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Objective: To make a distinction between Ludisia discolor and its relatives genus in molecular level, SCoT markers were employed to assess the genetic relationship and construct the DNA fingerprint. Methods: Orthogonal design method were carried out to optimize the suitable SCoT-PCR reaction system based on five factors. The optimum annealing temperature and SCoT primers were also screened. The 12 germplasm resources were used as materials, the screened primers were selected to analyze the genetic relationship of 12 materials. POPGENE was used to calculate the genetic diversity, NTSYS was performed to analyze cluster, and DNA map was constructed. Results: The optimized SCoT-PCR reaction system was constructed and a total of 12 rich bands were screened out as the primers of SCoT molecular marker with polymorphism ratio of 98.98%. According to Nei's genetic similarity coefficient, a total of 12 materials were divided into three cluster when coefficient was 0.45. Goodyera schlechtendaliana was in category I with seven L. discolor lines, indicating that these samples had close relationship. In category II, there were three samples came from Anoectochilus roxburghii. Moreover, a green L. discolor sample was alone clustered into category III. The DNA fingerprint map by the SC8 primer could identify the 12 materials. Conclusion: There are rich genetic diversities in 12 samples of L. discolor and its relatives genus, and the construction of DNA fingerprint map provides a theoretical basis for the identification of L. discolor and its relatives genus, which were tested in this study.
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OBJECTIVE： To establish the method for PCR identification of bullwhip， and to identify the authenticity of bullwhip at the molecular level. METHODS： DNA samples of bullwhip and its counterfeits （donkey whip， pig whip， sheep whip） were extracted and their integrity， purity and concentration were detected. Using GenBank related information， using mitochondrial cytochrome b （Cyt b） gene of bullwhip as target gene， Primer-BLAST online software was used to design specific primer. PCR amplification was performed for whips of different species， and electrophoretic analysis was conducted for the product. PCR products of bullwhip samples were cloned and confirmed by DNA sequencing. The specificity and repeatability of the established PCR method were verified. RESULTS： DNA purity of the bullwhip and its counterfeits was high， and there was no protein or RNA pollution. 1.5％ agarose gel electrophoresis showed that there were obvious target gene bands of bullwhip samples at 200-300 bp， while no corresponding bands appeared in other counterfeit products. The results of DNA sequencing showed that the nucleotide sequence of the gene fragment of bullwhip was 100％ similar to that of the bullwhip in GeneBank. Results of methodological validation showed that established method was specific and reproducible. CONCLUSIONS： The established PCR identification method based on Cyt b gene in the study is simple， rapid， accurate， specific and reproducible， and can meet the requirements of analysis and identification of bullwhip and its counterfeits.
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Abstract Erythrina × neillii Mabberley & Lorence, Fabaceae, is a sterile hybrid between E. herbacea L. and E. humeana Spreng. Nothing was traced about its genetic, macro and micromorphology. Therefore, it was deemed of interest to study its botanical characters, in addition to the DNA fingerprint to help in the identification of the plant. The anatomical characters of the old stem and its bark are characterized by the presence of cork cells, bast fibers and sclereids. Pericycle is sclerenchymatous forming crystal sheath. The epidermises of the leaf and young stem are characterized by the presence of anomocytic and paracytic stomata, non-glandular, unicellular and multicellular two armed hairs, and glandular club shaped hair. Calcium oxalate is present in the form of crystal sheath and prisms. Secretory cavities are distributed in the phloem and cortex. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used as one of the molecular methods to differentiate between the samples of Erythrina. The DNA of Erythrina was extracted and analyzed using seven-mer random primers. Randomly Amplified Polymorphic DNA were recognized. This characterization allows certification of the authenticity of Erythrina × neillii, in order to provide quality control for the plant.
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Objective To reveal the genetic diversity of provenances and populations of Taxus chinensis var. mairei in Fujian province from 23 orgins, providing certain theoretical foundation for the preservation and protection. Methods RAPD molecular marker technology was used to study the genetic diversity of T. chinensis var. mairei in Fujian province. Results The number of observed allele was 1.974 4, the number of effective alleles was 1.603 8, the average value of the Nei’s genetic diversity was 0.351 3 and the Shannon’s information index was 0.522 8. And there existed more accurate information on genetic relationship, diversity among different geographical provenances. The genetic distance of those 23 species ranged from 0.104 to 0.620 7, the genetic coefficients of 23 varieties ranged from 0.401 7 to 0.786 3. The genetic distance of seven T. chinensis var. mairei of different populations ranged from 0.021 0 to 0.379 4, the genetic coefficients ranged from 0.684 3 to 0.979 2. According to Nei’s method calculation of T. chinensis var. mairei, those seven different populations’ genetic diversity was Dst = 0.108 8, differentiation index Gst = 0.369 0, and gene flow coefficient Nm = 0.855 0. 36.9% of the total genetic variation existed among populations, the variation within populations was only 63.1%, and analysis showed high differentiation existed in provenances. Conclusion There is a certain heritable variation among different provenances of T. chinensis var. mairei, but there still exists more accurate information and high similarity coefficients among different populations.
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Objective: RAPD technique has been used to construct DNA fingerprints and analyze the genetic diversity in 14 batches of Forsythia suspensa from different origins. Methods: Primers with good polymorphism were screened from 45 RAPD primers combinations by PCR, and the DNA fingerprints for 14 batches of F. suspensa were constructed by RAPD technique. Results: Eleven RAPD primers with good polymorphism were screened from 45 RAPD primers by PCR amplification, and 80 bands were amplified by the 11 RAPD primers with average 7.27 bands for each primer, of which 67 were polymorphic and PPB was 83.8%. The genetic similarity (GS) ranged from 0.4875 to 0.9625 in 14 batches of F. suspensa from different origins, which showed they had rich genetic diversity. The result of cluster analysis showed that F. suspensa was correlated with sample origin and was consistent with species distribution. Conclusion: The DNA fingerprints for 14 batches of F. suspensa are established based on the RAPD amplified bands.
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Gokshura a well-known drug in Ayurveda which is extensively used in many disease conditions like dysuria, asthma, diabetes, cough, oedema, cardiac disorders etc. Tribulus terrestris (Family – Zygophyllaceae) is an official source of Gokshura as per API. Five species of genus Tribulus are found throughout India with a slight morphological difference. In this study, three different species of Tribulus genus from different regions were subjected for molecular characterization by RAPD method. Analysis showed that three different samples gave clearly similar banding pattern with each of the random primers used and 80% similarity between the three samples were observed when the results were subjected to band scoring and analysis with clustering. Even through the micromorpholgical observations showed differentiating characters in mature carpels and intrastaminal glands of the selected species.
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Objective: In order to correctly identify the different germplasm resources of Gastrodia elata and gain the excellent germplasm resources, SRAP molecular marker was used to analyze the genetic diversity of G. elata. Methods: G. elata was collected from seven different areas, which included 24 smaples of G. elata f. elata, G. elata f. g1auca and G. elata f. viridis, the DNA figerprint of G. elata was constructed with SRAP molecular marker, and the genetic diversity was analyzed. Results: Six huandred Thirty-seven belts were amplified by 33 primers pairs, and the polymorphic percentage was 73.16%. The range of genetic similarity coefficient was 0.404 0-0.908 0, the genetic similarity coefficient among G. elata f. elata was in the range of 0.906 6-0.996 4, and those of G. elata f. g1auca, G. elata f. viridis, and hybrid was in the range of 0.410 4-0.999 6, 0.541 0-0.950 4 and 0.578 2, respectively. They showed small genetic differences. The analysis of molecular variance showed higher percentages of genetic variation within population than those among populations in all species. Populations showed higher genetic structure (FST = 0.33, P < 0.05). In addition, artificial cultivation had influence to the genetic differentiation, but not significantly. So in terms of different cultivation conditions so far had no significant impact on the genetic differentiation of G. elata. Mantel's test has been used to detect the correlation between genetic distance and geographic distance of all sorts of germplasm resources, the result had no significant correlation, and due to the limited number of samples, the result is not representative. Conclusion: SRAP molecular marker method can more effectively reflect the genetic polymorphisms of G. elata. Compared with other two phenotypes, G. elata f. elata is more conservative and has lower genetic diversity. The other two variants have higher genetic diversity.
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Objective: To analyze the correlation of DNA fingerprints of Sarcandra glabra with different quality levels and its quality. Methods: Using ISSR-PCR, 100 ISSR primers (UBC801-UBC900) were screened, and 23 of them were polymorphic. The 23 ISSR primers were used to amplify 18 S. glabra samples from different habitats. Based on the 18 ISSR amplified bands, the data base of amplified bands fingerprint was established using Excel. Results: one hundred and ninty-eight bands were obtained. Each primer amplified eight bands on average. The number of polymorphic DNA bands was 184, and the polymorphic proportion of DNA bands was 92.9%. Seven sites in two primers (UBC811 and UBC825) screened from 23 polymorphic ISSR primers were in one group, and seven sites in three primers (UBC827, UBC834, and UBC842) were in another group. The DNA fingerprints of 18 S. glabra samples were established, and U844-6 bands were screened from 184 polymorphic bands to correlate with sample quality. Conclusion: ISSR molecular markers could identify the DNA fingerprints of 18 S. glabra samples, and screen one band that is related to the quality.
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For the protection of consumers and developments of relevant industry, authentication of medicinal plants is a critical issue.This review covers various aspects of authentication methods and techniques based on molecular biology and genomics with special emphasis on molecular biology techniques including genome-based authentication,microchip-based authentication, DNA barcoding, and their applications.
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Objective The DNA fingerprints of cultivated Pinellia ternata collected from different geographic regions were generated by using AFLP markers to find the feasibility in analyzing their genetic diversity, relationship, and germplasm identification. Methods The DNA polymorphism of 51 cultivated germplasm of P. ternata collected from 17 different habitats and four cultivars of P. pedatisecta (outgroup) were detected by AFLP molecular markers. Results The DNA fingerprints of 51 individuals of P. ternata were obviously distinguished by eight pairs of high polymorphic and efficient primer combinations screened from 64 primer combinations. The phylogenetic clustering results revealed that all the tested cultivars were fully differentiated, and individuals from the same regions were mainly clustered together. Moreover, cultivars from East-China, including Zhejiang and Jiangsu Provinces, displayed clear genetic distinction from other regions. The clustering results were strongly supported by Bootstrap test. Conclusion AFLP Markers can be potentially used in analyzing of genetic diversity, relationship, and germplasm identification of this medicinal plant, and the germplasm from regions of East-China, including Zhejiang and Jiangsu Provinces, displays the relative separate genetic characters from other regions.
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A novel fingerprint system of Chinese medicinal materials employing both DNA and chemical means has been proposed. To establish the fingerprint system is very effective to the quality control of Chinese medicinal materials, because it will be controlled from chemical substance and genetic information. The chemical components and DNA fingerprints will show the complex chemical components and biological source, respectively. DNA fingerprint could give a further proof to distinguish Chinese materia medica from the chemical. The combination of chemical and DNA fingerprints is constructive to quality control of Chinese materia medica, the cultivar identification and the selective breed of good varieties for medicinal use. To carry out the fingerprint system of Chinese medicinal materials, it is necessary to establish fingerprint database of Chinese medicinal materials, including chemical and DNA fingerprint, as well as many detection methods, and the pattern recognition system by computer is also necessary.
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Objective To identify seven species of Gynostemma BI.,including G.pentaphyllum,G. pentagynum,G.cardiospermum,G.longipes,G.yixingense,G.laxiflorum,and G.guangxiense,by in- ter-simple sequence repeat(ISSR)markers.Methods General DNA was isolated from leaves of the seven species in Gynostemma B1.by CTAB,57 primers constituted by ISSR were tested for PCR and sepharose electrophoresis.Results Fourteen primers amplified polymorphic bands,the amplification patterns of primers UBC-873 and UBC-895 were higher in terms of polymorphic and amplified band ratio.They are used to distinguish all the examined seven species.Conclusion ISSR-PCR Method provides a quick,reli- able molecular marker technique for the identification of different species of Gynostemma B1.
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Saliva and samples containing saliva were regarded as a kind of potential biological trace evidencefor individual identification in forensic science practice. In this paper, the authors have studied theDNA analysis of saliva and saliva containing samples. The results demonstrated that DNA can be extracted from saliva and saliva-containing samples. RFLPs analysis by DNA fingerprinting and PCRbased typing can be performed according to the amount of DNA extracted from these samples.
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Comparison between the DNA fingerprinting and the serological method used for paternity test is presented. 32 cases of disputed paternity were tested using DNA fingererints with MYO minisatellite DNA probe and blood typing. The results of DNA fingerprinting test correlated with those of blood typing. 8 alleged fathers were excluded while 24 were confirmed. The relationship between the results from two methods was discussed.