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.

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.

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.

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.

Proteome analysis of Pueraria mirifica tubers collected in different seasons.

Pueraria mirifica-derived tuberous powder has been long-term consumed in Thailand as female hormone-replacement traditional remedies. The protein profiles of tubers collected in different seasons were evaluated. Phenol extraction, 2D-PAGE, and mass spectrometry were employed for tuberous proteome analysis. Out of the 322 proteins detected, over 59% were functionally classified as being involved in metabolism. The rest proteins were involved in defense, protein synthesis, cell structure, transportation, stress, storage, and also unidentified function. The proteins were found to be differentially expressed with respect to harvest season. Importantly, chalcone isomerase, isoflavone synthase, cytochrome p450, UDP-glycosyltransferase, and isoflavone reductase, which are all involved in the biosynthesis pathway of bioactive isoflavonoids, were most abundantly expressed in the summer-collected tubers. This is the first report on the proteomic patterns in P. mirifica tubers in relevant with seasonal variation. The study enlights the understanding of variance isoflavonoid production in P. mirifica tubers.

The use of cycleave PCR for the differentiation of the rejuvenating herb species Pueraria candollei (White Kwao Khruea), Butea superba (Red Kwao Khruea), and Mucuna macrocarpa (Black Kwao Khruea), and the simultaneous detection of multiple DNA targets in a DNA admixture.

Kwao Khruea, the tuberous roots of Pueraria candollei Graham ex Benth. (White Kwao Khruea), Butea superba Roxb. (Red Kwao Khruea), and Mucuna macrocarpa Wall. (Black Kwao Khruea), are used as rejuvenating herbs in traditional medicine in many tropical countries. Although Kwao Khruea has attracted strong interest because of its rejuvenation properties, each species is used for specific purposes and effects. P. candollei shows estrogenic effects in females. In contrast, B. superba and M. macrocarpa show androgenic effects in males. The potential misidentification of dried tuberous roots of various Kwao Khruea species might cause problems in the drug market, especially when they are reduced into powders. A cycleave PCR, which is based on the sequence of chloroplast matK gene, was developed to differentiate P. candollei, B. superba, and M. macrocarpa. The results showed that cycleave PCR is able to identify specific Kwao Khruea species. A multiplex cycleave PCR was optimized for the simultaneous detection of two different DNA targets in a DNA admixture. The specificity of this technique was confirmed by its ability to distinguish M. macrocarpa from five related Mucuna species. Cycleave PCR can be a specific, sensitive, and rapid method for the identification of medicinal plants and crude plant samples.

Molecular cloning and functional characterization of two divergent 4-coumarate†:†coenzyme A ligases from Kudzu (Pueraria lobata).

As part of the efforts to understand isoflavonoid metabolism in Pueraria lobata at the molecular level, the cDNAs encoding two divergent 4-coumarate : coenzyme A ligases (4CLs, designated Pl4CL1 and Pl4CL2, respectively) were isolated from P. lobata roots. Sequence analysis revealed that Pl4CL1 had an N-terminal extension of twenty-one amino acid residues compared to Pl4CL2. Phylogenetic analysis showed that Pl4CL1 and Pl4CL2 fell into angiosperm Class II and Class I, respectively. Through in vitro biochemical assays, both Pl4CLs were found to have the capacity to utilize 4-coumarate and trans-cinnamate as substrates, while neither of them could convert sinapate. Pl4CL2 had a broader substrate specificity than Pl4CL1. The affinity of Pl4CL1 for 4-coumarate was 2.6-fold higher than that of Pl4CL2 (with the Km values of 3.5 µM and 9.1 µM, respectively). Combining the dataset including gene expression profiles, metabolites measurements, and biochemical properties, our results indicated that Pl4CL1, just as other angiosperm Class II 4CLs, might play a role in isoflavone biosynthesis in P. lobata, while Pl4CL2 belongs to angiosperm Class I, and may function as a housekeeping enzyme concerning lignification.

Botanical origin of dietary supplements labeled as "Kwao Keur", a folk medicine from Thailand.

In the course of our study on the quality of dietary supplements in Japan, both the internal transcribed spacer (ITS) sequence of nrDNA and the rps16 intron sequence of cpDNA of products labeled as "Kwao Keur" were investigated. As a result, the DNA sequence of Pueraria candollei var. mirifica, which is the source plant of Kwao Keur, was observed in only about half of the products. Inferred from the determined sequences, source plants in the other products included Medicago sativa, Glycyrrhiza uralensis, Pachyrhizus erosus, and Ipomoea batatas, etc. These inferior products are estimated to lack the efficacy implied by their labeling. In order to guarantee the quality of dietary supplements, it is important to identify the source materials exactly; in addition, an infrastructure that can exclude these inferior products from the market is needed for the protection of consumers from potential damage to their health and finances. The DNA analysis performed in this study is useful for this purpose.

Discrimination of the Thai rejuvenating herbs Pueraria candollei (White Kwao Khruea), Butea superba (Red Kwao Khruea), and Mucuna collettii (Black Kwao Khruea) using PCR-RFLP.

The tuberous roots of Pueraria candollei (White Kwao Khruea), Butea superba (Red Kwao Khruea) and Mucuna collettii (Black Kwao Khruea), which belong to the family Leguminosae, are used as rejuvenating herbs in traditional Thai medicine. Although all of these species have an indication for rejuvenation, each differs in its medicinal properties. Two varieties of P. candollei, var. mirifica and var. candollei, affect females, whereas B. superba and M. collettii exhibit effects on males. However, the identification of these roots according to the name "Kwao Khruea" is confusing due to the similarity in their features. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) was utilised to identify plant origin. The partial matK gene was amplified and subjected to restriction enzyme digestion with DdeI and TaqI. The restriction fragments generated differed in number and size. To test the reliability of the method, an admixture of the different Kwao Khruea species containing equal amounts of DNA was tested. The results showed combined restriction patterns, and each species could be detected in the background of the others. The method was also used to authenticate eight different crude drugs sold as various types of Kwao Khruea in Thai markets. The results showed that the misidentification of commercial drugs remains a problem in crude drug markets. The PCR-RFLP analysis developed here provides a simple and accurate discrimination of these rejuvenating "Kwao Khruea" species.

[Analysis of genetic relationships of Pueraria thomsonii based on SRAP markers].

OBJECTIVE: To determine the interspecies relationships of 18 Pueraria thomsonii cultivars in molecular level. METHOD: Eighteen P. thomsonii cultivars were evaluated by using sequence-related amplified polymorphism (SRAP) markers, with P. lobata and P. peduncularis as contrast species. Systematic relationships were constructed based on the UPGMA method by TREECONW software. RESULT: The results showed that 22 primer pairs produced 338 loci, out of which 216 were polymorphic, the percentage of polymorphic loci was 63.9%. An average of 15.4 loci and 9.8 polymorphic loci were generated by each pair of primers. Genetic distance was analyzed by TREECONW software. Genetic distance of 18 P. thomsonii were changed from 0.004 7 to 0.265 8, with an average of 0.316. Using cluster analysis (UPGMA) based on those polymorphism bands amplified with SRAP primers, the 22 cultivars were classified into three groups, groups 1 with 18 P. thomsonii, group 2 with 3 P. lobata, and group 3 with 1 P. peduncularis. Most of the P. thomsonii from the same region were not in the same group. CONCLUSION: SRAP markers could be a good marker for genetic relationship research in the P. thomsonii.

[Classification of Pueraria lobata in different geographical regions].

OBJECTIVE: To classify Pueraria lobata originated from different geographical regions based on ITS,psbK-psbI and trnH-psbA information. METHOD: Twenty-four samples of P. lobata were collected from northeast China, north China, central China and northwest China. DNA extraction, PCR, sequence and genotypes/haplotypes analysis were performed . RESULT: ITS1, 5.8S and ITS2 varied only 1 bp respectively, psbK-psbI 2 bps; trnH-psbA varied 1 bp and 10 bp deletion. CONCLUSION: Based on the variation of ITS,psbK-psbI and trnH-psbA, 4 genotypes and 2 haplotypes were identified, respectively.

Molecular authentication of Radix Puerariae Lobatae and Radix Puerariae Thomsonii by ITS and 5S rRNA spacer sequencing.

In the present study, we examined nuclear DNA sequences in an attempt to reveal the relationships between Pueraria lobata (Willd). Ohwi, P. thomsonii Benth., and P. montana (Lour.) Merr. We found that internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA are highly divergent in P. lobata and P. thomsonii, and four types of ITS with different length are found in the two species. On the other hand, DNA sequences of 5S rRNA gene spacer are highly conserved across multiple copies in P. lobata and P. thomsonii, they could be used to identify P. lobata, P. thomsonii, and P. montana of this complex, and may serve as a useful tool in medical authentication of Radix Puerariae Lobatae and Radix Puerariae Thomsonii.