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italic>Bulbophyllum orchids are popular for its ornamental appearance and great medicinal values. However, there is still a lack of research on phylogenetic relationship and species identification for this genus. In this study, the plastome sequences of three medicinal Bulbophyllum orchids (Bulbophyllum affine, Bulbophyllum pectinatum, Bulbophyllum funingense) were sequenced and analyzed. After assembly and annotation, it was found that the plastomes of Bulbophyllum plants encoded a total of 108 genes, including 74 protein-coding genes, 30 tRNA genes and 4 rRNA genes. Based on the analysis of mVISTA and comparison between junctions, it was found that the plastome structure of Bulbophyllum orchids was relatively conserved, and the variation mainly existed in the non-coding regions. Phylogenetic analysis showed that Bulbophyllum orchids were closely related to Dendrobium orchids. SSR analysis of Bulbophyllum showed that most SSRs were located in the intergenic spacer and had the most single nucleotide repeats. In addition, based on the comparative analysis of non-coding sequences, a total of 10 high-variability sequences were screened out, among which the combination of five non-coding region sequences, including psbI-trnS, psbC-trnS, clpP-ex1-psbB, psaJ-rpl33, rpl33-rps18, had the highest sequence variability and could be used in the species identification study of medicinal plants of Bulbophyllum. In conclusion, this study provides a theoretical basis for phylogenetic relationship and species identification of Bulbophyllum orchids through the comparative analysis of plastome sequences of three medicinal plants of the genus Bulbophyllum.
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Bletilla striata has been used as traditional Chinese medicine for several centuries. In recent years, the quality and quantity of wild B. striata plants have declined sharply due to habitat deterioration and human over-exploitation. Therefore, it is of great urgency to evaluate and protect B. striata wild plant resource. In this study, sequence-related amplified polymorphism (SRAP) markers were applied to assess the level and pattern of genetic diversity in twelve populations of B. striata. The results showed a high level of genetic diversity (PPB = 90.48%, H = 0.349 4, I = 0.509 6) and moderate genetic differentiation among populations (G(st) = 0.260 9). Based on the unweighted pair-group method with arithmetic average (UPGMA), twelve populations gathered in three clusters. The cluster 1 included four populations. There are Nanjing, Zhenjiang, Xuancheng and Hangzhou. The seven populations which come from Hubei Province, Hunan Province, Jiangxi Province and Guizhou Province belonged to the cluster 2. The cluster 3 only contained Wenshan population. Moreover, Mantel test revealed significant positive correlation between genetic distances and geographic distances (r = 0.632 9; P < 0.000 1). According to the results, we proposed a series of conservation consideration for B. striata.
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China , Marcadores Genéticos , Variación Genética , Genética de Población , Orchidaceae , Genética , Filogenia , Plantas Medicinales , GenéticaRESUMEN
Bletilla striata has been used as traditional Chinese medicine for several centuries. In recent years, the quality and quantity of wild B. striata plants have declined sharply due to habitat deterioration and human over-exploitation. Therefore, it is of great urgency to evaluate and protect B. striata wild plant resource. In this study, sequence-related amplified polymorphism (SRAP) markers were applied to assess the level and pattern of genetic diversity in twelve populations of B. striata. The results showed a high level of genetic diversity (PPB = 90.48%, H = 0.349 4, I = 0.509 6) and moderate genetic differentiation among populations (Gst = 0.260 9). Based on the unweighted pair-group method with arithmetic average (UPGMA), twelve populations gathered in three clusters. The cluster 1 included four populations. There are Nanjing, Zhenjiang, Xuancheng and Hangzhou. The seven populations which come from Hubei Province, Hunan Province, Jiangxi Province and Guizhou Province belonged to the cluster 2. The cluster 3 only contained Wenshan population. Moreover, Mantel test revealed significant positive correlation between genetic distances and geographic distances (r = 0.632 9; P B. striata.
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In this study, 17 kinds of Dendrobium species of Fengdous including 39 individuals were collected from 4 provinces. Mitochondrial gene sequences co I, nad 5, nad 1-intron 2 and chloroplast gene sequences rbcL, matK amd psbA-trnH were amplified from these materials, as well as nrDNA ITS. Furthermore, suitable sequences for identification of Dendrobium species of Fengdous were screened by K-2-P and P-distance. The results showed that during the mentioned 7 sequences, nrDNA ITS, nad 1-intron 2 and psbA-trnH which had a high degree of variability could be used to identify Dendrobium species of Fengdous. However, single fragment could not be used to distinguish D. moniliforme and D. huoshanense. Moreover, compared to other combined fragments, new type combined fragments nrDNA ITS+nad 1-intron 2 was more effective in identifying the original plants of Dendrobium species and could be used to identify D. huoshanense and D. moniliforme. Besides, according to the UPGMA tree constructed with nrDNA ITS+nad 1-intron 2, 3 inspected Dendrobium plants were identified as D. huoshanense, D. moniliforme and D. officinale, respectively. This study identified Dendrobium species of Fengdous by combined fragments nrDNA ITS+nad 1-intron 2 for the first time, which provided a more effective basis for identification of Dendrobium species. And this study will be helpful for regulating the market of Fengdous.
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ADN de Plantas , Genética , ADN Espaciador Ribosómico , Genética , Dendrobium , Clasificación , Genética , Genes del Cloroplasto , Genes de Plantas , Intrones , Plantas Medicinales , Clasificación , GenéticaRESUMEN
To identify adulterants from medicinal plants of Bletilla H. G. Reichenbach, the suitable candidate DNA barcoding of Bletilla was evaluated. In this study, the internal transcribed spacer (ITS) of nuclear ribosomal DNA, the LFY homologous gene intron 2 and chloroplast ycfl gene were amplified and sequenced from forty-one samples. The intra-specific and inter-specific divergences of Bletilla were calculated, and the identification efficiency was assessed using Barcoding Gap, NJ tree by K2P distance and BLAST1 method. The result showed the intra-specific divergence of nrDNA ITS and ycJfl (0.022-0.106 and 0.017-0.106) were obviously higher than the inter-specific divergence (0-0.012 and 0-0.015), and four species of Bletilla were also accurately distinguished in NJ trees. Whereas, there was no Barcoding Gap on LFY homologous gene intron 2, thus it cannot effectively identify species of Bletilla. Using NJ tree of nrDNA ITS and ycfl gene, powdery medicine and the adulterants of Bletilla were successfully unidentified. In conclusion, nrDNA ITS and ycfl can be used as a potential DNA barcoding to identify the medicinal plants in Bletilla and its adulterants. There were only three basic differences on nrDNA ITS between "Jujing baiji" and Bletilla striata of Lu'an in Anhui province, and two basic differences in ycfl. Based on morphological and molecular data, "Jujing baiji" could be recognized as the species of Bletilla striata.
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Secuencia de Bases , Código de Barras del ADN Taxonómico , ADN de Plantas , Genética , ADN Espaciador Ribosómico , Genética , Orchidaceae , Clasificación , Plantas Medicinales , ClasificaciónRESUMEN
The LEAFY (LFY) homologous gene of Dendrobium moniliforme (L.) Sw. was cloned by new primers which were designed based on the conservative region of known sequences of orchid LEAFY gene. Partial LFY homologous gene was cloned by common PCR, then we got the complete LFY homologous gene Den LFY by Tail-PCR. The complete sequence of DenLFY gene was 3 575 bp which contained three exons and two introns. Using BLAST method, comparison analysis among the exon of LFY homologous gene indicted that the DenLFY gene had high identity with orchids LFY homologous, including the related fragment of PhalLFY (84%) in Phalaenopsis hybrid cultivar, LFY homologous gene in Oncidium (90%) and in other orchid (over 80%). Using MP analysis, Dendrobium is found to be the sister to Oncidium and Phalaenopsis. Homologous analysis demonstrated that the C-terminal amino acids were highly conserved. When the exons and introns were separately considered, exons and the sequence of amino acid were good markers for the function research of DenLFY gene. The second intron can be used in authentication research of Dendrobium based on the length polymorphism between Dendrobium moniliforme and Dendrobium officinale.
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Secuencia de Aminoácidos , Secuencia de Bases , ADN de Plantas , Genética , Dendrobium , Genética , Exones , Intrones , Orchidaceae , Genética , Filogenia , Hojas de la Planta , Genética , Proteínas de Plantas , Genética , Plantas Medicinales , Genética , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Random amplified microsatellite polymorphism (RAMP) markers were used to access the genetic diversity among 112 samples of nine populations of Dendrobium officinale Kimura et Migo. Using 16 informative primers, 123 bands were amplified and 86 (69.92%) were polymorphic. The polymorphic bands from three to eight could be detected for each RAMP primer, with a mean of 5, indicating abundant genetic diversity among populations. Genetic similarity coefficients ranged from 0.250 to 0.813. UPGMA dendrogram illustrated 9 populations clustered into 3 groups, and the cluster pattern showed correlation with the locations of the D. officinale populations. These results were supported by the previous conclusions that were achieved by other molecular markers, and RAMP is proved to be effective for evaluating the genetic diversity of wild populations of Dendrobium officinale.
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Análisis por Conglomerados , Cartilla de ADN , ADN de Plantas , Genética , Dendrobium , Genética , Variación Genética , Repeticiones de Microsatélite , Filogenia , Plantas Medicinales , Genética , Reacción en Cadena de la Polimerasa , Métodos , Polimorfismo Genético , Técnica del ADN Polimorfo Amplificado Aleatorio , MétodosRESUMEN
Simple sequence repeat (SSR) was used to investigate the genetic diversity and structure of Dendrobium officinale. A total of 15 primer pairs with stable and repeatable polymorphism were screened out from 60 SSR primer pairs developed by the method of microsatellite enrichment by magnetic beads. Forty-eight samples of Dendrobium officinale were analyzed in genetic polymorphism. These loci were polymorphic and displayed 3 to 9 alleles per locus with a mean number of 6.1. The observed and expected heterozygosities ranged from 0.60 to 0.85 and from 0.49 to 0.85 respectively. The polymorphic information content (PIC) of each SSR locus varied from 0.437 to 0.829 with an average of 0.702. Fifteen primer pairs were used in Dendrobium cross-species amplification and totally 13 primer pairs were proved to have the transferability in D. officinale related species. In addition, 500 tissue culture plantlets of D. officinale were tested for purity identification by means of PCR amplification with four SSR primer pairs. The results showed that SSR technique is a feasible, simple and inexpensive method for determining adulterants in germplasm identification.
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Cartilla de ADN , ADN de Plantas , Genética , Dendrobium , Genética , Variación Genética , Repeticiones de Microsatélite , Plantas Medicinales , Genética , Reacción en Cadena de la Polimerasa , Métodos , Polimorfismo Genético , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
The psbA-trnH regions of Dendrobium species of Fengdous were sequenced by our research group. The psbA-trnH sequences of fifteen Dendrobium species were analyzed with software MEGA 4.0. The results showed that the lengths of sequences varied from 721 to 767 bp. The variable sites were 42 while the informative sites were 11. Genetic distances were calculated using the Kimura 2-parameter model. Genetic distances varied from 0.0013-0.0183 among fifteen species while the average genetic distance was 0.0148. The interspecies differences of psbA-trnH regions were demonstrated. Six indels happened in this fragment, which led to the great difference of sequence lengths among fifteen species. We found that there were no population differences in the psbA-trnH region of various species of Fengdous so far. By using the database of various Dendrobium species of Fengdous and two genetics software, the botanical origin of the inspected species of Fengdous was authenticated successfully by sequencing the psbA-trnH regions. The psbA-trnH region of cpDNA can be used as a candidate marker for authentication of Dendrobium species of Fengdous.
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Secuencia de Bases , Código de Barras del ADN Taxonómico , Métodos , ADN de Cloroplastos , Genética , ADN Intergénico , ADN Ribosómico , Genética , Dendrobium , Clasificación , Genética , Variación Genética , Datos de Secuencia Molecular , Filogenia , Plantas Medicinales , Clasificación , Genética , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
<p><b>OBJECTIVE</b>To establish and optimize ISSR-PCR system of Dendrobiwn officinale according to the ISSR-PCR characters of D. officinale.</p><p><b>METHOD</b>The effects of ISSR-PCRs was examined by selecting primers and designing different concentrations of the factors in the ISSR-PCRs, the reliable ISSR-PCR systems for D. officinale populations researching was established by analyzing the reasons for occurrence of differential bands and optimizing reaction conditions.</p><p><b>RESULT AND CONCLUSION</b>The optimal ISSR-PCR system in D. officinale was reported for the first time, and 25 15327012 microL ISSR-PCR system contained 10 x Taq buffer, 1.5 U Taq DNA polymerase, 1.2-1.8 mmol x L(-1) MgCl2, 80 micromol x L(-1) dNTP, 0.2 micro mol x L(-1) primer and 20 ng template DNA. The appropriate annealing temperature was among 52-60 degrees C. ISSR PCRs were significantly influenced by Taq DNA polymerase, template DNA quantity and annealing temperature, etc. The ISSR-PCR systems, which were established in this paper for studying D. officinale, could provide clear reliable abundant polymorphisms molecular markers and were proved suitable for studying population authentication and population molecular ecology of D. officinale.</p>
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Dermatoglifia del ADN , Cartilla de ADN , ADN de Plantas , Genética , Dendrobium , Genética , Marcadores Genéticos , Genética de Población , Plantas Medicinales , Genética , Reacción en Cadena de la Polimerasa , Secuencias Repetitivas de Ácidos Nucleicos , Reproducibilidad de los ResultadosRESUMEN
<p><b>AIM</b>To authenticate all the varieties of Perilla (single-species genus), to analyze sequences of rDNA ITS regions and single nucleotide polymorphism (SNP) within them and based on these, to design allele-specific diagnostic PCR primers.</p><p><b>METHODS</b>The rDNA ITS regions of the perilla varieties were sequenced and analyzed by Clustal X 1.8, MEGA 3.0. Allele-specific diagnostic PCR primers that can authenticate all the perilla varieties were designed based on SNPs loci.</p><p><b>RESULTS</b>The length of rDNA ITS sequences of perilla varieties ranged from 612 to 615 bp in size, including ITS1 (230 -232 bp), 5.8S (179 bp) and ITS2 (203 -204 bp). The GC content is about 61.5% - 61.9%. There is not only SNPs in non-coding region ITS1 and ITS2 (ncSNP), but also three coding SNPs (cSNP) loci in the conservative region of 5.8S. All the SNPs have only two allele loci polymorphism. The cSNP in 5.8S is related to the morphology variation among the varieties. Allele-specific diagnostic PCR primers have been designed according to SNPs loci to authenticate accurately all the seeds and leaves of Perilla varieties.</p><p><b>CONCLUSION</b>SNPs in rDNA ITS region can be used as an effective molecular markers to authenticate all the varieties of Perilla.</p>
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Alelos , ADN de Plantas , Química , Genética , ADN Espaciador Ribosómico , Química , Genética , Marcadores Genéticos , Perilla , Clasificación , Genética , Perilla frutescens , Genética , Hojas de la Planta , Genética , Plantas Medicinales , Genética , Reacción en Cadena de la Polimerasa , Métodos , Polimorfismo de Nucleótido Simple , Semillas , Genética , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
<p><b>AIM</b>Genetic diversity, relationship and molecular authentication of total 8 wild populations of Dendrobium officinale were investigated using RAPD markers.</p><p><b>METHODS</b>10 random decamer primers were screened for Random Amplified Polymophic DNA (RAPD) fragments. A DNA molecular dendrogram was established based on cluster analysis by UPGMA (unweighted pair-group method with arithmetic average), and the relationship of the wild populations were analyzed, and all the wild populations were authenticated.</p><p><b>RESULTS</b>A total of 439 loci with an average of 43.9 loci per primer and 54.9 loci per population were amplified from 8 wild populations by 10 effective primers. In the total 104 amplified bands, 95 were polymorphic, corresponding to 91.35% genetic polymorphism. The genetic distances were 0. 590 to 0. 727, with an average of 0. 686.</p><p><b>CONCLUSION</b>Distinct genetic differences and extensive genetic diversity were presented among the wild populations. RAPD markers were an informative and useful tool for the genetic diversity, evaluation and authentication of wild populations of Dendrobium officinale. Primer S412 could be used to authenticate 8 wild populations completely.</p>
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China , Análisis por Conglomerados , Dermatoglifia del ADN , Cartilla de ADN , ADN de Plantas , Genética , Dendrobium , Genética , Ecosistema , Marcadores Genéticos , Variación Genética , Filogenia , Plantas Medicinales , Genética , Técnica del ADN Polimorfo Amplificado AleatorioRESUMEN
<p><b>AIM</b>To study the difference of rDNA ITS sequences between Zanthexylum bungeanum populations and their adulterants in main habitants of China so as to provide molecular markers for identifying Zanthexylum bungeanum populations against adulterants.</p><p><b>METHODS</b>rDNA ITS regions (including ITS-1, 5.8S and ITS-2) of 7 populations of Zanthexylum bungeanum which are separate located in Gansu, Shanxi, Sichuan, Hebei provinces, and 3 adulterants were sequenced by PCR products sequencing method or clone sequencing method.</p><p><b>RESULTS</b>The sequences of rDNA ITS region of Zanthexylum bungeanum were reported for the first time, and the sequences of ITS region ranged from 619 to 620 bp, and the length difference amoung Zanthexylum bungeanum and their adulterants is 4 bp. There are 15 variable sites, 12 informative sites and 3 authenticable sites among Zanthexylum bungeanum populations. The difference of rDNA ITS regions amoung Zanthexylum bungeanum and their adulterants is obvious, the number of variable sites is 71.</p><p><b>CONCLUSION</b>The difference of rDNA ITS sequences can be used to authenticate accurately the populations of Zanthexylum bungeanum and their adulterants. These populations of Z. bungeanum which have close relationship always distribute in near geographic areas. The characteristics of rDNA ITS sequence can be used as good markers for authenticating Zanthexylum bungeanum populations form their adulterants.</p>
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Secuencia de Bases , China , ADN de Plantas , Genética , ADN Espaciador Ribosómico , Genética , Contaminación de Medicamentos , Ecosistema , Datos de Secuencia Molecular , Filogenia , Plantas Medicinales , Genética , Análisis de Secuencia de ADN , Especificidad de la Especie , Zanthoxylum , Clasificación , GenéticaRESUMEN
<p><b>OBJECTIVE</b>To select optimal media and conditions for the tissue culture of Dendrobium lituiforum.</p><p><b>METHOD</b>The basic media, light illumination condition, phytohormone, and natural aqueous extracts. were tested and compared. The chlorophyll contents and soluble protein contents were measured.</p><p><b>RESULT</b>N6 medium was suitable for embryo germination and growth. 1/2 MS + NAA 0.5 mg x L(-1) cordd be used as the optimal protocorm multiplying media. Culture 20 days in darkness in advance, and then in light is useful to embryo germinate. 1/2 MS+ NAA 0-0.5 mg x L(-1) is beneficial to protocorm multiplication largely. N6 + 6-BA 2.0 mg x L(-1) + NAA 0.5 mg x L(-1) + 10% potato juice is useful to protocorm differentiation. Phytohormone and potato juice added to the media promoted chlorophyll content and souble protein content. N6 + NAA 0.5 mg x L(-1) + 10% banana juice is the best medium for plantlet rootage and strengthening.</p><p><b>CONCLUSION</b>Concentration of inorganic salt, nitrogen source and illumination condition are important to embryo germination and growth. The nitrogen source type has effect on the protocorm multiplication. The chlorophyll contents and souble protein contents may be index to indicate the growth condition of plantlets, which can help to select the optimal media.</p>
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Clorofila , Medios de Cultivo , Dendrobium , Química , Luz , Reguladores del Crecimiento de las Plantas , Farmacología , Proteínas de Plantas , Plantas Medicinales , Química , Técnicas de Cultivo de TejidosRESUMEN
<p><b>AIM</b>To establish the whole rDNA ITS region sequence database of various Dendrobium species of "Fengdou" and to authenticate exactly the inspected species of "Fengdou".</p><p><b>METHODS</b>The rDNA ITS regions of various Dendrobium species of "Fengdou" were amplified and sequenced. The database of their rDNA ITS regions was established in order to authenticate the inspected species by means of the softwares of CLUSTRAL and MEGA which were used to analyze the rDNA ITS region.</p><p><b>RESULTS</b>A database of the rDNA ITS sequences of 21 species of Dendrobium has been established. The notable and stable differences of the interspecies of the rDNA ITS regions have been demonstrated. The numbers of transitions and transversions among 21 species are 11-122. The variable sites are 341 while the informative sites are 195. The ITS sequence differences between the outgroup species (Pholidota yunnanensis) and species of "Fengdou" are obvious. The numbers of transitions and transversions are 131-161. The population differences of the rDNA ITS region of various species of "Fengdou" are very small (0-6).</p><p><b>CONCLUSION</b>On the basis of the database of various Dendrobium species of "Fengdou" and two genetics software, the botanical origin of the inspected species of "Fengdou" has been authenticated successfully by sequencing the rDNA ITS regions.</p>
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Secuencia de Bases , ADN de Plantas , ADN Ribosómico , Bases de Datos de Ácidos Nucleicos , Dendrobium , Clasificación , Genética , Plantas Medicinales , Genética , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
<p><b>OBJECTIVE</b>To define molecular characters to distinguish D. chrysanthum from its allied species D. primulinum, D. lituiflorum, D. aphyllum, D. crepidatum.</p><p><b>METHOD</b>The molecular characteristics of D. chrysanthum and its allied species were compared. The sequences of rDNA ITS regions were exploited to explore the evidence for authentication D. chrysanthum and its allied species.</p><p><b>RESULT</b>Although the morphological difference was slight, the sequence difference of ITS regions among five rDNAs was obvious and stable. Fifteen sites of ITS region were defined as DNA character to identify D. chrysanthum from the other four allied species.</p><p><b>CONCLUSION</b>The difference of rDNA ITS sequences can be used to authenticate accurately D. chrysanthum from three allied species of Dendrobium.</p>
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Secuencia de Bases , ADN de Plantas , Genética , Dendrobium , Clasificación , Genética , Contaminación de Medicamentos , Datos de Secuencia Molecular , Plantas Medicinales , Genética , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
<p><b>OBJECTIVE</b>To study rDNA ITS sequence differences between F type and that of H type of Dendrobium officinale in main habitat of China.</p><p><b>METHOD</b>The population differences of the rDNA ITS region (including ITS1, ITS2, 5.8S) sequences of D. officinale were studied by the method of DNA sequences analysis.</p><p><b>RESULT</b>There were two different sites between the rDNA ITS sequence of F type and that of H type. One was in ITS1 region, and the other was in 5.8S region. It was proved that there was some relativity between the character of rDNA ITS region and the life type of the populations. The phenomenon of single nucleotide polymorphism (SNP) existed in 5.8S region of rDNA ITS region between F type and H type. The sequences of rDNA ITS region of D. officinale were reported for the first time, and the sequences of ITS region ranged 634 bp (ITS1 231 bp, ITS2 240 bp, 5.8S 163 bp).</p><p><b>CONCLUSION</b>The analysis of rDNA ITS of D. officinale deeply reveal the population differences of D. officinale of F type and H type.</p>