Gang Gao,Aminu Shehu Abubakar,Jikang Chen,Ping Chen,Kunmei Chen,Chunming Yu,Xiaofei Wang,Xiaojun Qiu,Xiaoyu Huang,Deyi Shao,Yue Wang,Yu Chen,Aiguo Zhu
Comparative genome and metabolome analyses uncover the evolution and flavonoid biosynthesis between Apocynum venetum and Apocynum hendersonii
Plant Genetics,Plant evolution,Transcriptomics
Summary Apocynum species have great application prospects in textile and phytoremediation of saline soil, are rich in flavonoids, and possess medicinal significance. Here, we report the draft genomes of Apocynum venetum and Apocynum hendersonii, and elucidate their evolutionary relationship. The high synteny and collinearity between the two suggested that they have experienced the same WGD event. Comparative analysis revealed that flavone 3-hydroxylase (ApF3H) and differentially evolved flavonoid 3-O-glucosyl transferase (ApUFGT) genes are critical for determining natural variation in flavonoid biosynthesis between the species. Overexpression of ApF3H-1 enhanced the total flavonoid content and promoted the antioxidant capacity of transformed plants compared to the wild-type. ApUFGT5 and 6 explained the diversification of flavonoids or their derivatives. These data provide biochemical insight and knowledge on the genetic regulation of flavonoid biosynthesis, supporting the adoption of these genes in breeding programs aimed at the multipurpose utilization of the plants.
Comparative genome and metabolome analyses uncover the evolution and flavonoid biosynthesis between Apocynum venetum and Apocynum hendersonii

Apocynum species have great application prospects in textile and phytoremediation of saline soil, are rich in flavonoids, and possess medicinal significance. Here, we report the draft genomes of Apocynum venetum and Apocynum hendersonii, and elucidate their evolutionary relationship. The high synteny and collinearity between the two suggested that they have experienced the same WGD event. Comparative analysis revealed that flavone 3-hydroxylase (ApF3H) and differentially evolved flavonoid 3-O-glucosyl transferase (ApUFGT) genes are critical for determining natural variation in flavonoid biosynthesis between the species. Overexpression of ApF3H-1 enhanced the total flavonoid content and promoted the antioxidant capacity of transformed plants compared to the wild-type. ApUFGT5 and 6 explained the diversification of flavonoids or their derivatives. These data provide biochemical insight and knowledge on the genetic regulation of flavonoid biosynthesis, supporting the adoption of these genes in breeding programs aimed at the multipurpose utilization of the plants.

Apocynum species are perennial plants with rhizomes extending 5–6 m underground. They are adapted to survive under extreme environments, including low rainfall, saline environments, 1alkaline deserts, riverbanks, alluvial plains, and the Gobi desert. These species are distributed worldwide and primarily grow in Asia, North America, and Eastern Europe. Two notable species (Apocynum venetum and Apocynum hendersonii), well known in China and Asia, are commonly used in herbal medicine and tea, in addition to their bast fiber. Their medicinal significance is linked to their rich flavonoid constituents, which confer a wide range of physiological functions., The two species have a distinguishing morphological difference, with A. venetum having characteristically more red flowers and stems, while A. hendersonii has a conspicuous white flower, so their common name of red hemp and white hemp, respectively., This variation in the flower and stem colors may be due to differences in the functional expression of flavonoid biosynthesis genes., Flavonoids are one of the largest groups of plant secondary metabolites. They play essential roles in a wide range of physiological processes and are becoming increasingly crucial for yield stability and ecological adaptability in the face of environmental deterioration. In addition, their beneficial effects on human health as dietary sources of antioxidants have attracted increasing attention. Because of their similar geographical distribution, A. venetum and A. hendersonii are usually confused; however, there are distinct differences between the two species, with the former having a red-colored stem and flower with relatively broader leaves. These stem and flower colors may be due to differences in the functional expression of anthocyanins, as recent findings have shown that A. venetum has the highest flavonoid content. The natural variation in flavonoids between A. venetum and A. hendersonii, implies that a comparative analysis of the two species could provide insight into the mechanistic themes. Therefore, elucidating the mechanism underlying flavonoid biosynthesis is critical for Apocynum breeding. However, the paucity of genomic information has restricted progress on the molecular modification of species. Flavonoids are biosynthesized through a chain of processes, beginning with malonyl-CoA, and 4-coumaroyl-CoA catalyzes by a multi-enzyme complex,,, including chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, flavonoid 3′-hydroxylase, flavonoid 3′,5′-hydroxylase, dihydroflavonol reductase, and UDP-glucose flavonoid3-O-glucosyl transferase. Flavonols, anthocyanins, and proanthocyanidins are the major classes of flavonoids and play significant roles in synthesizing other classes, physiology, and morphology in plants based on their structural diversity., Flavonoids are mostly glycosylated via the action of flavonoid O-glucosyltransferase (UFGTs) at either the C-3 or C-7 position with glucose or rhamnose sugar. They exhibit diverse physiological functions, are primarily involved in stress response, and serve as co-pigments. Flavonoids produced in response to developmental or environmental needs are often found in nature as derivatives of flavylium ions or 2-phenylbenzopyrilium, in the form of polyhydroxylated or methoxylated heterosides.,, Recently, combined technologies of bulked segregate analysis (BSA) coupled with randomly amplified polymorphic DNA have been developed to distinguish between the two species. While few studies have assessed the genetics of A. venetum and A. hendersonii, the inadequacy of gene sequence resources has limited their in-depth study. Chromosome assembly and higher-quality genomes facilitate comparative studies of the evolution of significant plant traits. Therefore, genome data of Apocynum species is imperative in providing valuable resources for elucidating flavonoid biosynthesis and related genes, which have always been linked to higher stress tolerance and may aid in the improvement of the species and economic values. Here, we report a chromosome-scale assembly to facilitate in-depth research on A. venetum and A. hendersonii. Multiomic techniques have provided insight into complex biological processes. Using the draft genomes of A. venetum and A. hendersonii coupled with transcriptomic, metabolomic, and experimental data, we investigated the evolutionary relationship and natural variation in the accumulation of total flavonoids in the two species. The findings will increase our understanding of the functions of structural genes and facilitate molecular breeding for genetic improvements.

The genomes of A. venetum and A. hendersonii were estimated by k-mer statistics based on a genome survey, followed by de novo assembly of long reads generated by the combined Oxford Nanopore and Hi-C technologies (Figures 1A and 1B, related to STAR Methods). A total of 32.48 Gb clean reads (∼ 139.53-fold coverage) for A. venetum and 60.48 Gb clean reads (∼ 269.97-fold coverage) for A. hendersonii were obtained. After correction based on error correction data, we obtained 232.77 Mb contig with an average contig N50 length of 9.68 Mp for A. venetum and 240.24 Mb contig with an average contig N50 length of 9.60 Mp for A. hendersonii (Tables 1 and S1. Statistics on assembly results and S2. Genome statistics after Hi-C assembly, related to STAR Methods). The chromosome numbers and ploidies of the two species were determined by karyotype analysis. The A. venetum and A. hendersonii genomes were composed of 22 chromosomes, and the number of chromosomes was confirmed by fluorescence in situ hybridization (FISH) and telomere repeat probes (Data S1. TS, Telomere-specific-repeat probe, related to STAR Methods). The ploidy of the two species was determined using FISH with 18S rDNA repeat sequence probes (Data S1. 18S rDNA repeat sequence probe, related to STAR Methods), and the 18S rDNA hybridization signal showed that the cells in the two samples contained two pairs of chromosomes, indicating that both A. venetum and A. hendersonii were diploid, with 2n = 22 (Figure 1C). The LACHESIS programs employed to investigate the Hi-C scaffolding reliability oriented and anchored the 232.77 Mb contig (representing 99.2% total length) and 240.24 Mb contig (representing 97.4% total length) into 11 pseudochromosomes for A. venetum and A. hendersonii, respectively (Table 1 and Figure 1A). The 11 chromosomes were clearly distinguished within each group, with the intensity of the interaction at the diagonal position higher than that at the non-diagonal position in both species (Figure 1B). This is consistent with the principle of HiC-assisted genome assembly, supporting the genome assembly effect.

Genome annotation revealed a total of 756,249,732 (96.49%) and 782,208,273 (99.49%) reads for A. venetum and A. hendersonii, respectively, mapping to the ref. (Table S3. Statistical information on second-generation data comparison). Gene repertoire completeness revealed 93.61% complete BUSCOs (91.88 single copies and 1.74% duplicated copies) for A. venetum. There were 93.40% complete BUSCOs for A. hendersonii (91.60% single copies and 1.81% duplicated copies) (Tables 1 and S4. BUSCO evaluation results), indicating higher quality of the annotation and assembly. The assembly covered 98.03 and 97.60% of the 458 core eukaryotic genes (CEG) for A. venetum and A. hendersonii, respectively (Table 1), reflecting completeness., These data indicated that the genome assemblies of A. venetum and A. hendersonii presented high contiguity and sequence quality. There were 20,292 protein-coding genes (PCGs), including 5,173 one-copy genes and 4,632 multi-copy genes clustered into 15,298 gene families in A. venetum. Whereas in A. hendersonii, 23,456 PCGs comprising 5,151 one-copy genes and 4,717 multi-copy genes clustered into 15,592 gene families were identified (Table S5. Statistics of gene prediction results). Among these, 15,274 orthologous gene families were common to all the 11 selected plant species (Figure 1D, and Table S6. Gene family statistics of Apocynum species and 9 others plant species). A total of 24 families containing 68 genes and 245 families containing 733 genes were unique to A. venetum and A. hendersonii, respectively (Figure S1. Venn diagram of unique gene families among Apocynum species and 9 other plant species), which may be related to environmental adaptation and metabolic pathways within the Apocynum lineage. These results indicated that the A. hendersonii genome contained more unclustered and unique genes than the A. venetum genome. Repetitive sequences and transposable elements (TEs) in A. venetum and A. hendersonii accounted for 35.21 and 34.80% of the total genome, respectively (Table S7. Statistics for repeat sequences in the Apocynum species), and the sequence distribution was more abundant in the LG04 and LG05 chromosomes in both species (Figure 1). Class I TEs formed the dominant repeats in A. venetum and A. hendersonii at 30.38 and 29.41%, respectively. Copia and Gypsy long terminal repeats (LTRs), as well as large retrotransposon derivatives (LARDs), were the most abundant TEs at 11.80, 10.52, and 6.65%, respectively, in A. venetum and 11.70, 10.60, and 5.82% in A. hendersonii.

Gene families that underwent discernible changes and were divergently involved along different branches, with emphasis on those involved in Apocynum traits, were analyzed (Figure 2A). The results showed that 921 and 929 gene families exhibited significant expansions (p < 0.001) in the A. venetum and A. hendersonii lineages, respectively, compared with their ancestors. No contraction of this gene family was observed in either species, similar to Vitis vinifera. Gene family annotation demonstrated that the primary enriched expansion genes belonged to secondary metabolites, such as those involved in flavonoid biosynthesis. WGD events were investigated by estimating 4-fold degenerate synonymous site (4DTv) mutations in collinear genes (Figure 2B). The peak around 0.53 4DTv indicated shared WGD among A. venetum, A. hendersonii, V. vinifera, and Arabidopsis thaliana before divergence. At 0.20–0.80, A. venetum and A. hendersonii had a shared WGD event consistent with the phylogenetic data of the two species (Figures 2B and 2C). The higher synteny and shared collinearity between A. venetum and A. hendersonii suggested that the two species experienced the same WGD event, indicating that divergence might be more recent, which is in line with the results of the Ks analysis (Data S1. LTRs in the species, related to STAR Methods). The evolutionary analysis confirmed the placement of A. venetum and A. hendersonii in Apocynaceae, sharing a monophyletic clade with Catharanthus roseus (a representative member of the Apocynaceae)., Both Apocynum species diverged successively and showed the closest evolutionary relationships among the 11 species investigated (Figure S2. Evolutionary analysis of A. venetum, A. hendersonii and other plant species, related to STAR Methods). These species shared a common ancestor approximately 140.8 million years ago (MYA).

The flavonoid biosynthesis pathway was predominantly enriched in A. venetum and A. hendersonii (Figure 3A). A total of 223 flavonoids or their derivatives, comprising flavones (74), flavonols (85), flavanones (23), isoflavones (13), and anthocyanins/proanthocyanins (28), were identified in the two species (Data S1. Identified flavonoid components in Apocynum, related to STAR Methods). Among these, 51 flavonoids were differentially accumulated between the two species, with 31 being predominant in A. venetum and 20 in A. hendersonii (Data S1. Differentially accumulated flavonoids, related to STAR Methods). Quantitative analysis by targeted LC/MS (Table 2) confirmed that in addition to the total flavonoid content and the candidate discrimination marker quercetin 3-D-galactoside, colored compounds, such as gallocatechin, proanthocyanidin, and delphinidin 3-glucoside were also significantly accumulated in A. venetum compared to A. hendersonii. Genome assembly allowed us to locate all functionally characterized flavonoid metabolism genes and their closely related homologs. Unlike specialized metabolites, such as momilactones and morphinan, which are produced in high plants by conserved biosynthetic gene clusters,, the potential genes involved in A. venetum and A. hendersonii flavonoid biosynthesis were widely distributed on all 11 chromosomes (Figure S3. Chromosomal gene arrangement of flavonoid biosynthesis candidate genes in A. venetum and A. hendersonii, related to STAR Methods). Gene synteny analysis of candidate flavonoid biosynthesis genes in A. venetum and A. hendersonii with those in V. vinifera (which showed close homologies and richly accumulated high flavonoids) revealed the presence of essential genes encoding enzymes (Figure 3B). Although some isoforms involved in flavonoid biosynthesis are missing in one Apocynum genome and vice versa, flavonoid metabolic pathways are well conserved in both. The expression patterns of potential synthetases involved in flavonoid biosynthesis (including PAL, C4H, 4CL, CHS, CHI, F3H, F3′5′H, FLS, DFR, LDOX/ANS, LAR, and UFGT) were analyzed. Among these, the ApF3H and ApUFGT gene families were significantly up/downregulated.

Three pairs of highly conserved F3H genes (with 100% sequence similarity between each pair) were identified in the A. venetum and A. hendersonii genome: ApF3H-1 (Av, EVM0009869; Ah, EVM0007724), ApF3H-2 (Av, EVM0017534; Ah, EVM0010080), and ApF3H-3(Av, EVM0019829; Ah, EVM0016058). ApF3H-2 and ApF3H-3, located on chromosome 04, were expressed at the same levels in different tissues of A. venetum and A. hendersonii. In contrast, ApF3H-1, located on chromosome 09, was significantly upregulated in A. venetum leaves compared to A. hendersonii leaves (Data S1. Fold changes of the F3H genes, related to STAR Methods). Combining metabolic and gene expression data obtained for A. venetum and A. hendersonii, we identified ApF3H-1 as a critical enzyme required for total flavonoid accumulation. This variation may significantly affect flavonoid biosynthesis in the duos. Thus, the ORF of ApF3H-1 under the control of the CaMV35S promoter (Figure 4A) was transformed into A. hendersonii and the transgenic lines were screened using kanamycin (100 mg/L) and identified by PCR analysis using a gene-specific primer (Data S1. ApF3H1OE, related to STAR Methods) and GUS staining (Figure 4B). The expression levels of ApF3H-1 in the calli and leaves were significantly higher in the transgenic lines than in the WT plants, resulting in a significant increase in total flavonoid content and enhanced antioxidant capacity of the plant (Figures 4C–4H).

After excluding the too-long and partial amino acid-encoding sequences, eight pairs of glycosylated family homologous genes (AvUFGT1-8 and AhUFGT1-8) from A. venetum and A. hendersonii were characterized. The phylogenetic analysis and unrooted tree obtained using MEGA 10 (neighbor-joining algorithm) categorized the ApUFGTs into unique clusters based on their region-specificity,, comprising 3 GT (AvUFGT1, AhUFGT1, AvUFGT6, AhUFGT6), 5 GT (AvUFGT3, AhUFGT3), GGT (AvUFGT2, AhUFGT2, AvUFGT4, AvUFGT4, AvUFGT7, AhUFGT7), and CGT (AvUFGT5, AhUFGT5, AhUFGT8, AhUFGT8) subfamilies (Figure 5). Eight pairs were highly correlated or differentially evolved ApUFGTs suggesting novel specificities. Due to the significance of UFGT glycosylation in the stability, solubility, and bioavailability of flavonoids, ApUFGTs were prokaryotic expressed, and recombinant proteins were used for the enzymatic assay. Six ApUFGTs were amplified and successfully expressed in Escherichia coli using the pET-30a prokaryotic vector (Data S1. Primers for prokaryotic expression, related to STAR Methods). Enzyme-substrate specificity was determined using UDP-glucose as a sugar donor and three typical flavonoid aglycones (flavonols—kaempferol and quercetin; flavones—apigenin, and flavanones naringenin) as substrates (Table 3). The ApUFGTs exhibited specific activity against kaempferol, quercetin, and apigenin aglycones but showed no activity against naringenin. AvUFGT3 and AhUFGT3 showed the highest catalytic efficiency, with kcat values of 0.198 ± 0.002 and 0.201 ± 0.002 s−1 for kaempferol and 0.097 ± 0.001 and 0.101 ± 0.002 s−1 for quercetin, respectively. In addition, UFGT3 and UFGT1 of both A. venetum and A. hendersonii showed similar enzymatic properties on the selected substrates. All ApUFGTs displayed higher activities with kaempferol than with quercetin, except for AvUFGT5 and AhUFGT6. AvUFGT5 from A. venetum exhibited higher catalytic activity for quercetin and lower activity for kaempferol compared to its AhUFGT5 ortholog from A. hendersonii. AhUFGT6 exhibited the highest catalytic activity, with a significantly higher kcat value of 0.224 ± 0.005 s−1. Regarding UFGT6, higher activities of flavonols (both kaempferol and quercetin) and considerably lower activities of flavones (apigenin) were observed in AvUFGT6 compared to AhUFGT6 (Figure 6).

Apocynum species (A. venetum and A. hendersonii) are perennial herbaceous plants with medicinal significance and excellent ecological adaptability.,, Quality genomes and chromosome assembly are crucial for the comparative analysis of significant traits in plants. High-quality genome and chromosome assembly data for the two species revealed substantial differences in repeat sequences, with a higher number of DNA transposons in A. hendersonii than A. venetum, which might have been attributed to the species’ morphology. In morning glory, many mutations that lead to morphological changes, including diversity of flower color, result from TEs., Due to the pleiotropic role of the flavonoid biosynthesis pathway in plants, any single mutation may lead to phenotypic differences. Integrated multi-omics analysis of the two species revealed the accumulation of total flavonoids. Overexpression of ApF3H-1 in A. hendersonii demonstrated potential for improving total flavonoid accumulation, suggesting its exploitation for molecular breeding targeted at enhancing flavonoid content. ApF3H-1, which catalyzes the early biosynthesis pathway, determines the differential accumulation of total flavonoids. F3H acts on flavonoids to yield dihydrokaempferol (colourless dihydroflavonol), which is subsequently hydroxylated by flavonoid 3′ hydroxylase (F3′H) or flavonoid 3′,5' hydroxylase (F3′5′H) to produce red and blue anthocyanins precursors (cyanidin and delphinidin), respectively. The ApF3H-1 gene was expressed at high levels in A. venetum leaves compared to A. hendersonii, despite no differences in amino acids between these paralogous copies. This may be because variations in chromosome location or regulatory genes affected gene expression. Consistent with previous studies, the F3H genes in Apocynum were highly conserved. Flavonoids endow Apocynum with crucial medicinal value, improving the species’ resistance and adaptability to adverse conditions. Elucidating the genetic basis underlying the natural variation of plant metabolites provides a better understanding of gene function within ecological and evolutionary contexts. It represents an effective way of determining gene function. This makes it useful for gain-of-function breeding approaches., Given the multi-step nature of flavonoid biosynthesis,, variation in flavonoids is also determined by tailoring enzymes. Natural variation and diversity of flavonoid constituents between A. venetum and A. hendersonii were determined by the differentially evolved UFGT, ApUFGT5, and ApUFGT6. These demonstrated variations in preference for the flavone apigenin and exhibited various kinetic parameters. Decorating enzymes are responsible for the final steps of metabolite synthesis, resulting in a more significant contribution to the natural variation in metabolite abundance. The glycosylation of flavonoids confers structural complexity, molecular stability, subcellular transportability, and biological activity.,, However, knowledge of flavonoid UGTs derives almost exclusively from research on flavonols, which accumulate in Arabidopsis and other plants.,, In this study, diverse catalytic activity in ApUFGT5 (CGT subfamily) and ApUFGT6 (3 GT subfamily) was observed between Apocynum species. The mRNA and amino acid analyses revealed that the homologous AvUFGT1-4 and AhUFGT1-4 exhibited almost 100% sequence similarity, with some nonsense amino acid mutations. There are nine single nucleotide polymorphisms for AvUFGT5 and AhUFGT5, whereas AhUFGT6 has a 48 aa deletion mutant at the N-terminus compared to AvUFGT6. These results suggested that the two UFGTs are major enzymes that control natural variation in downstream metabolites of the flavonoid biosynthesis pathway between A. venetum and A. hendersonii. Subsequent investigation of UFTG5 and UFGT6 activities on flavones in planta and flavonoid-targeted profiling between the WT and overexpression lines will further contribute to flavonoid biosynthesis in Apocynum species. Apocynum species are rich sources of flavonoids that have medicinal significance, including antimicrobial and antioxidant properties. This may explain their tolerance to adverse environments, such as saline-alkaline soils, where they naturally exist., As natural antioxidants, flavonoids protect plants from unfavorable environmental conditions and enhance tolerance to abiotic and biotic stresses. Thus, we correlated flavonoid accumulation with Apocynum’s free radical scavenging activity, transgenic lines overexpressing the F3H flavonoid biosynthesis gene, and the concentration-dependent activity of ABST+, DPPH, and Fe3+-TPTZ. Our data showed that ApF3H-1 promoted the accumulation of total flavonoids, which conferred enhanced tolerance to reactive oxygen species to Apocynum. Recent studies on Arabidopsis have demonstrated that glycosylated flavonoids can protect against UVB. Flavonoids improve drought tolerance,, suggesting that increasing flavonoid contents may enhance the species’ ecological adaptability and stress resistance. Thus, overexpression of F3H may be exploited for molecular breeding to improve flavonoid accumulation in Apocynum. This study has provided important information for future studies of the two species. And further studies are recommended to unravel the roles of repeat sequences in the flavonoid variation and other significant characteristics of A. venetum and A. hendersonii.

Although the genome data and genetic transformation report are of great significance for further study of Apocynum plants, investigating decorating enzymes ApUFTG5 and ApUFGT6 in planta and flavonoid-targeted profiling between the WT and overexpression lines will further give a clearer understanding of flavonoid biosynthesis in Apocynum species.



Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Zhu Aiguo (zhuaiguo@caas.cn)

This study did not generate new unique reagents.

Seeds of both A. venetum and A. hendersonii were initially germinated on MS media. The germinated seedlings and plantlets were used for the transformation. Shoots and leaves were excised, cut to appropriate sizes about 4 - 6 mm, and inoculated with fresh Agrobacterium tumefaciens strain, harbouring the pBI121-F3H construct (OD 600 = 0.6 - 0.8) for 30 minutes with continuous shaking. The explants were rinsed two times with double distilled water, and excess moisture removed using sterile filter paper. Subsequently, the explants were placed on MS supplemented with 1 mg/L naphthalene acetic acid (NAA) and incubated in dark at 25°C for two days. The explants were later transferred to MS media containing 1 mg/L NAA, 50 mg/L kanamycin and 100 mg/L Cefotaxime and incubated under 16/8 light/dark photoperiod for callus induction. Shoot and root were induced in MS supplemented with 0.04 mg/l NAA and 0.4 mg/l thidiazuron (TDZ). Hardening was done was done in a controlled chamber by transferring the plant to a potted organic soil mixture and watered using Hoagland solution (1.63 mg/L w/v).

DNA samples were extracted from fresh A. venetum and A. hendersonii leaves obtained from the Xinjiang Research Centre of the Chinese Academy of Agricultural Sciences using the CTAB method. The extracted DNA was fragmented, enriched, and purified, and the fragments were end-repaired to obtain the final library following the Nanopore library construction protocol. The DNA library was quantified using Qubit and Sequenced on the PromethION sequencer platform at Biomarker Biotechnology Co., Ltd. Beijing. Nanopore sequencing data were evaluated and assessed by filtering low-quality reads, removing the joint to obtain reads, and correcting the base error to get high-accuracy data for genome assembly, post-assembly evaluation, and other analyses.

The third generations of Nanopores data obtained were error-corrected using Canu. The integrity of the assembled genome was evaluated based on the second-generation sequencing read comparison rate, core genetic integrity, and BUSCO assessment. Second-generation read comparison refers to the proportion of clean reads in relation to the reference genome, and was performed using BWA. CEGMA v2.5 and BUSCO v2.0 were used to assess the integrity of the final genome assembly. Hi-C fragment libraries with 300–700 bp insert sizes were constructed and sequenced using the Illumina platform according, as previously described. HiC-Pro software was used for quality control, and RACKES was used to evaluate the assembly results. Accurate karyotyping was confirmed using fluorescence in situ hybridization. A telomere-specific repeat probe was used to verify the number of intact chromosomes, and an 18s rDNA repeat sequence probe was used to identify multiple copies of chromosomes. Chromosomes were stained using 4′,6-diamidino-2-phenylindole (DAPI), and the dispersed metaphase chromosome cells were counted under a fluorescence microscope.

The genetic structures of A. venetum and A. hendersonii were annotated based on de novo prediction, homologous species prediction, and unigene prediction., Genscan, August v2.4, GlimmerHMM v3.0.4, GeneID v1.4S and NAP (version 2006-07-28) set at default were used for the de novo prediction. GeMoMa, v1.3.1 with default parameters was used for homologous species based predictions with protein sequences of Arabidopsis thaliana, Catharanthus roseus, Coffea canephora and Solanum lycopersicum obtained from NCBI. Hisat v2.0.4 (set at: --max-intronlen 20000,--min-intronlen 20) and Stringtie v1.2.3 with default parameters were employed for the genome assembly based on reference transcripts. TransDecoder (http://transdecoder.github.io) v2.0 and GeneMarkS-T v5.1 both set to default for gene prediction while PASA v2.0.2 (parameters: -align_ tools gmap, -maxIntronLen 20000) was used in validating the predicted unigene sequences and finally EVM v1.1.1 was used to integrate the predicted results. Non-coding RNA (microRNAs, rRNA, and tRNA) were also predicted. Rfam with Blistn for genome-wide comparison was employed to identify microRNAs and rRNAs, and tRNAscan-SE in the identification of tRNA. At the same time, the genome of both species was annotated for gene function with Nr, KOG, GO and KEGG metabolic pathway. Functional annotations were performed on the gene set using BLASTP with default settings against the NCBI, NR, SwissProt, and KOG databases. LTR_FINDER and RepeatScout were used with default parameters to build a database of repeat genome sequences based on structural and de novo prediction principles. PASTEClassifier was used to classify the database, and together with Repbase formed the final repeating sequence database. Using RepeatMasker, we obtained repeat predictions for the genome based on a constructed repeat sequence database.

Orthologous gene families were constructed between A. venetum, A. hendersonii, and nine other species (Oryza sativa GCA_001648735.1, Arabidopsis thaliana GCA_000222325.1, Vitis vinifera GCA_002922885.1, Morus alba GCA_012066045.3, Cannabis sativa GCA_001865755.1, Gossypium barbadense GCA_001856525.1, Catharanthus roseus GCA_028651295.1, Boehmeria nivea GCA_002806895.1, and Corchorus capsularis GCA_001974805.1) using OrthoMCL. MUSCLE was used for multiple sequence alignment of single-copy orthologs for all 11 species. A maximum likelihood tree was drawn using RAxML. Subsequently, a phylogenetic tree was constructed using the GTRGAMMA model with V. vinifera as an out-group. The protein sequences were transformed into CDS, and MCMCTREE was used to estimate the divergence time of Apocynum species from other species. Expansions and contractions of gene families were computed using CAFÉ, and 4DTv obtained based on the CDS alignment was used to depict whole genome duplications (WGD). The 4DTv substitution per synonymous site (Ks) was computed for each homologous gene pairs within and between species. The age of LTR divergence time was calculated using DistMat software following Kimura model with a 7.3 × 10−9 molecular clock. Synteny analysis was conducted between Apocynum species and V. vinifera using MCscanX.

RNA was extracted from the stems and leaves of A. venetum and A. hendersonii using the AG21019 SteadyPure Plant RNA Extraction Kit (Accurate Biotechnology Co., Ltd., Hunan, China), according to the manufacturer's instructions. These RNA-seq libraries were then sequenced through the Illumina HiSeq 2000 platform and aligned to the already assembled and annotated Apocynum genomes. Gene expression levels were computed as the number of reads per kilobase of gene length per million mapped reads (FPKM) using RSEM software. Metabolites were profiled using targeted/untargeted metabolomics methods at Oebiotech Biotechnology Co., Ltd. (Shanghai, China) (http://www.oebiotech.com/), and six independent samples for each group were analyzed. A liquid chromatography-electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) system was used for relative metabolite quantification. The ACQUITY UHPLC system (Waters Corpo MA, Milford, USA) and Triple TOF 5600 System (AB SCIEX, Framingham, MA, USA) equipped with an UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm) in both positive and negative ion modes was used for the analysis according to previous studies., The binary gradient elution system were 0.1 % formic acid in water, v/v (A) and 0.1 % formic acid in acetonitrile, v/v (B) and separation done under the gradient: 0 min, 5% B; 2min, 20% B; 4min, 25% B; 9min, 60% B; 14min, 100% B; 18min, 100% B; 18.1 min, 5% B and 19.5min, 5%B. The flow rate was 0.4 mL/min and column temperature of 45 °C. The samples temperature was maintained at 4°C with an injection volume of 50 μL and a full scan mode (m/z ranges from 70 to 1000) was used for data acquisition.

The sequences of genes associated with flavonoid biosynthesis were downloaded from UniProt (https://www.uniprot.org) and identified using BLASTP against the database with an E-value cut-off of 1e-. These included genes related to enzymes potentially involved in flavonoid biosynthesis pathways, such as PAL, C4H, 4CL, CHS, CHI, F3H, F3'5'H, FLS, DFR, LDOX/ANS, LAR, and UFGT. Integrated analyses of comparative transcriptomic, genomic, and phytochemical data based on gene annotation were performed to elucidate the molecular mechanisms of flavonoid biosynthesis in A. venetum and A. hendersonii.

The ORF sequence of ApF3H was amplified using cDNA from A. hendersonii and cloned into the pBI121 overexpression vector. The recombinant vector was introduced into Agrobacterium strain LBA4404 and then transferred into A. hendersonii using tender stems as explants. The positively transformed plants were selected on kanamycin (100 mg/L)-supplemented MS media and identified by PCR using specific primers and GUS staining. Gene expression was quantified by qRT-PCR, and relative expression was calculated using the 2-ΔΔCT method. The primers used for qRT-PCR are listed in the supplementary file (Data S1. ApF3H1QR, ApF3H2QR, and ApF3H3QR).

The open reading frame (ORF) of ApUFGTs was amplified and inserted into the pET-30a vector. Recombinant pET-30a-ApUFGT plasmid DNA was then transformed into Escherichia coli BL21 cells for prokaryotic expression. Recombinant ApUFGT enzymes were purified using a Biologic DuoFlowTM chromatography system (Bio-Rad, USA), following the manufacturer's protocol, and then used for enzymatic assays. In vitro enzyme reactions were performed in a total volume of 100 μL containing 200 μM flavonoid substrates, flavonols (kaempferol and quercetin), flavones (apigenin), and flavanones (naringenin, 1.5 mM UDP glucose, 5 mM MgCl2, and 500 ng purified protein in Tris-HCl buffer (100 mM, pH 7.4). The mixture was incubated at 37°C for 20 min, and the reaction was stopped by adding 300 μL of ice-cold methanol. The reaction mixture was then passed through a 0.2 μm filter (Millipore) before use for LC-MS analysis.

Total flavonoids from wild and transformed plants were extracted and measured as previously described. Free radical scavenging capacity was measured in three test systems using the stable radical ABTS+ [2,2-azino-bis-(3-ehylbenzothiazoline-6-sulfonic acid)], DPPH (2,2′-diphenyl-1-picrylhydrazyl), and FRAP assays. The percentage inhibition of ABTS+ (I ABTS+ %), DPPH (I DPPH %), and FRAP (μmol.mL-1) was calculated as previously reported.Analysis of variation was performed with GenStat 17th edition and means separated using the Student’s Newman Keul method.

The raw metabolic analysis data were analyzed by the progqenesis QI software (Waters Corporation, USA). Standard internal detection parameters were deselected for peak RT alignment and isotopic peaks excluded for analysis. Noise elimination level and minimum intensity were set at 10.00 and 15 % of base peak intensity respectively. The matrix obtained was further reduced by eliminating any peaks with missing value (ion intensity = 0) realized in more than 60 % samples. The internal standard was used for data QC (reproducibility). 0.3 mg/ml 2-Chloro-L-phenylalanine (CAS: 103616-89-3) in Methanol solution was use as reference substance. The kinetic constants of ApUFGTs for flavonoid acceptance were determined using 0–400 μM of the different flavonoids at a fixed concentration of 1.5 mM UDP-glucose. Flavonoids and UDP sugars were purchased from Sigma–(Ald)rich USA. All kinetic parameters were calculated using the Michaelis-Menten model (Sigma Plot, version 12.5), and all reactions were performed in triplicate.