Development of SSR markers for genetic diversity analysis and species identification in Polygonatum odoratum (Mill.) Druce based on transcriptome sequences.

Polygonatum odoratum (Mill.) Druce is a well-known traditional Chinese herb belonging to the Polygonatum. However, the understanding of the genetic diversity of this species at the molecular level is limited due to the lack of transcriptomic and genomic information. In this study, 37,387 unigenes were assembled based on the transcriptome sequencing of the rhizome of Polygonatum odoratum (Mill.) Druce., and 11,021 single- sequence repeats (SSR) motifs, mainly consisting of single-nucleotide repeats (44.44%), dinucleotides (31.06%), and trinucleotides (22.59%), were identified. Based on these SSR motifs, 9,987 primer pairs of SSR markers were designed and 68 SSR markers were randomly selected for verification, of which 21 SSR markers showed polymorphisms among the 24 Polygonatum odoratum germplasms. Ninety-four alleles were detected: the observed alleles ranged from 2 to 11, the effective alleles varied from 1.086 8 to 4.916 8, the Shannon diversity index was 0.173 2~1.749 7, and the polymorphism information content PIC ranged from 0.076 7 to 0.803 9. Based on our analysis of genetic diversity (SSR genotypes) and population structure, we divided the 24 germplasm resources into two groups, indicating that the germplasm with similar geographical origins can be grouped together. In addition, the primers 'YZ14' and 'YZ47' could effectively distinguished the related species: Polygonatum kingianum Coll.et Hemsl., Polygonatum sibiricum Red., Polygonatum cyrtonema Hua, Polygonatum zanlanscianense Pamp. and Polygonatum odoratum (Mill.) Druce. This is the first study in which a dataset of expressed sequence tag (EST)-SSR markers is constructed for the Polygonatum odoratum (Mill.) Druce, and these newly developed EST-SSR markers provided a very efficient tool for genetic relationship analysis, species identification and marker-assisted selection breeding of Polygonatum odoratum (Mill.) Druce.

Genomic Investigation of a Rhizosphere Isolate, Streptomyces sp. JL1001, Associated with Polygonatum cyrtonema Hua.

In the present study, using genome mining, Streptomyces sp. JL1001, which possesses a leinamycin-type gene cluster, was identified from 14 strains of Streptomyces originating from the rhizosphere soil of Polygonatum cyrtonema Hua. The complete genome of Streptomyces sp. JL1001 was sequenced and analyzed. The genome of Streptomyces sp. JL1001 consists of a 7,943,495 bp chromosome with a 71.71% G+C content and 7315 protein-coding genes. We also identified 36 biosynthetic gene clusters (BGCs) for secondary metabolites in Streptomyces sp. JL1001. Twenty-seven BGCs had low (< 50%) or moderate (50-80%) similarity to other known secondary metabolite BGCs. In addition, a comparative analysis was conducted between the leinamycin-type gene cluster in Streptomyces sp. JL1001 and the biosynthetic gene clusters of leinamycin and largimycin. This study aims to provide a comprehensive analysis of the genomic features of rhizosphere Streptomyces sp. JL1001. It establishes the foundation for further investigation into experimental trials involving novel bioactive metabolites such as AT-less type I polyketides that have important potential applications in medicine and agriculture.

Integrative analysis of the transcriptome and metabolome provides insights into polysaccharide accumulation in Polygonatum odoratum (Mill.) Druce rhizome.

Background: Polygonatum odoratum (Mill.) Druce is a traditional Chinese herb that is widely cultivated in China. Polysaccharides are the major bioactive components in rhizome of P. odoratum and have many important biological functions. Methods: To better understand the regulatory mechanisms of polysaccharide accumulation in P. odoratum rhizomes, the rhizomes of two P. odoratum cultivars 'Y10' and 'Y11' with distinct differences in polysaccharide content were used for transcriptome and metabolome analyses, and the differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) were identified. Results: A total of 14,194 differentially expressed genes (DEGs) were identified, of which 6,689 DEGs were down-regulated in 'Y10' compared with those in 'Y11'. KEGG enrichment analysis of the down-regulated DEGs revealed a significant enrichment of 'starch and sucrose metabolism', and 'amino sugar and nucleotide sugar metabolism'. Meanwhile, 80 differentially accumulated metabolites (DAMs) were detected, of which 52 were significantly up-regulated in 'Y11' compared to those in 'Y10'. The up-regulated DAMs were significantly enriched in 'tropane, piperidine and pyridine alkaloid biosynthesis', 'pentose phosphate pathway' and 'ABC transporters'. The integrated metabolomic and transcriptomic analysis have revealed that four DAMs, glucose, beta-D-fructose 6-phosphate, maltose and 3-beta-D-galactosyl-sn-glycerol were significantly enriched for polysaccharide accumulation, which may be regulated by 17 DEGs, including UTP-glucose-1-phosphate uridylyltransferase (UGP2), hexokinase (HK), sucrose synthase (SUS), and UDP-glucose 6-dehydrogenase (UGDH). Furthermore, 8 DEGs (sacA, HK, scrK, GPI) were identified as candidate genes for the accumulation of glucose and beta-D-fructose 6-phosphate in the proposed polysaccharide biosynthetic pathways, and these two metabolites were significantly associated with the expression levels of 13 transcription factors including C3H, FAR1, bHLH and ERF. This study provided comprehensive information on polysaccharide accumulation and laid the foundation for elucidating the molecular mechanisms of medicinal quality formation in P. odoratum rhizomes.

Combined Metabolomics and Transcriptomics Analysis of the Distribution of Flavonoids in the Fibrous Root and Taproot of Polygonatum kingianum Coll.et Hemsl.

Polygonati rhizoma, known for its distinct yellow rhizomes, is a common therapeutic and culinary plant in Far East Asia. The hue of medicinal plants is closely tied to the flavonoid biosynthesis and content levels. In this research, the fibrous root and taproot of Polygonatum kingianum Coll.et Hemsl. were studied to explore the secondary metabolite expression and flavonoid biosynthesis mechanisms using transcriptomics and metabolomics. Metabolic analysis identified that the differentially accumulated metabolites (DAMs) in the fibrous root and taproot were predominantly flavonoids, steroids, alkaloids, and phenolic acids. Overall, 200 flavonoids were identified in P. kingianum Coll.et Hemsl., with 170 exhibiting variances between the fibrous root and taproot. The transcriptome analysis revealed that a total of 289 unigenes encoding 32 enzymes were annotated into four flavonoid biosynthesis pathways, which include phenylpropanoid biosynthesis pathway, flavonoid biosynthesis pathway, isoflavonoid biosynthesis pathway, and flavone and flavonol biosynthesis pathway. The integration of transcriptomic and metabolomic data elucidated that the 76 differentially expressed genes (DEGs) encoding 13 enzyme genes (HCT, CCOMT, C4H, C3'H, CHI, PGT1, FLS, F3'H, CHS, ANR, DFR, F3'5'H, and LAR) and 15 DAMs preferred to be regulated in the flavonoid biosynthesis pathway. The expression of 10 DEGs was validated by qRT-PCR, agreeing with the same results by RNA-Seq. These findings shed light into the biosynthesis of secondary metabolites in P. kingianum Coll.et Hemsl., offering valuable information for the sustainable utilization and enhancement of this plant species.

Integrative analyses of metabolome and transcriptome reveal the dynamic accumulation and regulatory network in rhizomes and fruits of Polygonatum cyrtonema Hua.

BACKGROUND: According to Chinese ancient books, both fruits and rhizomes of Polygonatum cyrtonema Hua have medicinal and edible values. Up to now, there is no report about the metabolite profiles and regulatory network in fruits and different year-old rhizomes of P. cyrtonema. RESULTS: In this study, we performed integrative analyses of metabolome and transcriptome to reveal the dynamic accumulation and regulatory network of fruits and different year-old rhizomes in P. cyrtonema. The relative content of phenolic acids, lignans and coumarins, flavonoids and alkaloids increased with growth years, while steroids and lipids decreased with it. In addition, the relative content of nucleotides and derivatives, flavonoids, organic acids, steroids and lipids in fruits were higher than rhizomes. Genes that might relate to the biosynthesis of polysaccharides, flavonoids, triterpene saponins and alkaloids biosynthesis were further analyzed by transcriptome analysis, including sacA, GMPP, PMM, CCoAOMT, CHI, ANR, CHS, DXS, GGPS, ZEP, CYP72A219 and so on, for their expressions were positively correlated with the relative content of the metabolites. Additionally, the correlation network in sugar and aromatic amino acids metabolites were constructed to further illustrate the biosynthesis of polysaccharides, flavonoids and alkaloids in P. cyrtonema, and some transcription factors (TFs) were screened, such as C2C2, MYB, bZIP, GRAS and NAC. CONCLUSIONS: This study can deepen our understanding of the accumulation patterns and molecular mechanism of the main compounds in P. cyrtonema, and provide reference for the standardize production of P. cyrtonema.

Integrated Metabolomics and Transcriptomics Analysis of Flavonoid Biosynthesis Pathway in Polygonatum cyrtonema Hua.

Flavonoids, a class of phenolic compounds, are one of the main functional components and have a wide range of molecular structures and biological activities in Polygonatum. A few of them, including homoisoflavonoids, chalcones, isoflavones, and flavones, were identified in Polygonatum and displayed a wide range of powerful biological activities, such as anti-cancer, anti-viral, and blood sugar regulation. However, few studies have systematically been published on the flavonoid biosynthesis pathway in Polygonatum cyrtonema Hua. Therefore, in the present study, a combined transcriptome and metabolome analysis was performed on the leaf, stem, rhizome, and root tissues of P. cyrtonema to uncover the synthesis pathway of flavonoids and to identify key regulatory genes. Flavonoid-targeted metabolomics detected a total of 65 active substances from four different tissues, among which 49 substances were first study to identify in Polygonatum, and 38 substances were flavonoids. A total of 19 differentially accumulated metabolites (DAMs) (five flavonols, three flavones, two dihydrochalcones, two flavanones, one flavanol, five phenylpropanoids, and one coumarin) were finally screened by KEGG enrichment analysis. Transcriptome analysis indicated that a total of 222 unigenes encoding 28 enzymes were annotated into three flavonoid biosynthesis pathways, which were "phenylpropanoid biosynthesis", "flavonoid biosynthesis", and "flavone and flavonol biosynthesis". The combined analysis of the metabolome and transcriptome revealed that 37 differentially expressed genes (DEGs) encoding 11 enzymes (C4H, PAL, 4CL, CHS, CHI, F3H, DFR, LAR, ANR, FNS, FLS) and 19 DAMs were more likely to be regulated in the flavonoid biosynthesis pathway. The expression of 11 DEGs was validated by qRT-PCR, resulting in good agreement with the RNA-Seq. Our studies provide a theoretical basis for further elucidating the flavonoid biosynthesis pathway in Polygonatum.

Transcriptome-wide identification of ARF gene family in medicinal plant Polygonatum kingianum and expression analysis of PkARF members in different tissues.

BACKGROUND: Polygonatum kingianum holds significant importance in Traditional Chinese Medicine due to its medicinal properties, characterized by its diverse chemical constituents including polysaccharides, terpenoids, flavonoids, phenols, and phenylpropanoids. The Auxin Response Factor (ARF) is a pivotal transcription factor known for its regulatory role in both primary and secondary metabolite synthesis. However, our understanding of the ARF gene family in P. kingianum remains limited. METHODS AND RESULTS: We employed RNA-Seq to sequence three distinct tissues (leaf, root, and stem) of P. kingianum. The analysis revealed a total of 31,558 differentially expressed genes (DEGs), with 43 species of transcription factors annotated among them. Analyses via gene ontology and the Kyoto Encyclopedia of Genes and Genomes demonstrated that these DEGs were predominantly enriched in metabolic pathways and secondary metabolite biosynthesis. The proposed temporal expression analysis categorized the DEGs into nine clusters, suggesting the same expression trends that may be coordinated in multiple biological processes across the three tissues. Additionally, we conducted screening and expression pattern analysis of the ARF gene family, identifying 12 significantly expressed PkARF genes in P. kingianum roots. This discovery lays the groundwork for investigations into the role of PkARF genes in root growth, development, and secondary metabolism regulation. CONCLUSION: The obtained data and insights serve as a focal point for further research studies, centred on genetic manipulation of growth and secondary metabolism in P. kingianum. Furthermore, these findings contribute to the understanding of functional genomics in P. kingianum, offering valuable genetic resources.

Accumulation mechanism of metabolite markers identified by machine learning between Qingyuan and Xiushui counties in Polygonatum cyrtonema Hua.

Polygonatum cyrtonema Hua is a traditional Chinese medicinal plant acclaimed for its therapeutic potential in diabetes and various chronic diseases. Its rhizomes are the main functional parts rich in secondary metabolites, such as flavonoids and saponins. But their quality varies by region, posing challenges for industrial and medicinal application of P. cyrtonema. In this study, 482 metabolites were identified in P. cyrtonema rhizome from Qingyuan and Xiushui counties. Cluster analysis showed that samples between these two regions had distinct secondary metabolite profiles. Machine learning methods, specifically support vector machine-recursive feature elimination and random forest, were utilized to further identify metabolite markers including flavonoids, phenolic acids, and lignans. Comparative transcriptomics and weighted gene co-expression analysis were performed to uncover potential candidate genes including CHI, UGT1, and PcOMT10/11/12/13 associated with these compounds. Functional assays using tobacco transient expression system revealed that PcOMT10/11/12/13 indeed impacted metabolic fluxes of the phenylpropanoid pathway and phenylpropanoid-related metabolites such as chrysoeriol-6,8-di-C-glucoside, syringaresinol-4'-O-glucopyranosid, and 1-O-Sinapoyl-D-glucose. These findings identified metabolite markers between these two regions and provided valuable genetic insights for engineering the biosynthesis of these compounds.

Comparative chloroplast genome analysis of four Polygonatum species insights into DNA barcoding, evolution, and phylogeny.

The Polygonatum genus represents a perennial herb with the Liliaceae family, boasting substantial economic and medicinal significance. The majority of Polygonatum plants exhibit notable similarity while lacking distinctive identifying characteristics, thus resulting in the proliferation of adulterated medicinal materials within the market. Within this study, we conducted an in-depth analysis of the complete chloroplast (cp) genomes of four Polygonatum plants and compared them with four closely akin species. The primary objectives were to unveil structural variations, species divergence, and the phylogenetic interrelations among taxa. The cp genomes of the four Polygonatum species were typified by a conventional quadripartite structure, incorporating a large single copy region (LSC), a small single copy region (SSC), and a pair of inverted repeat regions. In total, we annotated a range of 131 to 133 genes, encompassing 84 to 86 protein-coding genes, 38 transfer RNA (tRNA) genes, 8 ribosomal RNA (rRNA) genes, and 0 to 2 pseudogenes (ycf1, infA). Our comparative analyses unequivocally revealed a remarkable consistency in gene order and GC content within the Polygonatum genus. Furthermore, we predicted a potential 59 to 64 RNA editing sites distributed across 22 protein-coding genes, with the ndhB gene exhibiting the most prominent propensity for RNA editing sites, boasting a tally of 15 sites. Notably, six regions of substantial potential variability were ascertained, characterized by elevated Pi values. Noteworthy, molecular markers for species identification, population genetic scrutiny, and phylogenetic investigations within the genus were identified in the form of the psaJ-rpl33 and trnS + trnT-psaD barcodes. The resultant phylogenetic tree unequivocally depicted the formation of a monophyletic clade comprising species within the evolutionary framework of Liliaceae, demonstrating closer evolutionary affinities with Maianthemum, Dracaeneae, and Asparageae. This comprehensive compendium of findings collectively contributes to the advancement of molecular species identification, elucidation of phylogenetic interrelationships, and the establishment of DNA barcodes tailored to the Polygonatum species.

Comparative Transcriptome Profiling Reveals Two WRKY Transcription Factors Positively Regulating Polysaccharide Biosynthesis in Polygonatum cyrtonema.

Polygonatum cyrtonema (P. cyrtonema) is a valuable rhizome-propagating traditional Chinese medical herb. Polysaccharides (PCPs) are the major bioactive constituents in P. cyrtonema. However, the molecular basis of PCP biosynthesis in P. cyrtonema remains unknown. In this study, we measured the PCP contents of 11 wild P. cyrtonema germplasms. The results showed that PCP content was the highest in Lishui Qingyuan (LSQY, 11.84%) and the lowest in Hangzhou Lin'an (HZLA, 7.18%). We next analyzed the transcriptome profiles of LSQY and HZLA. Through a qRT-PCR analysis of five differential expression genes from the PCP biosynthesis pathway, phosphomannomutase, UDP-glucose 4-epimerase (galE), and GDP-mannose 4,6-dehydratase were determined as the key enzymes. A protein of a key gene, galE1, was localized in the chloroplast. The PCP content in the transiently overexpressed galE1 tobacco leaves was higher than in the wild type. Moreover, luciferase and Y1H assays indicated that PcWRKY31 and PcWRKY34 could activate galE1 by binding to its promoter. Our research uncovers the novel regulatory mechanism of PCP biosynthesis in P. cyrtonema and is critical to molecular-assisted breeding.

Genetic diversity and population structure of Polygonatum cyrtonema Hua in China using SSR markers.

Polygonatum cyrtonema Hua is a perennial herbaceous plant of the Polygonatum genus, belonging to the Liliaceae family, with significant medicinal and nutritional value. In China, this species is a traditional medicinal and edible herb with a long history of application and is widely appreciated by the people. However, as the demand for medicinal herbs continues to grow, excessive harvesting has led to the depletion of wild resources and the risk of genetic erosion. In addition, the chaotic cultivation of varieties and the lack of high quality germplasm resources have led to inconsistent quality of medical materials. Therefore, it is urgent to conduct genetic diversity evaluation of this species and establish a sound conservation plan. This study assessed the genetic diversity and population structure of 96 samples collected from seven regions in China using the simple sequence repeat (SSR) molecular marker technology. In this study, a total of 60 alleles (Na) were detected across the 10 polymorphic SSR markers used, with an average of 6.0 alleles generated per locus. The values of polymorphic information content (PIC) values ranged from 0.3396 to 0.8794, with an average value of 0.6430. The average value of the effective number of alleles (Ne) was 2.761, and the average value of the Shannon's information index (I) was 1.196. The population structure analysis indicates that the Polygonatum cyrtonema Hua germplasm can be classified into three subpopulations (JZ, QY, JD) at the molecular level, which corresponds to the previous subgroups identified based on individual plant phenotypic traits. Analysis of Molecular Variance (AMOVA) showed that 74% of the genetic variation was between individuals within populations in different regions. The phylogenetic analysis of the 96 germplasm samples divided them into three main populations. The QY and JD subpopulations are largely clustered together, which could be attributed to their mountainous distribution and the local climate environment. The genetic differentiation coefficient (Fst) value was low at 0.065, indicating relatively low population differentiation. The ratio of the genetic differentiation coefficient (Fst) between the JZ population and the other two populations (QY and JD) is much higher than the ratio between the QY and JD populations. Based on the clustering results and the ratio of the genetic differentiation coefficient (Fst), it can be inferred that the genetic relationship between the QY and JD subpopulations is closer, with a certain degree of genetic differentiation from the JZ subpopulation. This study supports the conservation of germplasm resources of Polygonatum cyrtonema Hua in China and provides new parental material for germplasm genetic improvement and breeding programs.

Machine learning uncovers accumulation mechanism of flavonoid compounds in Polygonatum cyrtonema Hua.

The compositions and yield of flavonoid compounds of Polygonatum cyrtonema Hua (PC) are important indices of the quality of medicinal materials. However, the flavonoids compositions and accumulation mechanism are still unclear in PC. Here, we identified 22 flavonoids using widely-targeted metabolome analysis in 15 genotypes of PC. Then weighted gene co-expression network analysis based on 45 transcriptome samples was performed to construct 12 co-expressed modules, in which blue module highly correlated with flavonoids was identified. Furthermore, 4 feature genes including PcCHS1, PcCHI, PcCHS2 and PcCHR5 were identified from 94 hub genes in blue module via machine learning methods support vector machine-recursive feature elimination (SVM-RFE) and random forest (RF), and their functions on metabolic flux of flavonoids pathway were confirmed by tobacco transient expression system. Our findings identified representative flavonoids and key enzymes in PC that provided new insight for elite breeding rich in flavonoids, and thus will be beneficial for rapid development of great potential economic and medicinal value of PC.

Comparative and phylogenetic analysis of the complete chloroplast genomes of six Polygonatum species (Asparagaceae).

Polygonatum Miller belongs to the tribe Polygonateae of Asparagaceae. The horizontal creeping fleshy roots of several species in this genus serve as traditional Chinese medicine. Previous studies have mainly reported the size and gene contents of the plastomes, with little information on the comparative analysis of the plastid genomes of this genus. Additionally, there are still some species whose chloroplast genome information has not been reported. In this study, the complete plastomes of six Polygonatum were sequenced and assembled, among them, the chloroplast genome of P. campanulatum was reported for the first time. Comparative and phylogenetic analyses were then conducted with the published plastomes of three related species. Results indicated that the whole plastome length of the Polygonatum species ranged from 154,564 bp (P. multiflorum) to 156,028 bp (P. stenophyllum) having a quadripartite structure of LSC and SSC separated by two IR regions. A total of 113 unique genes were detected in each of the species. Comparative analysis revealed that gene content and total GC content in these species were highly identical. No significant contraction or expansion was observed in the IR boundaries among all the species except P. sibiricum1, in which the rps19 gene was pseudogenized owing to incomplete duplication. Abundant long dispersed repeats and SSRs were detected in each genome. There were five remarkably variable regions and 14 positively selected genes were identified among Polygonatum and Heteropolygonatum. Phylogenetic results based on chloroplast genome strongly supported the placement of P. campanulatum with alternate leaves in sect. Verticillata, a group characterized by whorled leaves. Moreover, P. verticillatum and P. cyrtonema were displayed as paraphyletic. This study revealed that the characters of plastomes in Polygonatum and Heteropolygonatum maintained a high degree of similarity. Five highly variable regions were found to be potential specific DNA barcodes in Polygonatum. Phylogenetic results suggested that leaf arrangement was not suitable as a basis for delimitation of subgeneric groups in Polygonatum and the definitions of P. cyrtonema and P. verticillatum require further study.

Integrating Transcriptomics and Hormones Dynamics Reveal Seed Germination and Emergence Process in Polygonatum cyrtonema Hua.

Polygonatum cyrtonema Hua is a traditional Chinese herb propagated using rhizomes, and excessive demand for seedlings and quality deterioration caused by rhizome propagation has highlighted that seed propagation may be an ideal solution to address these issues. However, the molecular mechanisms involved in P. cyrtonema Hua seed germination and emergence stages are not well understood. Therefore, in the present study, we performed transcriptomics combined with hormone dynamics during different seed germination stages, and 54,178 unigenes with an average length of 1390.38 bp (N50 = 1847 bp) were generated. Significant transcriptomic changes were related to plant hormone signal transduction and the starch and carbohydrate pathways. Genes related to ABA(abscisic acid), IAA(Indole acetic acid), and JA(Jasmonic acid) signaling, were downregulated, whereas genes related to ethylene, BR(brassinolide), CTK(Cytokinin), and SA(salicylic acid) biosynthesis and signaling were activated during the germination process. Interestingly, GA biosynthesis- and signaling-related genes were induced during the germination stage but decreased in the emergence stage. In addition, seed germination significantly upregulated the expression of genes associated with starch and sucrose metabolism. Notably, raffinose biosynthesis-related genes were induced, especially during the emergence stage. In total, 1171 transcription factor (TF) genes were found to be differentially expressed. Our results provide new insights into the mechanisms underlying P. cyrtonema Hua seed germination and emergence processes and further research for molecular breeding.

Characterization of the chloroplast genome of medicinal herb Polygonatum cyrtonema and identification of molecular markers by comparative analysis.

Polygonatum cyrtonema Hua is a traditional Chinese herb medicine, and it is widely distributed in China. The intrageneric taxonomy and phylogenetic relationships within Polygonatum have long been controversial due to their morphological similarity and lacking special DNA barcodes. In this paper, the complete chloroplast genome is a relatively conserved quadripartite structure including a large single copy region of 84 711 bp, a small single copy region of 18 210 bp, and a pair of inverted repeats region of 26 142 bp. A total of 342 simple sequence repeats were identified, and most of them were found to be composed of A/T, including 126 mono-nucleotides and 179 di-nucleotides. Nucleotide diversity was analyzed and eight highly variable regions (psbl∼trnT-CGU, atpF∼atpH, trnT-GGU∼psbD, psaJ∼rps20, trnL-UAG∼ndhD, ndhG∼ndhl, ndhA, and rpl32∼ccsA) were identified as potential molecular markers. Phylogenetic analysis based on the whole chloroplast genome showed that P. cyrtonema, within the family Asparagaceae, is closely related to Polygonatum sibiricum and Polygonatum kingianum. The sequence matK, trnT-GGU & ccsA, and ndhG∼ndhA were identified as three DNA barcodes. The assembly and comparative analysis of P. cyrtonema complete chloroplast genome will provide essential molecular information about the evolution and molecular biology for further study.

Cloning and Characterization of the Gene Encoding HMGS Synthase in Polygonatum sibiricum.

The saponins of Polygonatum sibiricum had many pharmacological activities such as antitumor, antioxidation, and blood sugar lowering, which were synthesized by two pathways: mevalonate (MVA) and methylerythritol phosphate (MEP). 3-Hydroxy-3-methylglutaryl coenzyme A synthase (HMGS) was the key enzyme in the MVA synthesis pathway, and its expression level may affect the accumulation of saponins which were the main active ingredients of P. sibiricum. In this study, we successfully cloned HMGS1 and HMGS2 from P. sibiricum and their sequence similarity was 93.71% with 89 different sites. The multiple sequence alignment results indicated that the N-terminal sequences of HMGS were conserved. Phylogenetic analysis showed that P. sibiricum, A. officinalis, N. tazetta, D. nobile, and other relatives had a common evolutionary ancestor. The expression levels of both HMGSs and the total saponin content in different tissues revealed that HMGS expression in rhizomes was positively correlated with total saponin content. Further study of the abiotic stress effect of Methyl Jasmonate (MeJA) demonstrated that the expression of HMGS1 and HMGS2 genes was induced by MeJA, peaked at 24 h, and fell by 48 h. Our present findings would provide a blueprint for future studies of HMGS and its role in triterpenoid biosynthesis in P. sibiricum.

Carbohydrate regulation response to cold during rhizome bud dormancy release in Polygonatum kingianum.

BACKGROUND: The rhizome of Polygonatum kingianum Coll. et Hemsl (P. kingianum) is a crucial traditional Chinese medicine, but severe bud dormancy occurs during early rhizome development. Low temperature is a positive factor affecting dormancy release, whereas the variation in carbohydrates during dormancy release has not been investigated systematically. Therefore, the sugar content, related metabolic pathways and gene co-expression were analysed to elucidate the regulatory mechanism of carbohydrates during dormancy release in the P. kingianum rhizome bud. RESULTS: During dormancy transition, starch and sucrose (Suc) exhibited opposing trends in the P. kingianum rhizome bud, representing a critical indicator of dormancy release. Galactose (Gal) and raffinose (Raf) were increased in content and synthesis. Glucose (Glc), cellulose (Cel), mannose (Man), arabinose (Ara), rhamnose (Rha) and stachyose (Sta) showed various changes, indicating their different roles in breaking rhizome bud dormancy in P. kingianum. At the beginning of dormancy release, Glc metabolism may be dominated by anaerobic oxidation (glycolysis followed by ethanol fermentation). After entering the S3 stage, the tricarboxylic acid cycle (TCA) and pentose phosphate pathway (PPP) were may be more active possibly. In the gene co-expression network comprising carbohydrates and hormones, HYD1 was identified as a hub gene, and numerous interactions centred on STS/SUS were also observed, suggesting the essential role of brassinosteroids (BRs), Raf and Suc in the regulatory network. CONCLUSION: We revealed cold-responsive genes related to carbohydrate metabolism, suggesting regulatory mechanisms of sugar during dormancy release in the P. kingianum rhizome bud. Additionally, gene co-expression analysis revealed possible interactions between sugar and hormone signalling, providing new insight into the dormancy release mechanism in P. kingianum rhizome buds.

SSR Loci Analysis in Transcriptome and Molecular Marker Development in Polygonatum sibiricum.

To study the SSR loci information and develop molecular markers, a total of 435,858 unigenes in transcriptome of Polygonatum sibiricum were used to explore SSR. The distribution frequency of SSR and the basic characteristics of repeat motifs were analyzed using MISA software, and SSR primers were designed by Primer 3.0 software and then validated by PCR. Moreover, the gene function analysis of SSR Unigene was obtained by Blast. The results showed that 112,728 SSR loci were found in the transcriptome of Polygonatum sibiricum, which distributed in 435,858 unigenes with a distribution frequency of 25.86%. Mo-nucleotide and Di-nucleotide repeat were the main types, accounted for 83.83% of all SSRs. The repeat motifs of A/T and AC/GT were the predominant repeat types of Mo-nucleotide and Di-nucleotide, respectively. A total of 113,305 pairs of SSR primers with the potential to produce polymorphism were designed for maker development. One hundred and fifty-four of the 500 randomly selected primers not only produced fragments with expected molecular size but also had high polymorphism, which could accurately separate the tested varieties. The gene function of unigenes containing SSR was mostly related to the molecular function of Polygonatum sibiricum. The SSR markers in transcriptome of Polygonatum sibiricum show rich type, strong specificity, and high potential of polymorphism, which will benefit the candidate gene mining and marker-assisted breeding. The developed markers can also provide technical methods for molecular identification of intraspecific species of Polygonatum Mill. and maker-assisted breeding of superior varieties of Polygonatum Mill.

De novo assembly and analysis of Polygonatum cyrtonema Hua and identification of genes involved in polysaccharide and saponin biosynthesis.

BACKGROUND: The investigation of molecular mechanisms involved in polysaccharides and saponin metabolism is critical for genetic engineering of Polygonatum cyrtonema Hua to raise major active ingredient content. Up to now, the transcript sequences are available for different tissues of P. cyrtonema, a wide range scanning about temporal transcript at different ages' rhizomes was still absent in P. cyrtonema. RESULTS: Transcriptome sequencing for rhizomes at different ages was performed. Sixty-two thousand six hundred thirty-five unigenes were generated by assembling transcripts from all samples. A total of 89 unigenes encoding key enzymes involved in polysaccharide biosynthesis and 56 unigenes encoding key enzymes involved in saponin biosynthesis. The content of total polysaccharide and total saponin was positively correlated with the expression patterns of mannose-6-phosphate isomerase (MPI), GDP-L-fucose synthase (TSTA3), UDP-apiose/xylose synthase (AXS), UDP-glucose 6-dehydrogenase (UGDH), Hydroxymethylglutaryl CoA synthase (HMGS), Mevalonate kinase (MVK), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (ispF), (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase (ispG), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (ispH), Farnesyl diphosphate synthase (FPPS). Finally, a number of key genes were selected and quantitative real-time PCR were performed to validate the transcriptome analysis results. CONCLUSIONS: These results create the link between polysaccharides and saponin biosynthesis and gene expression, provide insight for underlying key active substances, and reveal novel candidate genes including TFs that are worth further exploration for their functions and values.

Transcriptome Analysis of Different Sections of Rhizome in Polygonatum sibiricum Red. and Mining Putative Genes Participate in Polysaccharide Biosynthesis.

As a traditional Chinese herb, the rhizomes of Polygonatum sibiricum Red. are rich in various compounds which have plenty of pharmacological applications and biological activities. Among them, Polygonatum sibiricum polysaccharides (PSP) are the main active ingredients and exhibit a broad range of pharmacological. Based on previous researches, identifying genes involved in PSP biosynthesis will help delineate such pathway at the molecular level. In that case, we performed RNA sequencing analysis for two sections of P. sibiricum Red.'s rhizomes significantly different in PSP content. A total of 435,858 unigenes were obtained by assembling transcripts from both sections and 29,548 (6.77%) ones were annotated in all seven public databases. Analyzing count data of RNA-seq, 13,460 differential expression genes (DEGs) between two sections of rhizomes were acquired. After DEGs were mapped to KEGG databases, twelve represented KEGG pathways related to PSP biosynthesis were summed up. And most DEGs were assigned to the pathway of "Starch and sucrose metabolism". Finally, seventeen candidate genes whose expression levels were related to the polysaccharide content, were considered involving PSP biosynthesis in P. sibiricum Red. The present study lays a foundation of researching the molecular mechanisms of PSP biosynthesis.