, the hybrid exhibited 45 metabolites with significant differential abundance compared to P. delenatii, of which 36 were down-regulated. In contrast, only 15 flavonoid metabolites showed significant differential abundance relative to P. primulinum, with 12 being up-regulated (Table 1). Table 1 The number of flavonoid metabolites in Paphiopedilum Compared Samples Num. of Total Ident. Num. of Total Sig. Num. of Sig. Up Num. of Sig. Down Hybrid vs Pd 168 45 9 36 Hybrid vs Pp 168 15 12 3 Pd vs Pp 168 47 40 7 Num. of Total Ident.: The number of total identified metabolites. Num. of Total Sig.: The number of totals significantly differential metabolites. Num. of Sig. Up: The number of totals significantly upregulated metabolites. Num. of Sig. Down: The number of totals significantly downregulated metabolites Analysis of transcriptome dataset The transcriptome data were compared with the annotation results of seven databases. 45.6 % of unigenes could be compared to NR database, only 8.28 % could be compared to KOG database. And more than 26% in GO, NT, PFAM and SwissProt databases, they were 26.82 %, 27.35 %, 26.82 % and 28.12 %, respectively (Fig. 3A, Table S2 ). When compared with the NR database maintained by NCBI, 20.3 % of the unigenes showed significant sequence alignment with Dendrobium catenatum , followed by Dendrobium nobile (14.7 %) and Dendrobium chrysotoxum (12.7 %) (Fig. 3B, Table S3 ). Transcriptomic data from three color morphs of Paphiopedilum floral tissues were subjected to pairwise sample comparisons to quantify differentially expressed genes (DEGs). Our analysis revealed 16,621 DEGs between P. delenatii (paternal parent) and P. primulinum (maternal parent), with comparable numbers of up-regulated (8,842) and down-regulated (7,779) genes in the yellowish-green P. primulinum relative to P. delenatii (Fig. 4A). The hybrid progeny exhibited 3,338 DEGs compared to P. delenatii and 4,623 DEGs relative to the P. primulinum (Fig. 4A). Obviously, the majority of DEGs in hybrid comparisons displayed up-regulation regardless of the parental line. Intersectional analysis identified 194 shared DEGs across all three Paphiopedilum plants (Fig. 4B). Analysis of differential expressed genes related flavonoid biosynthesis. KEGG (http://www.kegg.jp/ or http://www.genome.jp/kegg/) is an integrated database resource for biological interpretation of genome sequences and other high-throughput data. Molecular functions of genes and proteins are associated with ortholog groups and stored in the KEGG Orthology (KO) database [22]. Comparative analysis against the KO database revealed distinct unigene associations with flavonoid biosynthesis pathways in the hybrid progeny relative to P. delenatii (paternal parent) and P. primulinum (maternal parent). Specifically, 47 unigenes were annotated to the general flavonoid biosynthesis pathway (ko00941), 3 unigenes to anthocyanin biosynthesis (ko00942), and 14 unigenes to flavone/flavonols biosynthesis (ko00944). Notably, no unigenes correlated with isoflavonoid biosynthesis (ko00943) were identified in either hybrid-parent comparison (Fig. 5). Discussion Artificial hybridization breeding in the genus Paphiopedilum commenced in the 1860s [3]. Driven by market demand, the proliferation of artificially hybridized varieties accelerated significantly over time. This trend is quantitatively reflected in Sander's List of Orchid Hybrids , which documents a dramatic increase in registered orchid hybrids from 750 species in 1946 to over 130 000 by 2019 [23, 24]. Among numerous Paphiopedilum hybrids derived from P. delenatii as a parental species, the majority retain the characteristic large and symmetrical petals inherited from this species. A subset of these hybrids exhibits distinct floral features including magenta-purple labella or pure white petals, as exemplified by Paphiopedilum Magic Lantern 'Ralph' and Paphiopedilum Joyce Hasegawa 'Ruey-Hwa' [25]. The hybrid plant material utilized in this study was developed through artificial hybridization breeding techniques between P. primulinum and P. delenatii . The resulting hybrid exhibits a creamy-white coloration across its entire floral structure, with a slightly yellowish tint compared to the petals of P. delenatii . Morphological analysis revealed that the labellum and dorsal sepal closely resembled those of P. primulinum in morphology. Prominently, the petal morphology demonstrated an intermediate inheritance pattern: the undulate margins characteristic of P. primulinum were preserved, combined with the large, glabrous surface texture typical of P. delenatii (Fig. 1). This chimeric expression pattern suggests incomplete dominance in petal trait inheritance during interspecific hybridization. Analysis of flavonoid detection in floral tissues revealed that P. delenatii exhibited a higher abundance of flavonoid metabolites compared to P. primulinum . Similarly, the hybrid demonstrated flavonoid metabolite levels higher than those of P. primulinum but lower than those of P. delenatii when compared to their parental species (Fig. S2). In numerous studies on flower color, the content of flavonoids, especially anthocyanins, in dark flowers was higher than that in light flowers among the same species [26]. In this study, the anthocyanin content in P. delenatii , characterized by magenta-purple labella, was found to be significantly higher than that of the yellowish-green P. primulinum and hybrid cultivar, except for Petunidin 3-O-glucoside. Notably, Petunidin 3-O-glucoside, a compound which substantial accumulation induces deep red to purple pigmentation in plant tissues, was also detected at elevated levels in the Zikui tea plant ( Camellia sinensis cv. Zikui). Petunidin 3-O-glucoside, alongside cyanidin glycosides, is considered to co-regulate the expression of purple pigmentation traits in plant [27]. The Petunidin 3-O-glucoside in P. primulinum was significantly higher than that in the other two species, but did not show purple color, which may be due to the low content of cyanidin 3-O-glucoside. Beyond the direct chromatic effects of anthocyanins, other flavonoid metabolites, such as flavonoids and isoflavonoids, demonstrate substantially elevated levels in dark-pigmented floral tissues compared to lighter-colored counterparts [28], suggesting their synergistic or regulatory roles in pigmentation intensity [29]. In the transcriptome analysis, we also found three unigenes related to anthocyanin synthesis, 14 unigenes related to flavone and flavonol biosynthesis, and 47 unigenes related to flavonoid biosynthesis. Although it is not clear what role these genes play in the formation of the color of the Paphiopedilum. In this study, we performed transcriptome sequencing on the flowers of three Paphiopedilum plants, and it can be intuitively seen from the number of DEGs that most of the differentially expressed genes were up-regulated in the hybrid compared with its parents. In KEGG enrichment analysis, pairwise comparisons were conducted to examine the top 20 most significantly enriched pathways of differentially expressed genes among them. The DEGs enriched in flavonoid synthesis pathway and phenylpropanoid biosynthesis showed particularly significant enrichment (Fig. S3). The phenylpropanoid biosynthesis pathway and the flavonoid biosynthesis pathway share upstream pathways in phenylpropanoid metabolism but utilize distinct enzymes. In the flavonoid biosynthesis pathway, the phenylpropanoid biosynthesis pathway provides essential precursor substances [30]. In comparison with P. primulinum , the differentially expressed genes involved in the phenylpropanoid biosynthesis pathway and flavonoid biosynthesis pathway were all highly significant in hybrids. To visually explore the expression differences among the three Paphiopedilum plant within the flavonoid biosynthesis network, we first examined the pathway map of KEGG pathway ko00941 (Flavonoid Biosynthesis) (Fig. S4). Compared with P. primulinum , genes at most nodes in the flavonoid biosynthetic pathway of the hybrid were up-regulated (Fig. S4A). In contrast, when compared with P. delenatii , although genes at some nodes in the later stages of biosynthesis were up-regulated, the majority of genes at nodes in the early stages of biosynthesis were down-regulated. For example, multiple genes in pathways such as EC: 2.3.1.74 (CHS) and EC: 1.14.1491 (trans-cinnamate 4-monooxygenase) exhibited this pattern (Fig. S4B). The up-regulation and down-regulation of these genes might have influenced the differences in the final flavonoid metabolite content. With the application of multi-omics in botanical research, some scholars have conducted in-depth studies on the genomics and transcriptomics of Orchidaceae plants [31]. Researchers analyzed transcriptomic data of P. armeniacum , P. concolor , P. hirsutissimum and P. malabellumoense , revealing three whole-genome duplication (WGD) events during the evolutionary history of Paphiopedilum, the resultsuggested that Paphiopedilum belongs to the subfamily Cypripedioideae , which diverged from its sister clades within the subfamily approximately 29.9 Mya (14.6–39.1 Mya) [32]. On the other hand, in the Orchidaceae family, the earliest diverged subfamily is Apostasioideae , followed by Vanilloideae , Cypripedioideae , Orchidoideae , and finally Epidendroideae [1]. In this study, when comparing the transcriptome data of three Paphiopedilum species with the NR database, the results showed that 47.7% of sequences were aligned to the genus Dendrobium within the subfamily Epidendroideae , while 7.8% and 7.6% matched sequences from the subfamilies Vanilloideae and Apostasioideae , respectively. Noticeably, no significant matches were detected to other sister clades within the subfamily Cypripedioideae , which may be attributed to the insufficient genomic data coverage of Cypripedioideae plants in the NR database (Fig. 3A). On the other hand, when we aligned Paphiopedilum transcriptomic data to six other databases, the coverage rates were all below 30% (Fig. 3B). This phenomenon may primarily be due to Paphiopedilum being a non-model organism, coupled with a lack of molecular genetic research reports and the absence of its gene functional annotations in databases like KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology) [33]. Additionally, it is also possible that due to the uniqueness of Paphiopedilum , a large number of novel genes or species-specific genes exist in its transcriptome, which cannot be annotated through homology-based mapping to functional databases [34, 35]. Molecular analysis of Paphiopedilum is an essential approach to comprehensively understand this species, and it also provides a theoretical foundation for the conservation of Paphiopedilum populations and the breeding of new cultivars. Conclusion and future directions This study analyzed flavonoid metabolites in the floral organs of diverse Paphiopedilum species and their hybrid progeny, providing a critical foundation for understanding the molecular mechanisms underlying floral coloration in Paphiopedilum . Integrated with transcriptomics analysis, it offers novel insights into the regulatory mechanisms controlling the biosynthesis of color-related compounds in Paphiopedilum plants. The hybrid exhibited cream-colored floral colour, distinct from the purple and yellow-green phenotypes of the parental lines. In this process, anthocyanin content plays a pivotal role in purple pigmentation. Upregulation or downregulation of genes at specific nodes within the flavonoid biosynthesis pathway resulted in altered flavonoid accumulation, ultimately leading to the loss of coloration in the hybrids. Future research should further investigate the differentially expressed genes within the flavonoid biosynthesis pathway, integrating sequences and expression of related candidate genes to identify key regulatory genes control floral pigmentation in Paphiopedilum . Furthermore, metabolomic analysis revealed that only a minor subset of flavonoid metabolites showed significant content differences between the hybrid and P. primulinum . This result may be attributed to the yellow-green pigmentation of P. primulinum correlating with non-flavonoid metabolites rather than flavonoids. Consequently, we will extend our investigation to compare the contents of carotenoids, betalains, and chlorophyll in the floral organ of these three Paphiopedilum plants. Paphiopedilum cultivars are highly sought after in the floriculture market, particularly for their vibrant coloration. Breeding novel varieties with unique ornamental traits remains a primary objective for horticulturists. This study will continue to explore the regulatory genes associated with floral color and the dynamics of pigment-related metabolites, thereby providing a theoretical foundation for molecular breeding strategies in Paphiopedilum . Method and Materials Plant Materials The plant materials were cultivated in the tissue culture room and greenhouse located in Xingyi City, Guizhou Province (104°54´E, 25°09´N). The tissue culture room maintained a constant temperature of 25°C, while the greenhouse temperature fluctuated between 10–30°C. The Paphiopedilum varieties included P. delenatii, P. primulinum , and their hybrid (Fig. 1). P. delenatii plant and P. primulinum plant were initially sown in the tissue culture room in December 2017 and transplanted to the Xingyi greenhouse eight months later. For hybrid development, crosses were performed in April 2020 using P. delenatii as the paternal parent and P. primulinum as the maternal parent. The hybrid seeds were sown in tissue culture bottles in December 2020 and transplanted to the Xingyi greenhouse eight months post-germination. Sampling of floral tissues was conducted according to the respective flowering periods: P. delenatii plant and P. primulinum plant were sampled in April 2024, while the hybrid was sampled in May 2024. Biochemical analysis Flavonoid-targeted metabolite components were sampled from petals and labella and identified and quantified using a liquid chromatography-mass spectrometry (LC-MS) system. De novo transcriptome assembly and annotation RNA integrity was assessed using the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Low-quality reads were filtered from raw sequencing data to obtain clean reads. De novo transcriptome assembly was performed using Trinity software [36], generating unigenes with N50 and N90 values of 1205 and 393, respectively (Table S4). Transcriptome completeness was evaluated using BUSCO (Benchmarking Universal Single-Copy Orthologs), with the Trinity assembly achieving a BUSCO completeness score of 70.1% (Fig. S5). Gene function was annotated based on the following databases, such as NCBI non-redundant protein sequences (Nr), NCBI nucleotide sequences (Nt), Protein family (Pfam), Clusters of orthologous groups for eukaryotic complete genomes (KOG), Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). Coding sequences (CDS) were predicted by aligning unigenes against the NCBI non-redundant protein database (Nr) and Swiss-Prot database. For unigenes without hits in these databases, open reading frames (ORFs) were predicted using TransDecoder (v3.0.1), yielding putative CDS sequences. Analysis of differentially expressed genes RSEM and bowtie was used for comparing and normalizing the mapping read to calculating the gene expression levels by the fragments per kilobase of transcript per million mapped reads (FPKM). Differential expression of the two genes was analyzed using DESeq2, and the criteria for identification of differentially expressed genes were based on｜log2(FoldChange) |≥1 and the false discovery rate (FDR)≤0.05. GO and KEGG pathway enrichment analysis were performed from significant DEG using clusterProfiler R software. PPI analysis of differentially expressed genes was based on STRING database which contains known and predicted protein-protein interactions, via diamond (version 0.9.13). Declarations Acknowledgements Not applicable Author’s contributions Q. L. and Y. W. figured the original idea; J. C. and S. C. analyzed the data; C. W., L. L. and J. W. conducted experiments; L. Z. and K. D bred and cultivated the plant materials, including hybrid, P. delenatii and P. primulinum . Q. L. wrote the paper with input from other authors. Funding The financial assistance for this research was provided by the grants from the Guizhou Academy of Sciences project "Gene Screening and Identification for Flower Color Formation in Hybrid Paphiopedilum" (No. QKYRZ202401), directed by Q. L. Technical support for plant material development was obtained from the discipline construction project "Conservation and Development of Mountain Characteristic Plant Resources" (No. XKJS202312) at Minzu Normal University of Xingyi, led by L. Z. Data availability The raw sequencing data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under the BioProject accession number PRJNA1266858, which can be found at the following：https://dataview.ncbi.nlm.nih.gov/object/. Ethics approval and consent to participate All samples collected fully adhere to national and local legal requirements. The Paphiopedilum plant materials used in this study (including but not limited to seeds, seedlings, and tissue cultures) were derived exclusively from an aseptic artificial cultivation system, with no collection from wild resources whatsoever. This study is a genetic and physiological exploration of ornamental flowers, with plants as the research subjects. It does not involve any human participants, medical interventions, health outcome or patient data. According to the International Committee of Medical Journal Editors (ICMJE) definition of clinical trials and WHO registration guidelines, clinical trials require human subjects undergoing health-related interventions. Therefore, this study is exempt from clinical trial registration. Consent for publication Not applicable. Conflict of interest statement The authors declare no conflicts of interest. References Givnish TJ, Spalink D, Ames M, Lyon SP, Hunter SJ, Zuluaga A, et al. Orchid phylogenomics and multiple drivers of their extraordinary diversification. Proc Biol Sci. 2015;doi: 10.1098/rspb.2015.1553. Li X, Fan J, Luo S, Yin L, Liao H, Cui X, et al. Comparative transcriptome analysis identified important genes and regulatory pathways for flower color variation in Paphiopedilum hirsutissimum . BMC Plant Biol. 2021;21:495. Liu ZJ, Chen XQ, Chen LJ, Lei SP. 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Advances in the study of flower color variation in orchids. Chin Bull Bot 2021;56:699–714. Pruitt KD, Tatusova T, Maglott DR. NCBI Reference Sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res. 2007;35:61–62. Baker W, van den Broek A, Camon E, Camon E, Hingamp P, Sterk P, Stoesser G, et al. The EMBL nucleotide sequence database. Nucleic Acids Res 2000;28:19–23. Zhang Y, Zhang GQ, Zhang D, Liu XD, Xu XY, Sun WH, et al. Chromosome-scale assembly of the Dendrobium chrysotoxum genome enhances the understanding of orchid evolution. Hortic Res. 2021;8:183. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011;29:644– Additional Declarations No competing interests reported. Supplementary Files abbreviationlist.xlsx Supplementaryfigures.pdf TableS1.csv TableS2.csv TableS3.csv TableS4.csv Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6878590","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":485661038,"identity":"a840d9ea-3c0c-45f5-9181-13d0d330ef12","order_by":0,"name":"Quanshu Luo","email":"","orcid":"","institution":"Guizhou Institute of Biology","correspondingAuthor":false,"prefix":"","firstName":"Quanshu","middleName":"","lastName":"Luo","suffix":""},{"id":485661039,"identity":"56c07a38-9fbc-4f0c-a96a-12aad6caa723","order_by":1,"name":"Junming Chen","email":"","orcid":"","institution":"Guilin University of Aerospace Technology","correspondingAuthor":false,"prefix":"","firstName":"Junming","middleName":"","lastName":"Chen","suffix":""},{"id":485661040,"identity":"fb96cfbe-89a4-4a89-b78a-237a30d81e84","order_by":2,"name":"Cheng Wan","email":"","orcid":"","institution":"Guizhou Institute of Biology","correspondingAuthor":false,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Wan","suffix":""},{"id":485661041,"identity":"93b7b2f1-95aa-4263-8635-deb8a45fccb5","order_by":3,"name":"Leijia Li","email":"","orcid":"","institution":"Guizhou Institute of Biology","correspondingAuthor":false,"prefix":"","firstName":"Leijia","middleName":"","lastName":"Li","suffix":""},{"id":485661046,"identity":"50aac429-02e7-4c99-8efb-1461b8edaa78","order_by":4,"name":"Sheng Chen","email":"","orcid":"","institution":"Guizhou Institute of Biology","correspondingAuthor":false,"prefix":"","firstName":"Sheng","middleName":"","lastName":"Chen","suffix":""},{"id":485661047,"identity":"fc7bdff5-c967-4acd-9934-19f99c7f9bb8","order_by":5,"name":"Jihong Wang","email":"","orcid":"","institution":"Guizhou Institute of Biology","correspondingAuthor":false,"prefix":"","firstName":"Jihong","middleName":"","lastName":"Wang","suffix":""},{"id":485661048,"identity":"8e73bf2d-df62-444b-81b2-7823281f8faf","order_by":6,"name":"Li Zhou","email":"","orcid":"","institution":"Minzu Normal University of Xingyi","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Zhou","suffix":""},{"id":485661049,"identity":"5846e2f5-d53c-4ba1-8f4c-353ae0e45f55","order_by":7,"name":"Keyun Deng","email":"","orcid":"","institution":"Qianxinan Lvyuan Animal AND Plant Science and Technology Development Co. LTD","correspondingAuthor":false,"prefix":"","firstName":"Keyun","middleName":"","lastName":"Deng","suffix":""},{"id":485661050,"identity":"653ed6d8-7d14-49cd-830b-b891e5535881","order_by":8,"name":"Ying Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYBACPhjDgIH5wAGGAiDrAAEtbAgtbAkHgCRJWngMGIjTIpF88DZPzR17c4mcjwd+GNgk9h1gfvjoBl4tacnWPMeeJe6ckbvhYI9BWuLMA2zGxjl4teSYSfOwHU4wuJG74TCDweHEDQd42KQJa/l32N7gRs4DErTwth1m3HAjh4FILTzPki3n9gFVnnlmAPKL8czDBPzCz5588Mabb0CHHU9+/OFHhY1s3/Hmh4/xaQEBCVQuMwHlWLSMglEwCkbBKEADAK66T3eGZ9KjAAAAAElFTkSuQmCC","orcid":"","institution":"Guizhou Institute of Biology","correspondingAuthor":true,"prefix":"","firstName":"Ying","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-06-12 09:08:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6878590/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6878590/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86967642,"identity":"5174e4f9-2868-4094-950c-7f1142390025","added_by":"auto","created_at":"2025-07-17 17:53:46","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":64651,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypic comparison of three \u003cem\u003ePaphiopedilum L\u003c/em\u003e. \u003cem\u003ePaphiopedilum primulinum \u003c/em\u003e(A), \u003cem\u003ePaphiopedilum delenatii \u003c/em\u003e(B) and their hybrid progeny (C), Scale bars = 1cm.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6878590/v1/0476c5c2f318151277cb0196.jpg"},{"id":86966695,"identity":"994ef3f8-8fdb-4083-91b2-680aad10af2d","added_by":"auto","created_at":"2025-07-17 17:37:46","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":123835,"visible":true,"origin":"","legend":"\u003cp\u003eRelative quantitative values of flavonoid metabolites in three flowers of \u003cem\u003ePaphiopedilum\u003c/em\u003e. The figure shows only the relative quantification values of three flavonoid subclasses，flavanones (A), flavones and flavonols (B) and anthocyanins (C). Pp:\u003cem\u003e P. primulinum,\u003c/em\u003e Pd: \u003cem\u003eP. delenatii,\u003c/em\u003eHybrid: The hybrid progeny of \u003cem\u003eP. delenatii \u003c/em\u003eand \u003cem\u003eP. primulinum\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6878590/v1/2612857656eda49d4b1e3ed8.jpg"},{"id":86966696,"identity":"cff2a499-5a37-4b4b-81cd-3ebb3fe5669b","added_by":"auto","created_at":"2025-07-17 17:37:46","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":44715,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of transcriptome data with databases. A: Percentage of unigene numbers annotated in seven databases, GO: Gene Ontology, KO: Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology, KOG: Clusters of orthologous groups for eukaryotic complete genomes, Nr: NCBI non-redundant protein sequences, Nt: NCBI nucleotide sequences, Pfam: Protein family, Swiss-Prot: Swiss-Prot Protein Knowledgebase; B: Species distribution diagram of matched sequences in the Nr database.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6878590/v1/99839fdfebb60331ace516a1.jpg"},{"id":86966699,"identity":"7d93d766-a120-47f3-bcb3-fa04b5229399","added_by":"auto","created_at":"2025-07-17 17:37:46","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":37360,"visible":true,"origin":"","legend":"\u003cp\u003eStatistical Quantification of Differentially Expressed Genes. A：Statistical Bar Chart of Differentially Expressed Genes (DEGs). Compare denotes the names of comparative groups; All indicates the total number of DEGs identified in each comparison; Up and Down represent the counts of upregulated and downregulated DEGs, respectively. Threshold specifies the analytical software (e.g., DESeq2/edgeR); B: Venn Diagram of DEG Overlaps, Venn diagram visualizing overlaps of DEGs among multiple comparative groups.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6878590/v1/c23106e656e6fa5ba97ad4f8.jpg"},{"id":86967089,"identity":"90e1d7de-71ef-4016-b676-fa5b818fa037","added_by":"auto","created_at":"2025-07-17 17:45:46","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":49603,"visible":true,"origin":"","legend":"\u003cp\u003eKEGG metabolic pathway classification statistical diagram. The x-axis represents KEGG Orthology (KO) metabolic pathway names, while the y-axis indicates the number of genes annotated to each pathway. Box annotations specify the KEGG pathway names corresponding to their respective KO identifiers (e.g., ko00940).\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6878590/v1/8f4518ba583dc2464da149e6.jpg"},{"id":87930289,"identity":"903b7112-6ac3-42b4-8b26-e1d80d9caa57","added_by":"auto","created_at":"2025-07-30 13:32:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":846489,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6878590/v1/85212804-fb29-4500-899e-1b2ae4d7df6d.pdf"},{"id":86967087,"identity":"9366e038-e9ad-4ff4-b526-0e31be820953","added_by":"auto","created_at":"2025-07-17 17:45:46","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10629,"visible":true,"origin":"","legend":"","description":"","filename":"abbreviationlist.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6878590/v1/215c72f5b1656743574e72dd.xlsx"},{"id":86967091,"identity":"fb1e1615-599a-41a6-9f51-c9a569fbcee5","added_by":"auto","created_at":"2025-07-17 17:45:46","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":820821,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6878590/v1/c43c58b1f18260fc641aed2a.pdf"},{"id":86966700,"identity":"315d7d32-be9b-4f92-8ab7-4e7509700a3e","added_by":"auto","created_at":"2025-07-17 17:37:46","extension":"csv","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":8353,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.csv","url":"https://assets-eu.researchsquare.com/files/rs-6878590/v1/c4fef6ffc86dc03c07f7b5e2.csv"},{"id":86967643,"identity":"7fe28375-fc23-46f1-9565-f430bff8577b","added_by":"auto","created_at":"2025-07-17 17:53:46","extension":"csv","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":360,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.csv","url":"https://assets-eu.researchsquare.com/files/rs-6878590/v1/018cc535e668767f3c1bebca.csv"},{"id":86966702,"identity":"efe976a3-32ad-4a60-a4bc-170ce18db1bf","added_by":"auto","created_at":"2025-07-17 17:37:46","extension":"csv","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":15015,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.csv","url":"https://assets-eu.researchsquare.com/files/rs-6878590/v1/6246cba0f63f86c9e585a3ff.csv"},{"id":86966704,"identity":"dbc8951e-df82-474d-a383-fb81d63a9582","added_by":"auto","created_at":"2025-07-17 17:37:46","extension":"csv","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":176,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.csv","url":"https://assets-eu.researchsquare.com/files/rs-6878590/v1/8c024ad213f28ccef7d80780.csv"}],"financialInterests":"No competing interests reported.","formattedTitle":"Transcriptome and flavonoid metabolome analysis reveals the molecular basis of floral color divergence between the hybrid and their parental species in Paphiopedilum","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eOrchidaceae\u003c/em\u003e are the most diverse family of angiosperms, comprising 28000 species spread across 736 genera [1,2]. The\u0026nbsp;\u003cem\u003ePaphiopedilum\u003c/em\u003e genus (commonly known as \"\u0026nbsp;Lady's Slipper Orchid \") comprises 96-100 species globally and is primarily distributed in Southeast Asia, the Himalayan lowlands, and Southwest China [3]. Renowned for its slipper-shaped floral morphology and vibrant color variations, this genus holds significant horticultural value and is a prized ornamental plant in global markets [4]. China is the country with the richest diversity of \u003cem\u003ePaphiopedilum\u003c/em\u003e species in the world. There are 27 native species indigenous to China, accounting for one-third of the globally recorded wild \u003cem\u003ePaphiopedilum\u003c/em\u003e species, and most are distributed in Yunnan or Guangxi Province [3]. All\u0026nbsp;\u003cem\u003ePaphiopedilum\u0026nbsp;\u003c/em\u003especies are included in Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). The natural distribution range of\u0026nbsp;\u003cem\u003ePaphiopedilum delenatii\u003c/em\u003e Guill. (\u003cem\u003eOrchidaceae\u003c/em\u003e) spans northern Guangxi Province and southeastern Yunnan Province in China, as well as southern Vietnam [5]. Its floral morphology exhibits a diameter of 6-8 cm, with the dorsal sepal and synsepal presenting a pure white coloration, while the labellum displays a distinct pink pigmentation. This species displays notable adaptability to cultivation under controlled horticultural conditions [3]. On the other hand,\u0026nbsp;\u003cem\u003ePaphiopedilum primulinum\u0026nbsp;\u003c/em\u003eM. W. Wood \u0026amp; P. Taylor is native to Sumatra and Java, Indonesia [6]. The flower has a diameter of 6.5-7 cm, with the dorsal sepal and synsepal displaying greenish-yellow coloration, while the petals and labellum are yellow[7]. Because of their uniqueness in flower color and morphology, they are widely sought after by orchid lovers. Therefore, the hybrid varieties of\u0026nbsp;\u003cem\u003eP. delenatii\u003c/em\u003e or\u0026nbsp;\u003cem\u003eP. primulinum\u003c/em\u003e as their parents are also welcomed by\u0026nbsp;\u003cem\u003ePaphiopedilum\u003c/em\u003e lovers, and have won a large number of prizes in the orchid exhibition\u0026nbsp;[3].\u003c/p\u003e\n\u003cp\u003eFlowering plants typically rely on multi-sensory media, such as color, scent, and morphology, to attract pollinators, thereby ensuring successful reproduction [8]. Although some plants increase their attractiveness to pollinators through a combination of olfactory and visual signals [9], visual signals are still decisive for attracting pollinators [10]. The color of flowers is determined by secondary metabolites, including flavonoids, carotenoids, and betalains, which collectively regulate pigmentation based on their chemical structures and cellular environment [11]. Numerous studies on floral pigmentation have shown that flowers displaying red, purple, or blue colors exhibit high anthocyanin content, whereas pale or white flowers primarily contain flavonols or chalcones [12, 13]. Generally, within the same species, darker flowers colors are typically associated with higher flavonoid content, particularly anthocyanins [14], but the effect of total flavonoid content on flower color did not show obvious correlation [15].\u003c/p\u003e\n\u003cp\u003eThe flavonoid biosynthesis pathway constitutes a key branch of the phenylpropanoid pathway extended via chalcone synthase (CHS), through which a multitude of flavonoid secondary metabolites are generated. These metabolites are broadly categorized into six classes: flavones, flavonols, flavanones, flavanols, anthocyanins, and isoflavones [16]. The flavonoid biosynthesis pathway is governed by a series of critical enzymes and genes, including phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL) [17]. The enzymatic pathway for flavonoid biosynthesis as follows: 4CL generates flavanones, such as naringenin, through the catalytic actions of chalcone synthase (CHS) and chalcone isomerase (CHI). When catalyzed by dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS), flavanone can be converted into anthocyanins; under the catalysis of flavonol synthase (FLS) along with flavonoid 3'-hydroxylase (F3'H) or flavonoid 3',5'-hydroxylase (F3'5'H), it generates flavonols; additionally, isoflavone synthase (IFS) and flavone synthase (FNS) mediate the production of isoflavones and flavones respectively[17]. These enzymes are regulated by their corresponding genes [18]. With the development of science and technology, transcriptomics has been gradually applied to botanical research. Through RNA sequencing technology, the gene expression pattern of plants can be comprehensively analyzed, and the regulatory mechanism of genes can be explained [19], and the complex gene expression network can be revealed [20]. In research of orchid plants, using metabolomics-transcriptomics combined analysis, reveal \u003cem\u003eDendrobium nobile\u003c/em\u003e flavonoid metabolite content and metabolic pathway different genes on flower color diversity impact, and obtain regulatory gene network [21]. The three \u003cem\u003ePaphiopedilum\u003c/em\u003e plants used in this study exhibited yellow-green, purplish-red, and cream-white flower colors, respectively. Through integrated analysis of flavonoid metabolomics and transcriptomics, we performed a comparative analysis of the differences in flavonoid metabolism and gene expression between the hybrid and its parental lines. This approach revealed the molecular mechanisms underlying the segregation phenomenon of flower coloration between the hybrid plant and its parents.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eCharacteristics of flavonoid metabolizing substances in \u003cem\u003ePaphiopedilum L.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe floral structure of the native species \u003cem\u003eP. delenatii\u0026nbsp;\u003c/em\u003eexhibit two broad, smooth, and glabrous white petals. The dorsal sepal is nearly white, while both the synsepal and the labellum display a uniform purple coloration (Fig. 1a). \u003cem\u003eP. primulinum\u003c/em\u003e features petals with undulate margins and ciliate edges. The labellum is yellow-green, while the dorsal sepal and synsepal are green (Fig. 1b). The hybrid combines the broad petal morphology inherited from \u003cem\u003eP. delenatii\u003c/em\u003e and the undulate petal margins from \u003cem\u003eP. primulinum\u003c/em\u003e. The synsepal is yellow-green with sparse reddish pigmentation, while the dorsal sepal, petals, and labellum are uniformly cream-colored (Fig. 1c). Results of our determination of flavonoid metabolic content in these three \u003cem\u003ePaphiopedilum\u003c/em\u003e plants，a total of 29 flavanones were detected, among which Hesperetin 5-O-glucoside, Eriocitrin, Poncirin, Naringenin and Butin were much higher in \u003cem\u003eP. delenatii\u003c/em\u003e than in the other two. The contents of Naringenin and Butin in the cream-colored hybrid were higher than \u003cem\u003eP. primulinum\u003c/em\u003e (Fig. 2A, Table S1). 93 flavones and flavonols were detected. There was no significant difference in the content of most compounds between \u003cem\u003eP. primulinum\u003c/em\u003e and hybrid, while some metabolites, such as Kaempferol-3-O-rutinoside, Chrysoeriol 7-O-rutinoside, were found to be higher in \u003cem\u003eP. delenatii\u0026nbsp;\u003c/em\u003ethan in the others (Fig. 2B). Among the 13 anthocyanins detected, the content of Cyanidin in \u003cem\u003eP. delenatii\u003c/em\u003e was consistently higher than that in \u003cem\u003eP. primulinum\u003c/em\u003e and the hybrid. In contrast, the concentration of Procyanidin A2 in the hybrid significantly exceeded those in its parental species (Fig. 2C). Additionally, 23 isoflavonoids, 6 flavonoids, and 4 chalcones and dihydrochalcones were identified. Among 168 flavonoid metabolites, the hybrid exhibited 45 metabolites with significant differential abundance compared to \u003cem\u003eP. delenatii,\u003c/em\u003e of which 36 were down-regulated. In contrast, only 15 flavonoid metabolites showed significant differential abundance relative to \u003cem\u003eP. primulinum,\u003c/em\u003e with 12 being up-regulated (Table 1).\u003c/p\u003e\n\u003cp\u003eTable 1 The number of flavonoid metabolites in \u003cem\u003ePaphiopedilum\u003c/em\u003e\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"567\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003eCompared Samples\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003eNum. of Total Ident.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003eNum. of Total Sig.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003eNum. of Sig. Up\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 99px;\"\u003e\n \u003cp\u003eNum. of Sig. Down\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003eHybrid vs Pd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003e168\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 99px;\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003eHybrid vs Pp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003e168\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 99px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 130px;\"\u003e\n \u003cp\u003ePd vs Pp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003e168\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 99px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eNum. of Total Ident.: The number of total identified metabolites.\u003c/p\u003e\n\u003cp\u003eNum. of Total Sig.: The number of totals significantly differential metabolites.\u003c/p\u003e\n\u003cp\u003eNum. of \u0026nbsp;Sig. Up: The number of totals significantly upregulated metabolites.\u003c/p\u003e\n\u003cp\u003eNum. of Sig. Down: The number of totals significantly downregulated metabolites\u003c/p\u003e\n\u003cp\u003eAnalysis of transcriptome dataset\u003c/p\u003e\n\u003cp\u003eThe transcriptome data were compared with the annotation results of seven databases. 45.6 % of unigenes could be compared to NR database, only 8.28 % could be compared to KOG database. And more than 26% in GO, NT, PFAM and SwissProt databases, they were 26.82 %, 27.35 %, 26.82 % and 28.12 %, respectively (Fig. 3A, Table S2 ). When compared with the NR database maintained by NCBI, 20.3 % of the unigenes showed significant sequence alignment with \u003cem\u003eDendrobium catenatum\u003c/em\u003e, followed by \u003cem\u003eDendrobium nobile\u003c/em\u003e (14.7 %) and \u003cem\u003eDendrobium chrysotoxum\u0026nbsp;\u003c/em\u003e(12.7 %) (Fig. 3B, Table S3 ).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTranscriptomic data from three color morphs of \u003cem\u003ePaphiopedilum\u003c/em\u003e floral tissues were subjected to pairwise sample comparisons to quantify differentially expressed genes (DEGs). Our analysis revealed 16,621 DEGs between \u003cem\u003eP. delenatii\u003c/em\u003e (paternal parent) and \u003cem\u003eP. primulinum\u003c/em\u003e (maternal parent), with comparable numbers of up-regulated (8,842) and down-regulated (7,779) genes in the yellowish-green \u003cem\u003eP. primulinum\u003c/em\u003e relative to \u003cem\u003eP. delenatii\u003c/em\u003e (Fig. 4A). The hybrid progeny exhibited 3,338 DEGs compared to \u003cem\u003eP. delenatii\u003c/em\u003e and 4,623 DEGs relative to the \u003cem\u003eP. primulinum\u003c/em\u003e (Fig. 4A). Obviously, the majority of DEGs in hybrid comparisons displayed up-regulation regardless of the parental line. Intersectional analysis identified 194 shared DEGs across all three \u003cem\u003ePaphiopedilum\u003c/em\u003e plants (Fig. 4B).\u003c/p\u003e\n\u003cp\u003eAnalysis of differential expressed genes related flavonoid biosynthesis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eKEGG (http://www.kegg.jp/ or http://www.genome.jp/kegg/) is an integrated database resource for biological interpretation of genome sequences and other high-throughput data. Molecular functions of genes and proteins are associated with ortholog groups and stored in the KEGG Orthology (KO) database [22]. Comparative analysis against the KO database revealed distinct unigene associations with flavonoid biosynthesis pathways in the hybrid progeny relative to \u003cem\u003eP. delenatii\u003c/em\u003e (paternal parent) and \u003cem\u003eP. primulinum\u003c/em\u003e (maternal parent). Specifically, 47 unigenes were annotated to the general flavonoid biosynthesis pathway (ko00941), 3 unigenes to anthocyanin biosynthesis (ko00942), and 14 unigenes to flavone/flavonols biosynthesis (ko00944). Notably, no unigenes correlated with isoflavonoid biosynthesis (ko00943) were identified in either hybrid-parent comparison (Fig. 5).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eArtificial hybridization breeding in the genus \u003cem\u003ePaphiopedilum\u003c/em\u003e commenced in the 1860s [3]. Driven by market demand, the proliferation of artificially hybridized varieties accelerated significantly over time. This trend is quantitatively reflected in \u003cem\u003eSander's List of Orchid Hybrids\u003c/em\u003e, which documents a dramatic increase in registered orchid hybrids from 750 species in 1946 to over 130 000 by 2019\u0026nbsp;[23, 24]. Among numerous \u003cem\u003ePaphiopedilum\u003c/em\u003e hybrids derived from \u003cem\u003eP. delenatii\u003c/em\u003e as a parental species, the majority retain the characteristic large and symmetrical petals inherited from this species. A subset of these hybrids exhibits distinct floral features including magenta-purple labella or pure white petals, as exemplified by \u003cem\u003ePaphiopedilum\u003c/em\u003e Magic Lantern 'Ralph' and \u003cem\u003ePaphiopedilum\u003c/em\u003e Joyce Hasegawa 'Ruey-Hwa' [25]. The hybrid plant material utilized in this study was developed through artificial hybridization breeding techniques between \u003cem\u003eP. primulinum\u003c/em\u003e and \u003cem\u003eP. delenatii\u003c/em\u003e. The resulting hybrid exhibits a creamy-white coloration across its entire floral structure, with a slightly yellowish tint compared to the petals of \u003cem\u003eP. delenatii\u003c/em\u003e. Morphological analysis revealed that the labellum and dorsal sepal closely resembled those of \u003cem\u003eP. primulinum\u003c/em\u003e in morphology. Prominently, the petal morphology demonstrated an intermediate inheritance pattern: the undulate margins characteristic of \u003cem\u003eP. primulinum\u003c/em\u003e were preserved, combined with the large, glabrous surface texture typical of \u003cem\u003eP. delenatii\u0026nbsp;\u003c/em\u003e(Fig. 1). This chimeric expression pattern suggests incomplete dominance in petal trait inheritance during interspecific hybridization. Analysis of flavonoid detection in floral tissues revealed that \u003cem\u003eP. delenatii\u003c/em\u003e exhibited a higher abundance of flavonoid metabolites compared to \u003cem\u003eP. primulinum\u003c/em\u003e. Similarly, the hybrid demonstrated flavonoid metabolite levels higher than those of \u003cem\u003eP. primulinum\u003c/em\u003e but lower than those of \u003cem\u003eP. delenatii\u003c/em\u003e when compared to their parental species (Fig. S2). In numerous studies on flower color, the content of flavonoids, especially anthocyanins, in dark flowers was higher than that in light flowers among the same species [26]. In this study, the anthocyanin content in \u003cem\u003eP. delenatii\u003c/em\u003e, characterized by magenta-purple labella, was found to be significantly higher than that of the yellowish-green \u003cem\u003eP. primulinum\u003c/em\u003e and hybrid cultivar, except for Petunidin 3-O-glucoside. Notably, Petunidin 3-O-glucoside, a compound which substantial accumulation induces deep red to purple pigmentation in plant tissues, was also detected at elevated levels in the Zikui tea plant (\u003cem\u003eCamellia sinensis cv.\u003c/em\u003e Zikui). Petunidin 3-O-glucoside, alongside cyanidin glycosides, is considered to co-regulate the expression of purple pigmentation traits in plant [27]. The Petunidin 3-O-glucoside in \u003cem\u003eP. primulinum\u003c/em\u003e was significantly higher than that in the other two species, but did not show purple color, which may be due to the low content of cyanidin 3-O-glucoside. Beyond the direct chromatic effects of anthocyanins, other flavonoid metabolites, such as flavonoids and isoflavonoids, demonstrate substantially elevated levels in dark-pigmented floral tissues compared to lighter-colored counterparts [28], suggesting their synergistic or regulatory roles in pigmentation intensity [29]. In the transcriptome analysis, we also found three unigenes related to anthocyanin synthesis, 14 unigenes related to flavone and flavonol biosynthesis, and 47 unigenes related to flavonoid biosynthesis. Although it is not clear what role these genes play in the formation of the color of the \u003cem\u003ePaphiopedilum.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, we performed transcriptome sequencing on the flowers of three \u003cem\u003ePaphiopedilum\u003c/em\u003e plants, and it can be intuitively seen from the number of DEGs that most of the differentially expressed genes were up-regulated in the hybrid compared with its parents. In KEGG enrichment analysis, pairwise comparisons were conducted to examine the top 20 most significantly enriched pathways of differentially expressed genes among them. The DEGs enriched in flavonoid synthesis pathway and phenylpropanoid biosynthesis showed particularly significant enrichment (Fig. S3). The phenylpropanoid biosynthesis pathway and the flavonoid biosynthesis pathway share upstream pathways in phenylpropanoid metabolism but utilize distinct enzymes. In the flavonoid biosynthesis pathway, the phenylpropanoid biosynthesis pathway provides essential precursor substances [30]. In comparison with \u003cem\u003eP. primulinum\u003c/em\u003e, the differentially expressed genes involved in the phenylpropanoid biosynthesis pathway and flavonoid biosynthesis pathway were all highly significant in hybrids. To visually explore the expression differences among the three \u003cem\u003ePaphiopedilum\u003c/em\u003e plant within the flavonoid biosynthesis network, we first examined the pathway map of KEGG pathway ko00941 (Flavonoid Biosynthesis) (Fig. S4). Compared with \u003cem\u003eP. primulinum\u003c/em\u003e, genes at most nodes in the flavonoid biosynthetic pathway of the hybrid were up-regulated (Fig. S4A). In contrast, when compared with \u003cem\u003eP. delenatii\u003c/em\u003e, although genes at some nodes in the later stages of biosynthesis were up-regulated, the majority of genes at nodes in the early stages of biosynthesis were down-regulated. For example, multiple genes in pathways such as EC: 2.3.1.74 (CHS) and EC: 1.14.1491 (trans-cinnamate 4-monooxygenase) exhibited this pattern (Fig. S4B). The up-regulation and down-regulation of these genes might have influenced the differences in the final flavonoid metabolite content.\u003c/p\u003e\n\u003cp\u003eWith the application of multi-omics in botanical research, some scholars have conducted in-depth studies on the genomics and transcriptomics of \u003cem\u003eOrchidaceae\u0026nbsp;\u003c/em\u003eplants [31]. Researchers analyzed transcriptomic data of \u003cem\u003eP. armeniacum\u003c/em\u003e, \u003cem\u003eP. concolor\u003c/em\u003e, \u003cem\u003eP. hirsutissimum\u003c/em\u003e and \u003cem\u003eP. malabellumoense\u003c/em\u003e, revealing three whole-genome duplication (WGD) events during the evolutionary history of \u003cem\u003ePaphiopedilum,\u0026nbsp;\u003c/em\u003ethe resultsuggested that \u003cem\u003ePaphiopedilum\u003c/em\u003e belongs to the subfamily \u003cem\u003eCypripedioideae\u003c/em\u003e, which diverged from its sister clades within the subfamily approximately 29.9 Mya (14.6–39.1 Mya) [32]. On the other hand, in the \u003cem\u003eOrchidaceae\u003c/em\u003e family, the earliest diverged subfamily is \u003cem\u003eApostasioideae\u003c/em\u003e, followed by \u003cem\u003eVanilloideae\u003c/em\u003e, \u003cem\u003eCypripedioideae\u003c/em\u003e, \u003cem\u003eOrchidoideae\u003c/em\u003e, and finally \u003cem\u003eEpidendroideae\u0026nbsp;\u003c/em\u003e[1].\u003c/p\u003e\n\u003cp\u003eIn this study, when comparing the transcriptome data of three \u003cem\u003ePaphiopedilum\u0026nbsp;\u003c/em\u003especies with the NR database, the results showed that 47.7% of sequences were aligned to the genus \u003cem\u003eDendrobium\u003c/em\u003e within the subfamily\u003cem\u003e\u0026nbsp;Epidendroideae\u003c/em\u003e, while 7.8% and 7.6% matched sequences from the subfamilies \u003cem\u003eVanilloideae\u003c/em\u003e and \u003cem\u003eApostasioideae\u003c/em\u003e, respectively. Noticeably, no significant matches were detected to other sister clades within the subfamily \u003cem\u003eCypripedioideae\u003c/em\u003e, which may be attributed to the insufficient genomic data coverage of \u003cem\u003eCypripedioideae\u0026nbsp;\u003c/em\u003eplants in the NR database (Fig. 3A). On the other hand, when we aligned \u003cem\u003ePaphiopedilum\u003c/em\u003e transcriptomic data to six other databases, the coverage rates were all below 30% (Fig. 3B). This phenomenon may primarily be due to \u003cem\u003ePaphiopedilum\u003c/em\u003e being a non-model organism, coupled with a lack of molecular genetic research reports and the absence of its gene functional annotations in databases like KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology) [33]. Additionally, it is also possible that due to the uniqueness of\u0026nbsp;\u003cem\u003ePaphiopedilum\u003c/em\u003e, a large number of novel genes or species-specific genes exist in its transcriptome, which cannot be annotated through homology-based mapping to functional databases [34, 35]. Molecular analysis of \u003cem\u003ePaphiopedilum\u003c/em\u003e is an essential approach to comprehensively understand this species, and it also provides a theoretical foundation for the conservation of \u003cem\u003ePaphiopedilum\u003c/em\u003e populations and the breeding of new cultivars.\u003c/p\u003e"},{"header":"Conclusion and future directions","content":"\u003cp\u003eThis study analyzed flavonoid metabolites in the floral organs of diverse \u003cem\u003ePaphiopedilum\u003c/em\u003e species and their hybrid progeny, providing a critical foundation for understanding the molecular mechanisms underlying floral coloration in \u003cem\u003ePaphiopedilum\u003c/em\u003e. Integrated with transcriptomics analysis, it offers novel insights into the regulatory mechanisms controlling the biosynthesis of color-related compounds in \u003cem\u003ePaphiopedilum\u003c/em\u003e plants. The hybrid exhibited cream-colored floral colour, distinct from the purple and yellow-green phenotypes of the parental lines. In this process, anthocyanin content plays a pivotal role in purple pigmentation. Upregulation or downregulation of genes at specific nodes within the flavonoid biosynthesis pathway resulted in altered flavonoid accumulation, ultimately leading to the loss of coloration in the hybrids.\u003c/p\u003e\n\u003cp\u003eFuture research should further investigate the differentially expressed genes within the flavonoid biosynthesis pathway, integrating sequences and expression of related candidate genes to identify key regulatory genes control floral pigmentation in \u003cem\u003ePaphiopedilum\u003c/em\u003e. Furthermore, metabolomic analysis revealed that only a minor subset of flavonoid metabolites showed significant content differences between the hybrid and \u003cem\u003eP. primulinum\u003c/em\u003e. This result may be attributed to the yellow-green pigmentation of \u003cem\u003eP. primulinum\u003c/em\u003e correlating with non-flavonoid metabolites rather than flavonoids. Consequently, we will extend our investigation to compare the contents of carotenoids, betalains, and chlorophyll in the floral organ of these three \u003cem\u003ePaphiopedilum\u003c/em\u003e plants. \u003cem\u003ePaphiopedilum\u003c/em\u003e cultivars are highly sought after in the floriculture market, particularly for their vibrant coloration. Breeding novel varieties with unique ornamental traits remains a primary objective for horticulturists. This study will continue to explore the regulatory genes associated with floral color and the dynamics of pigment-related metabolites, thereby providing a theoretical foundation for molecular breeding strategies in \u003cem\u003ePaphiopedilum\u003c/em\u003e.\u003c/p\u003e"},{"header":"Method and Materials","content":"\u003cp\u003ePlant Materials\u003c/p\u003e\n\u003cp\u003eThe plant materials were cultivated in the tissue culture room and greenhouse located in Xingyi City, Guizhou Province (104°54´E, 25°09´N). The tissue culture room maintained a constant temperature of 25°C, while the greenhouse temperature fluctuated between 10–30°C. The Paphiopedilum varieties included \u003cem\u003eP. delenatii,\u003c/em\u003e \u003cem\u003eP. primulinum\u003c/em\u003e, and their hybrid (Fig. 1). \u003cem\u003eP. delenatii\u003c/em\u003e plant and \u003cem\u003eP. primulinum\u0026nbsp;\u003c/em\u003eplant were initially sown in the tissue culture room in December 2017 and transplanted to the Xingyi greenhouse eight months later. For hybrid development, crosses were performed in April 2020 using \u003cem\u003eP. delenatii\u0026nbsp;\u003c/em\u003eas the paternal parent and \u003cem\u003eP. primulinum\u0026nbsp;\u003c/em\u003eas the maternal parent. The hybrid seeds were sown in tissue culture bottles in December 2020 and transplanted to the Xingyi greenhouse eight months post-germination. Sampling of floral tissues was conducted according to the respective flowering periods: \u003cem\u003eP. delenatii\u003c/em\u003e plant and \u003cem\u003eP. primulinum\u003c/em\u003e plant were sampled in April 2024, while the hybrid was sampled in May 2024.\u003c/p\u003e\n\u003cp\u003eBiochemical analysis\u003c/p\u003e\n\u003cp\u003eFlavonoid-targeted metabolite components were sampled from petals and labella and identified and quantified using a liquid chromatography-mass spectrometry (LC-MS) system.\u003c/p\u003e\n\u003cp\u003eDe novo transcriptome assembly and annotation\u003c/p\u003e\n\u003cp\u003eRNA integrity was assessed using the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Low-quality reads were filtered from raw sequencing data to obtain clean reads. De novo transcriptome assembly was performed using Trinity software [36], generating unigenes with N50 and N90 values of 1205 and 393, respectively (Table S4). Transcriptome completeness was evaluated using BUSCO (Benchmarking Universal Single-Copy Orthologs), with the Trinity assembly achieving a BUSCO completeness score of 70.1% (Fig. S5). Gene function was annotated based on the following databases, such as NCBI non-redundant protein sequences (Nr), NCBI nucleotide sequences (Nt), Protein family (Pfam), Clusters of orthologous groups for eukaryotic complete genomes (KOG), Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). Coding sequences (CDS) were predicted by aligning unigenes against the NCBI non-redundant protein database (Nr) and Swiss-Prot database. For unigenes without hits in these databases, open reading frames (ORFs) were predicted using TransDecoder (v3.0.1), yielding putative CDS sequences.\u003c/p\u003e\n\u003cp\u003eAnalysis of differentially expressed genes\u003c/p\u003e\n\u003cp\u003eRSEM and bowtie was used for comparing and normalizing the mapping read to calculating the gene expression levels by the fragments per kilobase of transcript per million mapped reads (FPKM). Differential expression of the two genes was analyzed using DESeq2, and the criteria for identification of differentially expressed genes were based on｜log2(FoldChange) |≥1 and the false discovery rate (FDR)≤0.05. GO and KEGG pathway enrichment analysis were performed from significant DEG using clusterProfiler R software. PPI analysis of differentially expressed genes was based on STRING database which contains known and predicted protein-protein interactions, via diamond (version 0.9.13).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQ. L. and Y. W. figured the original idea; J. C. and S. C. analyzed the data; C. W., L. L. and J. W. conducted experiments; L. Z. and K. D bred and cultivated the plant materials, including hybrid, \u003cem\u003eP. delenatii\u003c/em\u003e and \u003cem\u003eP. primulinum\u003c/em\u003e. Q. L. wrote the paper with input from other authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe financial assistance for this research was provided by the grants from the Guizhou Academy of Sciences project \u0026quot;Gene Screening and Identification for Flower Color Formation in Hybrid Paphiopedilum\u0026quot; (No. QKYRZ202401), directed by Q. L. Technical support for plant material development was obtained from the discipline construction project \u0026quot;Conservation and Development of Mountain Characteristic Plant Resources\u0026quot; (No. XKJS202312) at Minzu Normal University of Xingyi, led by L. Z.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw sequencing data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under the BioProject accession number PRJNA1266858, which can be found at the following：https://dataview.ncbi.nlm.nih.gov/object/.\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eAll samples collected fully adhere to national and local legal requirements. The \u003cem\u003ePaphiopedilum\u003c/em\u003e plant materials used in this study (including but not limited to seeds, seedlings, and tissue cultures) were derived exclusively from an aseptic artificial cultivation system, with no collection from wild resources whatsoever. This study is a genetic and physiological exploration of ornamental flowers, with plants as the research subjects. It does not involve any human participants, medical interventions, health outcome or patient data. According to the International Committee of Medical Journal Editors (ICMJE) definition of clinical trials and WHO registration guidelines, clinical trials require human subjects undergoing health-related interventions. Therefore, this study is exempt from clinical trial registration.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eConflict of interest statement\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGivnish TJ, Spalink D, Ames M, Lyon SP, Hunter SJ, Zuluaga A, et al. Orchid phylogenomics and multiple drivers of their extraordinary diversification. Proc Biol Sci. 2015;doi: 10.1098/rspb.2015.1553.\u003c/li\u003e\n\u003cli\u003eLi X, Fan J, Luo S, Yin L, Liao H, Cui X, et al. 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Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011;29:644\u0026ndash;\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Paphiopedilum, floral color, Transcriptome, flavonoid metabolome","lastPublishedDoi":"10.21203/rs.3.rs-6878590/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6878590/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e The artificial hybridization of \u003cem\u003ePaphiopedilum\u003c/em\u003e began in 1869. With the breakthrough in aseptic sowing techniques, the number of hybrids surged. Over 30,000 hybrids have been registered by the Royal Horticultural Society (RHS). Hybrids derived from \u003cem\u003ePaphiopedilum delenatii\u003c/em\u003e typically retain its prominent characteristics: large, symmetrical petals and a magenta-purple labellum. In this study, the hybrid of \u003cem\u003eP. delenatii\u003c/em\u003e and \u003cem\u003eP. primulinum\u003c/em\u003e exhibited an overall creamy-white coloration. This study focuses on the divergence in floral coloration between the hybrid and their parental lines. Through comparative transcriptome analysis and flavonoid metabolic profiling across three distinct \u003cem\u003ePaphiopedilum\u003c/em\u003e, we aim to elucidate the molecular mechanisms underlying how hybrids deviate from the red and yellow-green phenotypes of their parents to develop a cream-colored pigmentation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e The labellum and dorsal sepal morphology closely resembled \u003cem\u003eP. primulinum\u003c/em\u003e, while the petal traits combined the wavy margins of \u003cem\u003eP. primulinum\u003c/em\u003e and smooth surface of \u003cem\u003eP. delenatii\u003c/em\u003e. Flavonoid metabolome analysis of the floral tissues, a total of 29 flavanones, 93 flavones and flavonols, 13 anthocyanins, 23 isoflavonoids, six flavonoids, and four chalcones and dihydrochalcones were detected. Remarkably, anthocyanin content is significantly higher in \u003cem\u003eP. delenatii\u003c/em\u003e compared to \u003cem\u003eP. primulinum\u003c/em\u003e and the hybrid. Transcriptome analysis highlighted significant up-regulation of genes in the flavonoid biosynthesis pathway and phenylpropanoid biosynthesis pathway when comparing hybrids to \u003cem\u003eP. primulinum\u003c/em\u003e. Conversely, early-stage biosynthetic genes were down-regulated in hybrid relative to \u003cem\u003eP. delenatii\u003c/em\u003e, likely contributing to metabolite content differences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion \u003c/strong\u003eThis study employed multi-omics analysis to compare the floral tissues of \u003cem\u003eP. delenatii\u003c/em\u003e and \u003cem\u003eP. primulinum\u003c/em\u003e, along with their hybrid progeny. It identified differences in flavonoid metabolite content among them. By analyzing differentially expressed genes (DEGs) within the KEGG flavonoid biosynthesis pathway, the molecular mechanism underlying the divergent floral pigmentation pattern of the hybrid compared to both parental species was explored. Analysis of transcriptomic data, through comparison with the non-redundant (NR) protein database, revealed a closer phylogenetic relationship between \u003cem\u003ePaphiopedilum\u003c/em\u003e and the genus \u003cem\u003eDendrobium\u003c/em\u003e, whereas morphologically \u003cem\u003ePaphiopedilum\u003c/em\u003e is classified under \u003cem\u003eCypripedium\u003c/em\u003e-related taxa. These findings provide novel insights and molecular evidence for understanding \u003cem\u003ePaphiopedilum\u003c/em\u003e floral coloration and phylogenetic relationships.\u003c/p\u003e","manuscriptTitle":"Transcriptome and flavonoid metabolome analysis reveals the molecular basis of floral color divergence between the hybrid and their parental species in Paphiopedilum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-17 17:37:42","doi":"10.21203/rs.3.rs-6878590/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"31925d2e-4703-435e-9824-0f6253e2ca33","owner":[],"postedDate":"July 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-30T13:23:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-17 17:37:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6878590","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6878590","identity":"rs-6878590","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}