High-integrity Pueraria montana var. lobata genome and population analysis revealed the genetic diversity of Pueraria genus.

Pueraria montana var. lobata (P. lobata) is a traditional medicinal plant belonging to the Pueraria genus of Fabaceae family. Pueraria montana var. thomsonii (P. thomsonii) and Pueraria montana var. montana (P. montana) are its related species. However, evolutionary history of the Pueraria genus is still largely unknown. Here, a high-integrity, chromosome-level genome of P. lobata and an improved genome of P. thomsonii were reported. It found evidence for an ancient whole-genome triplication and a recent whole-genome duplication shared with Fabaceae in three Pueraria species. Population genomics of 121 Pueraria accessions demonstrated that P. lobata populations had substantially higher genetic diversity, and P. thomsonii was probably derived from P. lobata by domestication as a subspecies. Selection sweep analysis identified candidate genes in P. thomsonii populations associated with the synthesis of auxin and gibberellin, which potentially play a role in the expansion and starch accumulation of tubers in P. thomsonii. Overall, the findings provide new insights into the evolutionary and domestication history of the Pueraria genome and offer a valuable genomic resource for the genetic improvement of these species.

Integrated Metabolomic and Transcriptomic Analysis of Puerarin Biosynthesis in Pueraria montana var. thomsonii at Different Growth Stages.

Puerarin, a class of isoflavonoid compounds concentrated in the roots of Puerarias, has antipyretic, sedative, and coronary blood-flow-increasing properties. Although the biosynthetic pathways of puerarin have been investigated by previous researchers, studies focusing on the influence of different growth stages on the accumulation of metabolites in the puerarin pathway are not detailed, and it is still controversial at the last step of the 8-C-glycosylation reaction. In this study, we conducted a comprehensive analysis of the metabolomic and transcriptomic changes in Pueraria montana var. thomsonii during two growing years, focusing on the vigorous growth and dormant stages, to elucidate the underlying mechanisms governing the changes in metabolite and gene expression within the puerarin biosynthesis pathway. In a comparison of the two growth stages in the two groups, puerarin and daidzin, the main downstream metabolites in the puerarin biosynthesis pathway, were found to accumulate mainly during the vigorous growth stage. We also identified 67 common differentially expressed genes in this pathway based on gene expression differences at different growth stages. Furthermore, we identified four candidate 8-C-GT genes that potentially contribute to the conversion of daidzein into puerarin and eight candidate 7-O-GT genes that may be involved in the conversion of daidzein into daidzin. A co-expression network analysis of important UGTs and HIDs along with daidzein and puerarin was conducted. Overall, our study contributes to the knowledge of puerarin biosynthesis and offers information about the stage at which the 8-C-glycosylation reaction occurs in biosynthesis. These findings provide valuable insights into the cultivation and quality enhancement of Pueraria montana var. thomsonii.

Characterization of a Putative New Member of the Genus Potyvirus from Kudzu (Pueraria montana var. lobata) in Mississippi.

Kudzu (Pueraria montana var. lobata), a plant native to Southeastern Asia, has become a major noxious weed covering millions of hectares in the Southern United States. A kudzu patch displaying virus-like symptoms located in Ackerman, northeastern Mississippi (MS), was used as a source for virus isolation and characterization involving mechanical and vector transmission, ultrastructural observation, surveys, Sanger and high-throughput genome sequencing, and sequence analyses. The results revealed the presence of a new potyvirus in infected kudzu, closely related to wisteria vein mosaic virus (WVMV) and provisionally named kudzu chlorotic ring blotch virus (KudCRBV). Genome features and pairwise comparison with six WVMV genomes currently available in GenBank and three additional isolates from MS sequenced in this work suggest that KudCRBV is likely a member of a new species in the genus Potyvirus. Furthermore, under experimental conditions, KudCRBV was successfully transmitted by cotton and potato aphids and mechanically to soybean and beans. A state-wide survey revealed several kudzu patches infected by the virus in northern MS.

Comparative plastomes of Pueraria montana var. lobata (Leguminosae: Phaseoleae) and closely related taxa: insights into phylogenomic implications and evolutionary divergence.

BACKGROUND: Pueraria montana var. lobata (kudzu) is an important food and medicinal crop in Asia. However, the phylogenetic relationships between Pueraria montana var. lobata and the other two varieties (P. montana var. thomsonii and P. montana var. montana) remain debated. Although there is increasing evidence showing that P. montana var. lobata adapts to various environments and is an invasive species in America, few studies have systematically investigated the role of the phylogenetic relationships and evolutionary patterns of plastomes between P. montana var. lobata and its closely related taxa. RESULTS: 26 newly sequenced chloroplast genomes of Pueraria accessions resulted in assembled plastomes with sizes ranging from 153,360 bp to 153,551 bp. Each chloroplast genome contained 130 genes, including eight rRNA genes, 37 tRNA genes, and 85 protein-coding genes. For 24 newly sequenced accessions of these three varieties of P. montana, we detected three genes and ten noncoding regions with higher nucleotide diversity (π). After incorporated publically available chloroplast genomes of Pueraria and other legumes, 47 chloroplast genomes were used to construct phylogenetic trees, including seven P. montana var. lobata, 14 P. montana var. thomsonii and six P. montana var. montana. Phylogenetic analysis revealed that P. montana var. lobata and P. montana var. thomsonii formed a clade, while all sampled P. montana var. montana formed another cluster based on cp genomes, LSC, SSC and protein-coding genes. Twenty-six amino acid residues were identified under positive selection with the site model. We also detected six genes (accD, ndhB, ndhC, rpl2, rpoC2, and rps2) that account for among-site variation in selective constraint under the clade model between accessions of the Pueraria montana var. lobata clade and the Pueraria montana var. montana clade. CONCLUSION: Our data provide novel comparative plastid genomic insights into conservative gene content and structure of cp genomes pertaining to P. montana var. lobata and the other two varieties, and reveal an important phylogenetic clue and plastid divergence among related taxa of P. montana come from loci that own moderate variation and underwent modest selection.

Transcriptome profiling reveals the genes involved in tuberous root expansion in Pueraria (Pueraria montana var. thomsonii).

BACKGROUND: Pueraria is a dry root commonly used in Traditional Chinese Medicine or as food and fodder, and tuberous root expansion is an important agronomic characteristic that influences its yield. However, no specific genes regulating tuberous root expansion in Pueraria have been identified. Therefore, we aimed to explore the expansion mechanism of Pueraria at six developmental stages (P1-P6), by profiling the tuberous roots of an annual local variety "Gange No.1" harvested at 105, 135, 165, 195, 225, and 255 days after transplanting. RESULTS: Observations of the tuberous root phenotype and cell microstructural morphology revealed that the P3 stage was a critical boundary point in the expansion process, which was preceded by a thickening diameter and yield gain rapidly of the tuberous roots, and followed by longitudinal elongation at both ends. A total of 17,441 differentially expressed genes (DEGs) were identified by comparing the P1 stage (unexpanded) against the P2-P6 stages (expanded) using transcriptome sequencing; 386 differential genes were shared across the six developmental stages. KEGG pathway enrichment analysis showed that the DEGs shared by P1 and P2-P6 stages were mainly involved in pathways related to the "cell wall and cell cycle", "plant hormone signal transduction", "sucrose and starch metabolism", and "transcription factor (TF)". The finding is consistent with the physiological data collected on changes in sugar, starch, and hormone contents. In addition, TFs including bHLHs, AP2s, ERFs, MYBs, WRKYs, and bZIPs were involved in cell differentiation, division, and expansion, which may relate to tuberous root expansion. The combination of KEGG and trend analyses revealed six essential candidate genes involved in tuberous root expansion; of them, CDC48, ARF, and EXP genes were significantly upregulated during tuberous root expansion while INV, EXT, and XTH genes were significantly downregulated. CONCLUSION: Our findings provide new insights into the complex mechanisms of tuberous root expansion in Pueraria and candidate target genes, which can aid in increasing Pueraria yield.

Genome-wide identification and expression pattern analysis of R2R3-MYB transcription factor gene family involved in puerarin biosynthesis and response to hormone in Pueraria lobata var. thomsonii.

BACKGROUND: R2R3-MYB transcription factors regulate secondary metabolism, stress responses and development in various plants. Puerarin is a bioactive ingredient and most abundant secondary metabolite isolated from Pueraria lobata. The biosynthesis of puerarin proceeds via the phenylpropanoid pathway and isoflavonoids pathway, in which 9 key enzymes are involved. The expression of these structural genes is under control of specific PtR2R3-MYB genes in different plant tissues. However, how PtR2R3-MYB genes regulates structural genes in puerarin biosynthesis remains elusive. This study mined the PtR2R3-MYB genes involved in puerarin biosynthesis and response to hormone in Pueraria lobata var. thomsonii. RESULTS: A total of 209 PtR2R3-MYB proteins were identified, in which classified into 34 subgroups based on the phylogenetic topology and the classification of the R2R3-MYB superfamily in Arabidopsis thaliana. Furtherly physical and chemical characteristics, gene structure, and conserved motif analysis were also used to further analyze PtR2R3-MYBs. Combining puerarin content and RNA-seq data, speculated on the regulated puerarin biosynthesis of PtR2R3-MYB genes and structural genes, thus 21 PtR2R3-MYB genes and 25 structural genes were selected for validation gene expression and further explore its response to MeJA and GSH treatment by using qRT-PCR analysis technique. Correlation analysis and cis-acting element analysis revealed that 6 PtR2R3-MYB genes (PtMYB039, PtMYB057, PtMYB080, PtMYB109, PtMYB115 and PtMYB138) and 7 structural genes (PtHID2, PtHID9, PtIFS3, PtUGT069, PtUGT188, PtUGT286 and PtUGT297) were directly or indirectly regulation of puerarin biosynthesis in ZG11. It is worth noting that after MeJA and GSH treatment for 12-24 h, the expression changes of most candidate genes were consistent with the correlation of puerarin biosynthesis, which also shows that MeJA and GSH have the potential to mediate puerarin biosynthesis by regulating gene expression in ZG11. CONCLUSIONS: Overall, this study provides a comprehensive understanding of the PtR2R3-MYB and will paves the way to reveal the transcriptional regulation of puerarin biosynthesis and response to phytohormone of PtR2R3-MYB genes in Pueraria lobata var. thomsonii.

Comparative Transcriptome Analysis of Pueraria lobata Provides Candidate Genes Involved in Puerarin Biosynthesis and Its Regulation.

Pueraria lobata is a traditional Chinese herb in which an isoflavone C-glucoside, namely puerarin, has received the utmost interest due to its medicinal properties. To date, the biogenesis of puerarin, especially its C-glucosyl reaction in the pathway, remains poorly understood. Moreover, the transcription factors (TFs) that regulate puerarin biosynthesis in P. lobata have not been reported. Here, we performed phytochemical studies on the different developmental stages of the root, stem, and leaf tissues of two P. lobata cultivars, which suggested that both the roots and stems of P. lobata were the sites of puerarin biosynthesis. RNA-sequencing was conducted with the root and stem tissues of the two cultivars under different stages, and the clean reads were mapped to the recently published genome of P. lobata var. thomsonii, yielding the transcriptome dataset. A detailed analysis of the gene expression data, gene coexpression network, and phylogeny proposed several C-GTs that likely participate in puerarin biosynthesis. The first genome-wide analysis of the whole MYB superfamily in P. lobata presented here identified a total of 123 nonredundant PlMYB genes that were significantly expressed in the analyzed tissues. The phylogenetic analysis of PlMYBs with other plant MYB proteins revealed strong PlMYB candidates that may regulate the biosynthesis of isoflavones, such as puerarin.

Daidzein Hydroxylation by CYP81E63 Is Involved in the Biosynthesis of Miroestrol in Pueraria mirifica.

White Kwao Krua (Pueraria candollei var. mirifica), a Thai medicinal plant, is a rich source of phytoestrogens, especially isoflavonoids and chromenes. These phytoestrogens are well known; however, their biosynthetic genes remain largely uncharacterized. Cytochrome P450 (P450) is a large protein family that plays a crucial role in the biosynthesis of various compounds in plants, including phytoestrogens. Thus, we focused on P450s involved in the isoflavone hydroxylation that potentially participates in the biosynthesis of miroestrol. Three candidate P450s were isolated from the transcriptome libraries by considering the phylogenetic and expression data of each tissue of P. mirifica. The candidate P450s were functionally characterized both in vitro and in planta. Accordingly, the yeast microsome harboring PmCYP81E63 regiospecifically exhibited either 2' or 3' daidzein hydroxylation and genistein hydroxylation. Based on in silico calculation, PmCYP81E63 had higher binding energy with daidzein than with genistein, which supported the in vitro result of the isoflavone specificity. To confirm in planta function, the candidate P450s were then transiently co-expressed with isoflavone-related genes in Nicotiana benthamiana. Despite no daidzein in the infiltrated N. benthamiana leaves, genistein and hydroxygenistein biosynthesis were detectable by liquid Chromatography with tandem mass spectrometry (LC-MS/MS). Additionally, we demonstrated that PmCYP81E63 interacted with several enzymes related to isoflavone biosynthesis using bimolecular fluorescence complementation studies and a yeast two-hybrid analysis, suggesting a scheme of metabolon formation in the pathway. Our findings provide compelling evidence regarding the involvement of PmCYP81E63 in the early step of the proposed miroestrol biosynthesis in P. mirifica.

Chromosome-Level Genome Assembly and Multi-Omics Dataset Provide Insights into Isoflavone and Puerarin Biosynthesis in Pueraria lobata (Wild.) Ohwi.

Pueraria lobata (wild.) Ohwi is a leguminous plant and one of the traditional Chinese herbal medicines. Its puerarin extract is widely used in the pharmaceutical industry. This study reported a chromosome-level genome assembly for P. lobata and its characteristics. The genome size was ~939.2 Mb, with a contig N50 of 29.51 Mbp. Approximately 97.82% of the assembled sequences were represented by 11 pseudochromosomes. We identified that the repetitive sequences accounted for 63.50% of the P. lobata genome. A total of 33,171 coding genes were predicted, of which 97.34% could predict the function. Compared with other species, P. lobata had 757 species-specific gene families, including 1874 genes. The genome evolution analysis revealed that P. lobata was most closely related to Glycine max and underwent two whole-genome duplication (WGD) events. One was in a gamma event shared by the core dicotyledons at around 65 million years ago, and another was in the common ancestor shared by legume species at around 25 million years ago. The collinearity analysis showed that 61.45% of the genes (54,579 gene pairs) in G. max and P. lobata had collinearity. In this study, six unique PlUGT43 homologous genes were retrieved from the genome of P. lobata, and no 2-hydroxyisoflavanone 8-C-glucoside was found in the metabolites. This also revealed that the puerarin synthesis was mainly from the glycation of daidzein. The combined transcriptome and metabolome analysis suggested that two bHLHs, six MYBs and four WRKYs were involved in the expression regulation of puerarin synthesis structural genes. The genetic information obtained in this study provided novel insights into the biological evolution of P. lobata and leguminous species, and it laid the foundation for further exploring the regulatory mechanism of puerarin synthesis.

Inhibition of LPS-Induced Microglial Activation by the Ethyl Acetate Extract of Pueraria mirifica.

Microglial activation has been found to play a crucial role in various neurological disorders. Proinflammatory substances overproduced by activated microglia, such as cytokines, chemokines, reactive oxygen species, and nitric oxide (NO), can result in neuroinflammation that further exacerbates the course of the diseases. This study aimed to explore the anti-inflammatory effect of the ethyl acetate extract of Pueraria mirifica on microglial activation. Lipopolysaccharide (LPS)-induced inflammation was used as a model to investigate the effects of P. mirifica on HAPI (highly aggressive proliferating immortalized), a rat microglial cell line. Administration of ethyl acetate extract from the tuberous roots of P. mirifica to HAPI cells dose-dependently reduced NO production and iNOS expression induced by LPS. Attenuation of IRF-1 (interferon regulatory factor-1) induction, one of the transcription factors governing iNOS expression, suggested that the inhibitory effect on NO production by the plant extract was at least partially mediated through this transcription factor. In addition, LPS-stimulated mRNA expression of MCP-1 (monocyte chemoattractant protein-1), IL-6 (interleukin-6), and TNF-α (tumor necrosis factor-α) was also suppressed with P. mirifica extract pretreatment. This study indicates that the ethyl acetate extract of P. mirifica could potentially serve as an anti-inflammatory mediator and may be useful in relieving the severity of neurological diseases where microglia play a role.

Chromosome-level and graphic genomes provide insights into metabolism of bioactive metabolites and cold-adaption of Pueraria lobata var. montana.

Pueraria lobata var. montana (P. montana) belongs to the genus Pueraria and originated in Asia. Compared with its sister P. thomsonii, P. montana has stronger growth vigour and cold-adaption but contains less bioactive metabolites such as puerarin. To promote the investigation of metabolic regulation and genetic improvement of Pueraria, the present study reports a chromosome-level genome of P. montana with length of 978.59 Mb and scaffold N50 of 80.18 Mb. Comparative genomics analysis showed that P. montana possesses smaller genome size than that of P. thomsonii owing to less repeat sequences and duplicated genes. A total of 6,548 and 4,675 variety-specific gene families were identified in P. montana and P. thomsonii, respectively. The identified variety-specific and expanded/contracted gene families related to biosynthesis of bioactive metabolites and microtubules are likely the causes for the different characteristics of metabolism and cold-adaption of P. montana and P. thomsonii. Moreover, a graphic genome was constructed based on 11 P. montana accessions. Total 92 structural variants were identified and most of which are related to stimulus-response. In conclusion, the chromosome-level and graphic genomes of P. montana will not only facilitate the studies of evolution and metabolic regulation, but also promote the breeding of Pueraria.

Biogeographic pattern and relevant environmental factors for rhizobial communities in the rhizosphere and root nodules of kudzu (Pueraria lobata).

Kudzu (Pueraria lobata) is an important medicinal plant, which can associate with rhizobia for nitrogen fixation. The mutualistic symbiosis between rhizobium and kudzu is not well understood, but it is necessary to fully utilize kudzu. Nodules and rhizosphere soils collected from 16 sampling sites were characterized based on phylogenetic analyses of the rpoB gene; 16S rRNA gene; the housekeeping genes SMc00019, truA, and thrA; and the symbiotic genes nodA and nifH. The relationships between biogeographic pattern, nitrogenase activity, and environmental factors were studied. Results indicated that a clear biogeographic pattern of rhizobial communities in the kudzu rhizosphere existed in southern China; latitude and soil pH were found to be the most important factors affecting the biogeographic pattern. Bradyrhizobium diazoefficiens and Bradyrhizobium erythrophlei were the dominant species in kudzu rhizosphere. The symbiotic rhizobia in kudzu nodules mainly belonged to B. lablabi, B. elkanii, B. pachyrhizi, and B. japonicum. Nitrogenase activities in the nodules of kudzu in the Jiangxi sampling region were significantly higher than those in the Guangxi and Hunan sampling regions, and they were significantly negatively correlated to pH and exchangeable Ca. These results constitute the first report of the existence of symbiotic genes in kudzu bradyrhizobia, which are similar to those in B. elkanii and B. pachyrhizi. Our findings could improve the understanding of kudzu-rhizobium symbiosis and could advance the application of rhizobial inoculation in medicinal legumes in terms of increasing the content of active ingredients.

Identification of Pueraria spp. through DNA barcoding and comparative transcriptomics.

BACKGROUND: Kudzu is a term used generically to describe members of the genus Pueraria. Kudzu roots have been used for centuries in traditional Chinese medicine in view of their high levels of beneficial isoflavones including the unique 8-C-glycoside of daidzein, puerarin. In the US, kudzu is seen as a noxious weed causing ecological and economic damage. However, not all kudzu species make puerarin or are equally invasive. Kudzu remains difficult to identify due to its diverse morphology and inconsistent nomenclature. RESULTS: We have generated sequences for the internal transcribed spacer 2 (ITS2) and maturase K (matK) regions of Pueraria montana lobata, P. montana montana, and P. phaseoloides, and identified two accessions previously used for differential analysis of puerarin biosynthesis as P. lobata and P. phaseoloides. Additionally, we have generated root transcriptomes for the puerarin-producing P. m. lobata and the non-puerarin producing P. phaseoloides. Within the transcriptomes, microsatellites were identified to aid in species identification as well as population diversity. CONCLUSIONS: The barcode sequences generated will aid in fast and efficient identification of the three kudzu species. Additionally, the microsatellites identified from the transcriptomes will aid in genetic analysis. The root transcriptomes also provide a molecular toolkit for comparative gene expression analysis towards elucidation of the biosynthesis of kudzu phytochemicals.

Subacute oral toxicity and bacterial mutagenicity of a mixture of Puerariae radix and Hizikia fusiforme extracts.

The study aims to identify the safety profile of a mixed extract (KGC-02-PS) from two traditional medicinal herbs, Puerariae radix and Hizikia fusiforme. In a subacute oral toxicity study, KGC-02-PS was administered orally for 28 days by gavage to Sprague Dawley rats (both sexes) at a daily dose of 0, 500, 1000, and 2000 mg/kg body weight. Bodyweight, food consumption, and clinical signs were monitored during the experimental period. After administering the final dose, this study conducted hematology, serum biochemistry, and pathological evaluations. In addition, the study performed a bacterial reverse mutation test with varying concentrations of KGC-02-PS (312.5 µg - 5,000 µg/plate) following OECD guideline No. 471, before testing five bacterial strains (Salmonella typhimurium TA98, TA100, TA1535, TA1537, and Escherichia coli WP2) in the presence or absence of metabolic activation. The preclinical evaluation of KGC-02-PS's subacute oral toxicity yielded no associated toxicological effects or any changes in clinical signs, body weight, and food consumption. Moreover, examining KGC-02-PS's hematological and serum biochemical characteristics and pathology yielded no toxicological changes in terms of organ weight measurements and gross or histopathological findings. KGC-02-PS neither increased the number of revertant colonies in all bacterial strains used in the bacterial reverse mutation test, nor did it induce genotoxicity related to bacterial reverse mutations under the study's conditions. Also, KGC-02-PS's no-observed-adverse-effect level was greater than 2000 mg/kg.

Chloroplast genomes of two Pueraria DC. species: sequencing, comparative analysis and molecular marker development.

Puerariae lobatae radix (Ge-Gen in Chinese) and Puerariae thomsonii radix (Fen-Ge) are widely used as medicine and health products, particularly in Chinese medicine. Puerarin and daidzein are the primary bioactive compounds in Puerariae radix. These isoflavones have been used to treat cardiovascular and cerebrovascular diseases, hypertension, diabetes, and osteoporosis. The content of puerarin in Ge-Gen is about six times higher than that in Fen-Ge, so its use has a higher pharmacological effect. It is therefore of great importance to effectively distinguish between these two species. However, because their basal plants, P. lobata (Willd.) Ohwi and P. thomsonii Benth., possess an extremely similar appearance, and detecting the level of chemical constituents is just a rough distinction, it is necessary to develop more efficient identification approaches. Here the complete chloroplast genomes of P. lobata and P. thomsonii were deciphered, including sequencing, assembly, comparative analysis, and molecular marker development. The results showed that they are 153,393 and 153,442 bp in length, respectively; both contain 124 annotated genes, including eight encoding rRNA, 29 encoding tRNA, and 87 encoding proteins. Phylogenetic analysis showed that they form a clade, indicating that they originate from the same ancestor. After obtaining 10 intergenic/intronic regions with a genetic distance greater than 0.5 cm, primers were designed to amplify regions of high variability in P. lobata and P. thomsonii. Finally, a 60-bp differential base fragment, located in the intron of rpl16, was developed as a molecular marker to efficiently distinguish between these two species.

Regulatory protein genes and microRNAs in response to selenium stimuli in Pueraria lobata (Willd.) Ohwi.

Regulatory protein genes and microRNAs (miRNAs) play important roles in response to abiotic and biotic stress, and the biosynthesis of secondary metabolites in plants. However, their responses to selenium (Se) stimuli have not been comprehensively studied in Pueraria lobata (Willd.) Ohwi, a selenocompound-rich medicinal and edible plant. In this study, we identified a total of 436/556/1161/624 transcription factors, 134/157/308/172 transcriptional regulators, and 341/456/250/518 protein kinases, which were co-expressed with at least one selenocompound-related structural gene/sulfate transporter or phosphate transporter/reactive oxygen species (ROS) scavenging structural gene/isoflavone-related structural gene, respectively. Then, we identified a total of 87 expressed miRNAs by Se disposure, in which 11 miRNAs, including miR171f-3p, miR390b-3P, miR-N111b, miR-N118, miR-N30, miR-N38-3P, miR-N61a, miR-N61b, miR-N80-3p, miR-N84-3P, and miR-N90.2-3P, were significantly upregulated. We also identified a total of 1172 target genes for the 87 expressed miRNAs. Gene Ontology enrichment analysis of these target genes showed that regulation of transcription, DNA-templated, integral component of membrane, nucleus, ATP binding, and plasma membrane are the top five subclassifications. Finally, we revealed that 5 miRNAs targeted 10 regulatory protein genes, which are highly correlated with at least one selenocompound-related structural gene or transporter gene; 5 miRNAs targeted 10 regulatory protein genes, which are highly correlated with at least one ROS scavenging structural gene; and 5 miRNAs targeted 9 regulatory protein genes, which are potentially involved in the isoflavone biosynthesis. Overall, the study provides us the comprehensive insight into the roles of regulatory proteins and miRNAs in response to Se stimuli in P. lobata.

Evaluation of white Kwao Krua (Pueraria candollei Grah. ex Benth.) products sold in Thailand by molecular, chemical, and microscopic analyses.

The tuberous roots of Pueraria candollei Grah. ex Benth. (Fabaceae), commonly known as white Kwao Krua, are used to relieve menopausal symptoms in Thai traditional medicine because they contain phytoestrogens. Black and red Kwao Krua crude drugs exist as well, but they have different botanical origins and pharmacological activities. There is a high demand for white Kwao Krua products, but because of the limited availability of the plant material, it is suspected that the adulteration and misidentification of white Kwao Krua crude drugs and products occur. In this study, we authenticated white Kwao Krua products collected from Thai herbal markets by molecular, chemical, and microscopic analyses. The nucleotide sequences in the internal transcribed spacer (ITS) and trnH-psbA regions of 23 samples of authentic P. candollei were analyzed, and both regions were found to have intraspecific DNA polymorphisms. Based on the single nucleotide polymorphisms in the ITS1 region, species-specific primer sets of P. candollei were designed to authenticate white Kwao Krua and differentiate it from red and black Kwao Krua. Only the PCR products of KWP02 were not amplified by the primer sets. Isoflavonoid contents and microscopic features were used to support the results of molecular analysis to clarify the botanical origin of white Kwao Krua. Molecular, chemical and microscopic methods confirmed that all the Thai Kwao Krua products examined in this study contained authentic "white Kwao Krua" as claimed on their labels.

Transcriptome analysis of Pueraria candollei var. mirifica for gene discovery in the biosyntheses of isoflavones and miroestrol.

BACKGROUND: Pueraria candollei var. mirifica, a Thai medicinal plant used traditionally as a rejuvenating herb, is known as a rich source of phytoestrogens, including isoflavonoids and the highly estrogenic miroestrol and deoxymiroestrol. Although these active constituents in P. candollei var. mirifica have been known for some time, actual knowledge regarding their biosynthetic genes remains unknown. RESULTS: Miroestrol biosynthesis was reconsidered and the most plausible mechanism starting from the isoflavonoid daidzein was proposed. A de novo transcriptome analysis was conducted using combined P. candollei var. mirifica tissues of young leaves, mature leaves, tuberous cortices, and cortex-excised tubers. A total of 166,923 contigs was assembled for functional annotation using protein databases and as a library for identification of genes that are potentially involved in the biosynthesis of isoflavonoids and miroestrol. Twenty-one differentially expressed genes from four separate libraries were identified as candidates involved in these biosynthetic pathways, and their respective expressions were validated by quantitative real-time reverse transcription polymerase chain reaction. Notably, isoflavonoid and miroestrol profiling generated by LC-MS/MS was positively correlated with expression levels of isoflavonoid biosynthetic genes across the four types of tissues. Moreover, we identified R2R3 MYB transcription factors that may be involved in the regulation of isoflavonoid biosynthesis in P. candollei var. mirifica. To confirm the function of a key-isoflavone biosynthetic gene, P. candollei var. mirifica isoflavone synthase identified in our library was transiently co-expressed with an Arabidopsis MYB12 transcription factor (AtMYB12) in Nicotiana benthamiana leaves. Remarkably, the combined expression of these proteins led to the production of the isoflavone genistein. CONCLUSIONS: Our results provide compelling evidence regarding the integration of transcriptome and metabolome as a powerful tool for identifying biosynthetic genes and transcription factors possibly involved in the isoflavonoid and miroestrol biosyntheses in P. candollei var. mirifica.

[Analysis of genomic SSRs in Pueraria lobata and P. thomsonii and establishment of DNA identity card for different germplasms of P. thomsonii of Jiangxi province].

The type and frequency of simple sequence repeats( SSRs) in the genomes was investigated using the DNA sequence data of Pueraria lobata and P. thomsonii. Based on these SSRs,20 pairs of SSR primers were designed and 5 high polymorphism primer pairs were selected to analyze genetic diversity of 9 cultivars of P. thomsonii in Jiangxi province. The results showed that the 5 pairs of primers could generate 16 polymorphic alleles bands. The average polymorphism information content( PIC) of each SSR primer pair was 0. 600 7.According to the genetic similarity coefficients,the 9 cultivars of P. thomsonii can be classified into 6 germplasms. This study established DNA identity cards with 5 pairs of SSR primers for different germplasm resources of P. thomsonii in Jiangxi province,which provided reference information for the selection of fine germplasms of P. thomsonii and the theoretical basis for the study of Dao-di herbs.

Comprehensive transcriptome analysis reveals genes potentially involved in isoflavone biosynthesis in Pueraria thomsonii Benth.

Pueraria thomsonii Benth is an important medicinal plant. Transcriptome sequencing, unigene assembly, the annotation of transcripts and the study of gene expression profiles play vital roles in gene function research. However, the full-length transcriptome of P. thomsonii remains unknown. Here, we obtained 44,339 nonredundant transcripts of P. thomsonii by using the PacBio RS II Isoform and Illumina sequencing platforms, of which 43,195 were annotated genes. Compared with the expression levels in the plant roots, those of transcripts with a |fold change| ≥ 4 and FDR < 0.01 in the leaves or stems were assigned as differentially expressed transcripts (DETs). In total, we found 9,225 DETs, 32 of which came from structural genes that were potentially involved in isoflavone biosynthesis. The expression profiles of 8 structural genes from the RNA-Seq data were validated by qRT-PCR. We identified 437 transcription factors (TFs) that were positively or negatively correlated with at least 1 of the structural genes involved in isoflavone biosynthesis using Pearson correlation coefficients (r) (r > 0.8 or r < -0.8). We also identified a total of 32 microRNAs (miRNAs), which targeted 805 transcripts. These miRNAs caused enriched function in 'ATP binding', 'defense response', 'ADP binding', and 'signal transduction'. Interestingly, MIR156a potentially promoted isoflavone biosynthesis by repressing SBP, and MIR319 promoted isoflavone biosynthesis by repressing TCP and HB-HD-ZIP. Finally, we identified 2,690 alternative splicing events, including that of the structural genes of trans-cinnamate 4-monooxygenase and pullulanase, which are potentially involved in the biosynthesis of isoflavone and starch, respectively, and of three TFs potentially involved in isoflavone biosynthesis. Together, these results provide us with comprehensive insight into the gene expression and regulation of P. thomsonii.