The effect of different LED wavelengths on the components and biosynthesis of isoflavonoid in sprout Astragalus membranaceus.

An artificial light source is the optimal element for studying the usability of the medicinal plant Astragalus membranaceus as a sprout vegetable. Based on artificial light source conditions, formononetin (FO) level was the highest (2.6 mg/L) in A. membranaceus exposed to white light emitting diode (LED) light, and calycosin (CA) level was the highest (3.09 mg/L) in the plant exposed to red LED light. According to the publicly available transcriptome data of LED-exposed sprout A. membranaceus LED, reference genes related to the content enhancement of FO, an isoflavone compound, and those related to the content enhancement of CA were selected. The expression patterns of these genes were assayed using qPCR. Among the genes related to FO enhancement, Gene-225190T showed the highest mRNA levels in cells of LED-white light-exposed sprout A. membranaceus; among the genes related to CA enhancement, Gene_042770T showed the highest expression under red LED light. Most genes related to the overall biosynthesis regulation of flavonoids of the upper concept of isoflavone were highly expressed in response to red LED light, and the transcriptional level of 4CL in response to red LED light was the highest. Based on these results, the artificial light sources that regulated the FO and CA contents in sprouts A. membranaceus were white and red LED lights, and the selected reference genes were capable of regulating isoflavone biosynthesis.

A reference-grade genome assembly for Astragalus mongholicus and insights into the biosynthesis and high accumulation of triterpenoids and flavonoids in its roots.

Astragalus membranaceus var. mongholicus (AMM), a member of the Leguminosae, is one of the most important medicinal plants worldwide. The dried roots of AMM have a wide range of pharmacological effects and are a traditional Chinese medicine. Here, we report the first chromosome-level reference genome of AMM, comprising nine pseudochromosomes with a total size of 1.47 Gb and 27 868 protein-encoding genes. Comparative genomic analysis reveals that AMM has not experienced an independent whole-genome duplication (WGD) event after the WGD event shared by the Papilionoideae species. Analysis of long terminal repeat retrotransposons suggests a recent burst of these elements at approximately 0.13 million years ago, which may explain the large size of the AMM genome. Multiple gene families involved in the biosynthesis of triterpenoids and flavonoids were expanded, and our data indicate that tandem duplication has been the main driver for expansion of these families. Among the expanded families, the phenylalanine ammonia-lyase gene family was primarily expressed in the roots of AMM, suggesting their roles in the biosynthesis of phenylpropanoid compounds. The functional versatility of 2,3-oxidosqualene cyclase genes in cluster III may play a critical role in the diversification of triterpenoids in AMM. Our findings provide novel insights into triterpenoid and flavonoid biosynthesis and can facilitate future research on the genetics and medical applications of AMM.

Characterization and protein engineering of glycosyltransferases for the biosynthesis of diverse hepatoprotective cycloartane-type saponins in Astragalus membranaceus.

Although plant secondary metabolites are important source of new drugs, obtaining these compounds is challenging due to their high structural diversity and low abundance. The roots of Astragalus membranaceus are a popular herbal medicine worldwide. It contains a series of cycloartane-type saponins (astragalosides) as hepatoprotective and antivirus components. However, astragalosides exhibit complex sugar substitution patterns which hindered their purification and bioactivity investigation. In this work, glycosyltransferases (GT) from A. membranaceus were studied to synthesize structurally diverse astragalosides. Three new GTs, AmGT1/5 and AmGT9, were characterized as 3-O-glycosyltransferase and 25-O-glycosyltransferase of cycloastragenol respectively. AmGT1G146V/I variants were obtained as specific 3-O-xylosyltransferases by sequence alignment, molecular modelling and site-directed mutagenesis. A combinatorial synthesis system was established using AmGT1/5/9, AmGT1G146V/S and the reported AmGT8 and AmGT8A394F . The system allowed the synthesis of 13 astragalosides in Astragalus root with conversion rates from 22.6% to 98.7%, covering most of the sugar-substitution patterns for astragalosides. In addition, AmGT1 exhibited remarkable sugar donor promiscuity to use 10 different donors, and was used to synthesize three novel astragalosides and ginsenosides. Glycosylation remarkably improved the hepatoprotective and SARS-CoV-2 inhibition activities for triterpenoids. This is one of the first attempts to produce a series of herbal constituents via combinatorial synthesis. The results provided new biocatalytic tools for saponin biosynthesis.

[Molecular mechanism underlying difference of astragaloside â £ content in imitating wild and cultivated Astragalus mongholicus].

The difference of astragaloside Ⅳ content and the expression of its biosynthesis related genes in imitating wild Astragalus mongolicus(IWA) and cultivated A.mongolicus(CA) under different growth years were systematically compared and analyzed.Then the key enzyme genes affected the difference of astragaloside Ⅳ content in the above two A.mongolicus were screened.High-perfo-rmance liquid chromatography(HPLC)was used to determine the content of astragaloside Ⅳ in A.mongolicusunderthe above two diffe-rent growth patterns.Based on the Illumina HiSeq and PacBio high-throughput sequencing platforms, thesecond-and third-generation transcriptome sequencing(RNA-Seq)databaseof the two A.mongolicuswas constructed.The related enzyme genes in the biosynthetic pathway of astragaloside Ⅳ were screened and verified byquantitative reverse transcriptase polymerase chain reaction(RT-qPCR).The RNA-sequencing(RNA-Seq) and RT-qPCR data of each gene were subjected to correlation analysis and trend analysis.The results showed that the variation trend of astragaloside Ⅳ contentby HPLC wasthe same as that of genes by RNA-Seq and RT-qPCR in 1-4 year IWA and 1-2 year CA.The trend level of astragaloside Ⅳ contentwas lower in 2-year IWA than 1-year IWA.Compared with 2-year IWA, 3-year IWA had an upward trend, while 4-year IWA hada downward trend versus 3-year IWA.Additionally, 1-year CA had increased trendthan 2-year CA.However, the content of astragaloside Ⅳ in 5-year IWA was higher than that of 6-year IWA, which wasinconsistent with the findings of RNA-Seq and RT-qPCR.This study preliminarily clarifiedthat the difference of astragaloside Ⅳ contentin 1-4 year IWA and 1-2 year CA wasclosely related to the expression of the upstream and midstream genes(MVK, CMK, PMK, MVD, SS) in the biosynthetic pathway.The results facilitate the production and planting of Radix Astragali seu Hedysari.

[Effects of water regulation on biosynthesis of calycosin-7-O-ß-D-glucoside in Astragalus membranaceus var. mongholicus].

The effects of water regulation on the biosynthesis of calycosin-7-O-ß-D-glucoside in 2-year-old Astragalus membranaceus var. mongholicus were studied,and the mechanism was explained from the aspects of key enzyme gene expression and antioxidant enzyme system. The content of calycosin-7-O-ß-D-glucoside was determined by HPLC,and the expression levels of six key enzyme genes( PAL,4 CL,CHS,CHI,IFS,13'H) in the synthesis pathway were analyzed by q RT-PCR. The activities of protective enzymes and contents of osmoregulation substances and malondialdehyde were also determined. In the water deficit group,the maximum concentration of calycosin-7-O-ß-D-glucoside was 0. 49 mg·g-1 on the 24 th day of treatment. In the whole water regulation,the water deficit group outweighed the water adequate group in osmoregulation substance and MDA contents. The activities of A. membranaceus var.mongholicus antioxidant enzymes SOD,POD,and CAT increased during the initial period of water regulation,but decreased with time.The expression of PAL,CHS,and 13'H in the water deficit group was at a low level,and the 4 CL had active expression,slightly lower than that in the water adequate group. The expression of CHI and IFS elevated rapidly when water deficit occurred. Correlation analysis showed that the content of calycosin-7-O-ß-D-glucoside was positively correlated with CHI expression( P<0. 01) and IFS expression( P<0. 05). Therefore,water regulation can change the accumulation pattern of calycosin-7-O-ß-D-glucoside,and water deficit may be an effective way to increase its content. CHI and IFS are the key genes in response to water deficit.

Transcriptome sequencing and characterization of Astragalus membranaceus var. mongholicus root reveals key genes involved in flavonoids biosynthesis.

BACKGROUND: Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao is a traditional medicinal herb of Leguminosae since it contains bioactive compounds such as flavonoids, which have significant pharmacological effects on immunity and antioxidant. However, the scanty genomic and transcriptome resources of Astragalus membranaceus have hindered further exploration of its biosynthesis and accumulation mechanism. OBJECTIVE: This project aim to further improve our understanding of the relationship between transcriptional behavior and flavonoids content of A. mongholicus. METHODS: The accumulation of flavonoids and related gene expression in five different developmental stages (A: vegetative, B: florescence, C: fruiting, D: fruit ripening and E: defoliating stages) of A. mongholicus root were studied by combining UV spectrophotometry and transcriptomic techniques. The de novo assembly, annotation and functional evaluation of the contigs were performed with bioinformatics tools. RESULTS: After screening and assembling the raw data, there were a total of 158,123 unigenes with an average length of 644.89 bp were finally obtained, which has 8362 unigenes could be jointly annotated by NR, SwissProt, eggNOG, GO, KEGG and Pfam databases. KEGG enrichment analysis was performed on differentially expressed genes(DEGs)in the four groups (A vs. B, B vs. C, C vs. D, D vs. E). The results showed that many DEGs in each group were significantly enriched to flavonoids biosynthesis related pathways. Among them, a number of 86 were involved in the biosynthesis of isoflavonoid (12), flavonoid (5) and phenylpropanoid (69). Further analysis of these DEGs revealed that the expression levels of key genes such as PAL, 4CL, CCR, COMT, DFR, etc. were all down-regulated at the fruiting stage, and then raised at the fruit ripening stage. This expression pattern was similar to the accumulation trend of total flavonoids content. CONCLUSIONS: In summary, this comprehensive transcriptome dataset allowed the identification of genes associated with flavonoids metabolic pathways. The results laid a foundation for the biosynthesis and regulation of flavonoids. It also provided a scientific basis for the most suitable harvest time and resource utilization of A. mongholicus.

UV-B Radiation Largely Promoted the Transformation of Primary Metabolites to Phenols in Astragalus mongholicus Seedlings.

: Ultraviolet-B (UV-B) radiation (280-320 nm) may induce photobiological stress in plants, activate the plant defense system, and induce changes of metabolites. In our previous work, we found that between the two Astragalus varieties prescribed by the Chinese Pharmacopoeia, Astragalus mongholicus has better tolerance to UV-B. Thus, it is necessary to study the metabolic strategy of Astragalus under UV-B radiation further. In the present study, we used untargeted gas chromatography-mass spectrometry (GC-MS) and targeted liquid chromatography-mass spectrometry (LC-MS techniques) to investigate the profiles of primary and secondary metabolic. The profiles revealed the metabolic response of Astragalus to UV-B radiation. We then used real-time polymerase chain reaction (RT-PCR) to obtain the transcription level of relevant genes under UV-B radiation (UV-B supplemented in the field, λmax = 313 nm, 30 W, lamp-leaf distance = 60 cm, 40 min·day-1), which annotated the responsive mechanism of phenolic metabolism in roots. Our results indicated that supplemental UV-B radiation induced a stronger shift from carbon assimilation to carbon accumulation. The flux through the phenylpropanoids pathway increased due to the mobilization of carbon reserves. The response of metabolism was observed to be significantly tissue-specific upon the UV-B radiation treatment. Among phenolic compounds, C6C1 carbon compounds (phenolic acids in leaves) and C6C3C6 carbon compounds (flavones in leaves and isoflavones in roots) increased at the expense of C6C3 carbon compounds. Verification experiments show that the response of phenolics in roots to UV-B is activated by upregulation of relevant genes rather than phenylalanine. Overall, this study reveals the tissues-specific alteration and mechanism of primary and secondary metabolic strategy in response to UV-B radiation.

Identification of miRNAs and Their Response to Cold Stress in Astragalus Membranaceus.

Astragalus membranaceus is an important medicinal plant widely cultivated in East Asia. MicroRNAs (miRNAs) are endogenous regulatory molecules that play essential roles in plant growth, development, and the response to environmental stresses. Cold is one of the key environmental factors affecting the yield and quality of A. membranaceus, and miRNAs may mediate the gene regulation network under cold stress in A. membranaceus. To identify miRNAs and reveal their functions in cold stress response in A. membranaceus, small RNA sequencing was conducted followed by bioinformatics analysis, and quantitative real time PCR (qRT-PCR) analysis was performed to profile the expression of miRNAs under cold stress. A total of 168 conserved miRNAs belonging to 34 families and 14 putative non-conserved miRNAs were identified. Many miRNA targets were predicted and these targets were involved in diversified regulatory and metabolic pathways. By using qRT-PCR, 27 miRNAs were found to be responsive to cold stress, including 4 cold stress-induced and 17 cold-repressed conserved miRNAs, and 6 cold-induced non-conserved miRNAs. These cold-responsive miRNAs probably mediate the response to cold stress by regulating development, hormone signaling, defense, redox homeostasis, and secondary metabolism in A. membranaceus. These cold-corresponsive miRNAs may be used as the candidate genes in further molecular breeding for improving cold tolerance of A. membranaceus.

Intraspecific and heteroplasmic variations, gene losses and inversions in the chloroplast genome of Astragalus membranaceus.

Astragalus membranaceus is an important medicinal plant in Asia. Several of its varieties have been used interchangeably as raw materials for commercial production. High resolution genetic markers are in urgent need to distinguish these varieties. Here, we sequenced and analyzed the chloroplast genome of A. membranaceus (Fisch.) Bunge var. mongholicus (Bunge) P.K. Hsiao using the next generation DNA sequencing technology. The genome was assembled using Abyss and then subjected to gene prediction using CPGAVAS and repeat analysis using MISA, Tandem Repeats Finder, and REPuter. Finally, the genome was subjected phylogenetic and comparative genomic analyses. The complete genome is 123,582 bp long, containing only one copy of the inverted repeat. Gene prediction revealed 110 genes encoding 76 proteins, 30 tRNAs, and four rRNAs. Five intra-specific hypermutation loci were identified, three of which are heteroplasmic. Furthermore, three gene losses and two large inversions were identified. Comparative genomic analyses demonstrated the dynamic nature of the Papilionoideae chloroplast genomes, which showed occurrence of numerous hypermutation loci, frequent gene losses, and fragment inversions. Results obtained herein elucidate the complex evolutionary history of chloroplast genomes and have laid the foundation for the identification of genetic markers to distinguish A. membranaceus varieties.

[Study on quality evaluation of sequence and SSR information in transcriptome of Astragalus membranacus].

In this study, 454/Roche GS FLX sequencing technology was used to obtain the data of the Astragalus membranaceus. Four hundred and fifty-four Sequencing System Software was applied to carry out the transcription of the group from scratch. Using MISA tools, 9 893 unigenes were selected for the sequence of the genome of A. membranaceus, and the information of SSR locus was analyzed. According to the result, the average length of reads was 413 bp, about 86% of the reads was involved in the splicing, the length of the N50 was 1 205 bp, the number of unigenes was measured by the whole transcript. 1 729 SSR loci in the A. membranaceus transcriptome were searched, the occurrence frequency of SSR was 9.24%, the frequency of SSR in the whole transcriptome was 13.42%, the average length of SSR was 7.97 kb. One hundred and twenty-seven kinds of core repeat sequences were found, the dominant type was TG/AC type of dinucleotide, it appeared to account for 4.25% of the total SSR locus. The results of the sequence of the transcription of the A. membranaceus transcriptome revealed the overall expression, and a large number of unigenessequence was obtained, and the SSR locus in the genome of the A. membranaceus is high, and the type is diverse, and the polymorphism of the gene is high.

[Comparison between Astragalus membranaceus var. mongholicus and Hedysarum polybotrys based on ITS sequences and metabolomics].

Astragalus membranaceus var. mongholicus and Hedysarum polybotrys belong to different genera, but have similar drug efficacy in traditional Chinese medicine theory, and H. polybotrys was used as the legal A. membranaceus var. mongholicus previously. In this study, similarities and differences between them were analyzed via their ITS/ITS2 fragments information. The ITS (internal transcribed spacer) regions were amplified using polymerase chain reaction and then sequenced in two-way. The alignment lengths of ITS regions were 616 bp, in which 508 loci were consistent, and 103 loci were different, accounting for 82.47% and 16.72% of the total ITS nucleotides in length, respectively. As genotype determines phenotype, 1HNMR-based metabolomic approach was further used to reveal the chemical similarities and differences between them. Thirty-four metabolites were identified in the 1H NMR spectra, and twenty-seven metabolites were the common components. Amino acids, carbohydrates and other primary metabolites were similar, while a large difference existed in the flavonoids and astragalosides. This study suggests that A. membranaceus var. mongholicus and H. polybotrys show similarities and differences from molecular and chemical perspectives, which has laid a foundation for elucidating the effective material basis of drug with similar efficacy and resources utilization.

Accumulation of astragalosides and related gene expression in different organs of Astragalus membranaceus Bge. var mongholicus (Bge.).

Astragalus membranaceus is one of the most important traditional Korean and Chinese medicinal herbs because it contains triterpenoid saponins (astragaloside I, II, III, and IV), which have beneficial and pharmacological effects on health. In this study, we analyzed 10 mevalonate pathway genes that are involved in astragaloside biosynthesis using the Illumina/Solexa HiSeq2000 platform. We determined the expression levels of the 10 genes using quantitative real-time PCR, and analyzed the accumulation of astragalosides in different organs using high-performance liquid chromatography. Genes related to the mevalonate pathway were expressed in different levels in different organs. Almost all genes showed high transcript levels in the stem and leaf, with the lowest transcript levels being recorded in the root. In contrast, most astragalosides accumulated in the root. In particular, the astragaloside IV content was distributed in the following order: root (0.58 mg/g DW) > flower (0.27 mg/g DW) > stem (0.23 mg/g DW) > leaf (0.04 mg/g DW). In the root, astragaloside II exhibited the highest content (2.09 mg/g DW) compared to astragaloside I, III, and IV. Notably, gene expression did not follow the same pattern as astragaloside accumulation. We suggest carefully that astragalosides are synthesized in the leaves and stem and then translocated to the root. This study contributes towards improving our understanding of astragaloside biosynthesis in A. membranaceus.

Accumulation of flavonoids and related gene expressions in different organs of Astragalus membranaceus Bge.

Astragalus membranaceus is one of the important medicinal plant in China and Korea. It is used to increase metabolism and digestion, enhance the immune system, and promote the healing of wounds and injuries. In the present study, we used quantitative real-time PCR to investigate the expression of genes related to the biosynthesis of flavonoids, in addition to high-performance liquid chromatography to assess calycosin and calycosin-7-O-ß-D-glucoside accumulation, in the different plant organs of A. membranaceus. The transcript levels of all genes (AmPAL, AmC4H, Am4CL, AmCHS, AmCHR, AmCHI, AmIFS, AmI3'H, and AmUCGT) involved in calycosin and calycosin-7-O-ß-D-glucoside biosynthesis were the highest in the flower. Calycosin content was ordered as follows: leaf (145.56 µg/g dry weight [DW]) > stem (18.3 µg/g DW) > root (1.64 µg/g DW) > flower (0.09 µg/g DW), whereas calycosin-7-O-ß-D-glucoside content was ordered as follows: root (4.88 µg/g DW) > stem (3.86 µg/g DW) > leaf (2.0 µg/g DW) > flower (not detected). All genes exhibited the highest transcription levels in the flower, whereas calycosin and its glycoside content were the highest in the leaf and root, respectively. Our results indicate that the enhancement of calycosin-7-O-ß-D-glucoside in the roots may originate from high calycosin accumulation in the stem and leaf. Thus, the mechanisms regulating calycosin and calycosin-7-O-ß-D-glucoside content differ in the different organs of A. membranaceus. The results are expected to provide baseline information from which the mechanism of flavonoid biosynthesis in the different organs of A. membranaceus may be elucidated.

High-throughput analysis and characterization of Astragalus membranaceus transcriptome using 454 GS FLX.

Astragalus membranaceus (Fisch.) Bge (AR), one of the most important medicinal plants in Asia, was found to exhibit various bioactivities. Due to limited genomic and transcriptomic data, the biosynthetic pathway of the major bioactive compound in AR, is currently unclear. In this study, 454 GS FLX technology was employed to produce a substantial expressed sequence tag (EST) dataset from the AR. In all, 742721 high-quality reads from the AR were produced using Roche GS FLX Titanium. A total of 9893 unique sequences were obtained and annotated by a similarity search against the public databases, and involved in the secondary metabolic pathway, which would facilitate deciphering the molecular mechanism of secondary metabolism in AR. The assembled sequences were annotated with gene names and Gene Ontology (GO) terms. GO revealed the unique sequences that could be assigned to 34 vocabularies. In the KEGG mapping, unique sequences were established as associated with 46 biochemical pathways. These results provided the largest EST collections in AR and will contribute to biosynthetic and biochemical studies that lead to drug improvement. With respect to the genes related to metabolism and biosynthesis pathway were also found. Our work demonstrated the utility of 454 GS FLX as a method for the rapid and cost-effective identification of AR transcriptome, and this EST dataset will be a powerful resource for further studies such as taxonomy, molecular breeding, and secondary metabolism in AR.

[Between Hengshanhuangqi and Chuanhuangqi based on metabolomics and ITS2 sequences].

To compare the differences between Hengshanhuangqi (HH) and Chuanhuangqi (CH) at molecular level, 1H NMR based plant metabolomics approach was used to reveal the chemical difference between HH and CH. Then, the contents of astragaloside IV and calycosin-7-O-beta-D-glucoside, the marker compounds specified in China Pharmacopoeia, were determined. In addition, the ITS2 fragments of HH and CH were sequenced. Twenty-three metabolites were identified in the 1H NMR spectrum, and the principal component analysis showed CH and HH could be separated clearly. HH contained more aspartic acid, GABA, citric acid, astragaloside IV and calycosin-7-O-beta-D-glucoside, while CH contained more threonine, alanine, acetic acid, choline, arginine, fructose and sucrose. And the astragaloside IV is almost undetectable in CH. In addition, the ITS2 fragment sequences of HH and CH were different at eight bases. Thus, the HH and CH showed significant differences chemically and genetically.

[Molecular identification of astragali radix and its adulterants by ITS sequences].

OBJECTIVE: To explore a new method for identification Astragali Radix from its adulterants by using ITS sequence. METHOD: Thirteen samples of the different Astragali Radix materials and 6 samples of the adulterants of the roots of Hedysarum polybotrys, Medicago sativa and Althaea rosea were collected. ITS sequence was amplified by PCR and sequenced unidirectionally. The interspecific K-2-P distances of Astragali Radix and its adulterants were calculated, and NJ tree and UPGMA tree were constructed by MEGA 4. RESULT: ITS sequences were obtained from 19 samples respectively, there were Astragali Radix 646-650 bp, H. polybotrys 664 bp, Medicago sativa 659 bp, Althaea rosea 728 bp, which were registered in the GenBank. Phylogeny trees reconstruction using NJ and UPGMA analysis based on ITS nucleotide sequences can effectively distinguish Astragali Radix from adulterants. CONCLUSION: ITS sequence can be used to identify Astragali Radix from its adulterants successfully and is an efficient molecular marker for authentication of Astragali Radix and its adulterants.

[Regulation of Vitreoscilla hemoglobin on biosynthesis of astragaloside IV].

In the present study, the regulation of Vitreoscilla hemoglobin (VHb) on astragaloside IV biosynthesis was investigated. An intermediate expression vector consisting of the CaMV35S promoter fused to the vgb and nopaline synthase terminator was transferred into Astragalus membranaceus via Agrobacterium rhizogenes. The transgenic hairy roots were confirmed by PCR amplification and Southern blot hybridization. The expression of vgb in transgenic hairy roots was confirmed by RT-PCR. After 15 days cultivation, the dry weight and growth rate of transgenic hairy roots were higher than that of the non-transgenic hairy root. ELSD-HPLC analysis showed that astragaloside IV content of transgenic hairy roots was 5 to 6 times of non-transgenic hairy root control and 10 to 12 times of Radix Astragali from Shanxi Province. These results suggested that the expression of vgb promoted the growth of transgenic hairy roots, and increased the content of astragaloside IV.

[Study on genetic relationship of Astragalus membranaceus var mongholicus in different producing area using SRAP].

OBJECTIVE: To study the genetic relationship of Astragalus membranaceus var. mongholicus in different producing area and provide theoretical basis for the evaluation of Astragalus germplasm resources. METHOD: Through quence-related amplified polymorphism (SRAP) analysis, the systematic diagram of genetic relationship was constructed by UPGMA method. RESULT: A total of 141 SRAP markers were scored. By the use of UPGMA cluster analysis with genetic distance, Astragalus could be divided into two provenance plots of Gansu and Shanxi. CONCLUSION: The genetic differentiation among populations of A. membranaceus var. mongholicus is remarkable. SRAP marker could be efficiently used for the study of the genetic relationship of Astragalus.

[A taxonomic study on the original plant of radix astragali].

There are dispute about the status of taxonomy among Astragalus membranaceus (Fisch.) Bge, A. membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao. and A. pallidipurpureus stat. nov. The varieties and taxa of the complex are still in need of revision. With molecular biology study used trnH-psbA intergenic region, the taxonomic revision of Radix Astragali has been made. A. pallidipurpureus stat. nov is suggested as a new species.

Molecular identification of Astragalus membranaceus at the species and locality levels.

Astragalus membranaceus (Fisch.) Bunge, a commonly used Chinese medicinal material, from certain localities contains more favourable trace elements and fewer harmful trace elements than those from other localities. Therefore, there is a need to distinguish Astragalus membranaceus from different localities. Internal transcribed spacer 1 (ITS1) of the nuclear ribosomal RNA gene of 23 Astragalus membranaceus samples were sequenced to confirm the species of the samples. Arbitrarily primed polymerase chain reaction (APPCR) was then used to obtain unique fingerprints for each sample using several primers. The presence and absence of bands were used for calculating mean similarity indexes among the samples. It was found that the Heilongjiang samples were markedly distinguishable from samples of other localities. In addition, bands common for samples from the same locality were also identified and used to distinguish samples from Neimengu and Shanxi. Therefore, Astragalus membranaceus from these provinces, the major cultivation places in China, can be differentiated.