Development and application of chloroplast molecular markers in Panax notoginseng / 中国中药杂志

The molecular markers(cpSSR, cpSNP and cpIndel) were developed based on the whole genome sequence of Panax notoginseng chloroplast genome, which provide a powerful tool for the evaluation and analysis of the future P. notoginseng germplasm resources. The 89 P. notoginseng samples from 9 groups were used for the experiment, and the data for the study were derived from NCBI and the GenBank numbers were: KJ566590, KP036468, KR021381 and KT001509. Through sequence alignment, 30 polymorphic sites(SNP and Indel) were identified, including 16 cpSNP and 14 cpIndel; cpSNP and cpIndel accounted for far more than the gene region in the intergenic region. The developed cpSSR reached 87-89, the repeat unit was mainly composed of trinucleotide, accounting for 70%-71%, and the dinucleotide was the least, accounting for 7%. Eighteen cpDNA molecular markers were developed, including 7 cpSSR primers, 6 cpIndel primers, and 5 cpSNP primers. The MatK gene and ycf1 primers were chosen as control. According to the results of DNA gel electrophoresis, cpSSR-5, pgcpir019 and pncp08 can be used to distinguish different cultivated populations of P. notoginseng. Among them, cpSSR-5 and pgcpir019 can also be used to distinguish the inter-species resources of ginseng by comprehensive sequence length, population π value and average nucleotide difference. However, pncp08 can only be used to distinguish different populations of P. notoginseng. In addition, the effect of distinguishing the groups of P. notoginseng, which the primer pncp-M(based on the MatK gene) is weaker than the cpSSR-5, pgcpir019 and pncp08.

Phylogeny and circumscription of the genus Orinus (Poaceae: Eragrostideae): evidence from NRDNA its sequences / Filogenia ITS e a circunscrição do gênero Orinus (Poaceae: Eragrostideae): evidência das sequências its do NRDNA

In this study, ITS sequences of the nuclear ribosomal DNA were conducted for six putative species of Orinus (O. alticulmus, O. anomala, O. kokonorica, O. longiglumis, O. thoroldii and O. tibeticus) with 572 individuals from 73 populations. The results found that the six species formed two monophyletic groups: the one (O. anomala Keng ex Keng f. and L. Liou) with O. alticulmus, O. anomala and O. kokonorica, and another (O. thoroldii (Stapfex Hemsl.) Bor) with O. longiglumis, O. thoroldii and O. tibeticus. The taxonomic data from ITS sequences were not congruent with those from morphological characteristics, likely resulting from rapid speciation trigged by the uplifts of the Qinghai-Tibetan Plateau and its adjacent regions and the extensive selection pressure under the alpine environments. The ITS data suggest that the classification of the six species within the genus Orinus should be reduced to two species. We include a complete taxonomic revision of the genus and a key to distinguish the two species. Therefore, it is very necessary to make the comprehensive revision and arrangement of these taxa, strictly regulate the taxa confusion of genus and species order, and do new reports on belonging to the genus taxa.

Neste estudo, sequências ITS do DNA ribossômico nuclear foram conduzidas para seis espécies putativas de Orinus (O. alticulmus, O. anomala, O. kokonorica, O. longiglumis, O. thoroldii and O. tibeticus) com 572 indivíduos de 73 populações. Os resultados revelaram que as seis espécies formaram dois grupos monofiléticos: o primeiro (O. anomala Keng ex Keng f. and L. Liou) com O. alticulmus, O. anomala and O. kokonorica, e o segundo (O. thoroldii (Stapfex Hemsl.) Bor) com O. longiglumis, O. thoroldii and O. tibeticus. Os dados taxonômicos das sequências ITS não foram congruentes com aqueles das características morfológicas, provavelmente resultantes da rápida especiação provocada pelas elevações do Planalto do Tibete e das suas regiões adjacentes e da grande pressão de seleção nos ambientes alpinos. Os dados ITS sugerem que a classificação das seis espécies dentro do gênero Orinus deveriam ser reduzidas para duas espécies. Nós incluímos uma revisão taxonômica completa do gênero e uma chave para distinguir as duas espécies. Portanto, é absolutamente necessário fazer uma revisão abrangente e um arranjo destes táxons, regular estritamente a confusão de táxons de gêneros e ordem de espécies, e fazer novos relatórios sobre pertencer aos táxons de gêneros.

Identification of medicinal plant Dendrobium based on the chloroplast psbK-psbI intergenic spacer / 药学学报

In this paper, the chloroplast psbK-psbI intergenic spacers of 18 species of Dendrobium and their adulterants were amplified and sequenced, and then the sequence characteristics were analyzed. The sequence lengths of chloroplast psbK-psbI regions of Dendrobium ranged from 474 to 513 bp and the GC contents were 25.4%-27.6%. The variable sites were 71 while the informative sites were 46. The inter-specific genetic distances calculated by Kimura 2-parameter (K2P) of Dendrobium were 0.006 1-0.058 1, with an average of 0.028 4. The K2P genetic distances between Dendrobium species and Bulbophyllum odoratissimum were 0.093 2-0.120 4. The NJ tree showed that the Dendrobium species can be easily differentiated from each other and 6 samples of the inspected Dendrobium species were identified successfully through sequencing the psbK-psbI intergenic spacer. Therefore, the chloroplast psbK-psbI intergenic spacer can be used as a candidate marker to identify Dendrobium species and its adulterants.

Molecular identification of raw materials from lian qiao bai du wan / 药学学报

Lian Qiao Bai Du Wan was used to study the identification of Chinese patent medicine by molecular marker technique. DNA was extracted through modified CTAB method. The psbA-trnH and rbcL sequences were gradient amplified, and PCR products were ligated with the pEASY-T5 vector and then transformed into Trans1-T1 cells, respectively. Clones were selected randomly and sequenced. All sequences were analyzed by BlastN and the neighbor-joining (NJ) phylogenetic tree was constructed by MEGA 4.0. The results showed that nine kinds of medicinal materials can be identified by psbA-trnH sequences, and six kinds of medicinal materials by rbcL sequences from Lian Qiao Bai Du Wan. Molecular marker technique can stably and accurately distinguish multi-origin medicinal materials in Chinese patent medicine.

High-throughput pyrosequencing of the complete chloroplast genome of Magnolia officinalis and its application in species identification / 药学学报

Chloroplast genome sequences have comprehensive application prospects in DNA barcoding and chloroplast engineering in traditional Chinese medicine. The complete chloroplast genome of Magnolia officinalis sequenced by high-throughput pyrosequencing and a sequencing procedure was established. Fourteen contigs were obtained after de nove assembly. The sequencing percent of coverage was 99.99%. The chloroplast genome is 160 183 bp in size, and has a typical quadripartite structure with the large (LSC, 88 210 bp) and small copy (SSC, 18 843 bp) regions separated by two copies of an inverted repeat (IRs, 26 565 bp each). chloroplast genes were successfully annotated, of which 17 genes located in each IR region. The chloroplast genome features in Magnolia officinalis are nearly identical to those from other Magnoliid chloroplast genomes. Phylogenetic analyses were performed based on 81 shared coding-genes for a total of 9 Magnolia samples of 5 closely related species. Results showed that distinguishing among species was generally straightforward at the species and population level. This study confirmed the effectiveness of our chloroplast genome sequencing procedure. The chloroplast genome can provide distinguishing differences to help identify Magnolia officinalis and its closely related plants.

Analysis on chloroplast DNA sequences of Polygonum capitatum of different geographical population / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the relationship between the variation of chloroplast DNA gene sequences and the geographical origins of Polygonum capitatum in order to provide the molecular evidence for its excellent germplasm resources.</p><p><b>METHOD</b>PCR direct sequencing was applied to detect the chloroplast psbA-trnH, trnL-trnF gene sequence of 11 samples collected from 11 populations of P. capitatum.</p><p><b>RESULT</b>The psbA-trnH gene sequence of P. capitatum from different populations was 402 bp in length, there were 6 variable sites. TrnL-F gene sequence was 875 bp, there were 5 variable sites. The clusters diagram by UPGMA method showed that P. capitatum groups in Yunnan and Guizhou existed a considerable variation.</p><p><b>CONCLUSION</b>P. capitaturni which is located in the east of Yunnan and the west of Guizhou is helpful of screening the germplasm resources.</p>

DNA barcoding the medicinal plants of the genus Paris / 药学学报

DNA barcoding is a technique in which species identification and discovery are performed by using short and standard fragments of DNA sequences. In this study, eleven species of Paris, including seven varieties, were sampled. Five chloroplast sequences, psbA-trnH, rpoB, rpoC1, rbcL, matK, and one nuclear marker, the second internal transcribed spacer (ITS2) of ribosomal DNA, were amplified and sequenced. The PCR amplification and sequencing efficiency, intra- and inter-specific divergence and barcoding gap were used to evaluate different loci, and the identification efficiency was assessed using BLAST1 and Nearest Distance methods. The ITS2 sequences in the studied samples of Paris were amplified and sequenced successfully using primers designed by our group, while matK showed low level in the amplification and psbA-trnH was difficult for sequencing because of over 800 bp and poly (A) structure. Analysis of the intra- and inter-specific divergence and barcoding gap showed ITS2 was superior to other loci. The ITS2 showed a much higher percentage of success (100%) in identification than other five loci, none of which indicated more than 50% except matK (52.9%). The 2-locus combination of rbcL+matK didn't improve ability of authentication. In addition, the rate of successful identification with ITS2 kept 100% when the samples were expanded to 67 samples of 29 species. In conclusion, ITS2 can be used to correctly identify medicinal plants of Paris, and it will be a potential DNA barcode for identifying medicinal plants of other taxa.

Authentication of Curcuma species (Zingiberaceae) based on nuclear 18S rDNA and plastid trnK sequences / 药学学报

Curcuma drugs have been used discriminatingly for invigorating blood circulation, promoting digestion, and as a cholagogic in China. However, there is confusion about the drug's botanical origins and clinical uses because of morphological similarity of Curcuma plants and drugs. Comparative sequencing of the 18S rRNA gene in nuclear ribosomal DNA (rDNA) and trnK gene in chloroplast DNA (cpDNA) was carried out in order to examine interspecies phylogeny and to identify ultimately Curcuma species. A total of a hundred of accessions of eighteen species were analyzed. This resulted in an aligned matrix of 1810 bp for 18S rDNA and 2 800 bp for trnK. 18S rDNA sequence divergence within the ingroup ranged from 0-0.05%, trnK ranged from 0-0.19%. One base transversion-substituted site (from cytosine to thymine) was observed from the upstream of 18S rDNA at nucleotide position 234 in C. kwangsiensis and Japanese population of C. zedoaria which have separated genetic distance to other Curcuma taxa. Two noncoding regions embedded in trnK intron showed higher variability, including nucleotide substitutions, repeat insertion and deletions. Based on consensus of relationship, eighteen major lineages within Curcuma are recognized at the species level. The results suggest that Curcuma is monophyletic with 100% bootstrap support and sister to the genera Hedychium and Zingiber. The trnK sequences showed considerable variations between Curcuma species and thus were revealed as a promising candidate for barcoding of Curcuma species, which provide valuable characters for inferring relationship within species but are insufficient to resolve relationships among closely related taxa.

Analysis and authentication of cpDNA psbA-trnH regions of Dendrobium species of fengdous / 药学学报

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.

Analysis and authentication of chloroplast matK gene sequences of Herba Dendrobii / 药学学报

To compare the characteristic of chloroplast matK gene sequences of different Herba Dendrobium species and to authenticate inspected species, the matK gene sequences of 12 species (including 22 materials) and outgroup were amplified, cloned, and sequenced. Genomic DNA of Dendrobium plants was extracted using modified cetyltrimethyl ammonium bromide (CTAB) method. The matK gene sequences were about 1 410 bp in length. The variable sites were 51 while the parsim-informative sites were 11. There were nucleotides insertions and deletions in some species, in addition to transitions and transversions, such as in D. denneanum and D.chrysotoxum. Interspecies and different populations (varieties) of Dendrobium could be distinguished on phylogeny tree. The average genetic distance was 0.008, and the maximal and minimal genetic distances between Dendrobium species were 0.014 and 0.003, respectively. There were 8-20 variable sites between Dendrobium species. The genetic distance between populations (varieties) was 0.001, and there were 1-5 variable sites. Moreover, the 4 inspected materials were successfully authenticated. The database of chloroplast matK gene sequences of 12 species of Herba Dendrobii and inspected species could be used for the molecular authentication between Dendrobium species and populations. The matK gene sequence could be used as molecular maker for authentication of Herba Dendrobium.

Identification of Gynostemma from their adulterant Cayratia japonica by PCR-RFLP / 中国中药杂志

<p><b>OBJECTIVE</b>To establish a convenient and effective method for the identification of Gynostemma and Cayratia japonica.</p><p><b>METHOD</b>Eight species, including Gynostemm pentaphyllum, G. pentagynum, G. cardiospermum, G. longipe, G. yixingense, G. laxiflorum, G. guangxiense and C. japonica were investigated through PCR - RFLP of six chloroplast DNA fragments. The six gene fragments were digested by six restriction endonuclease respectively, including Taq I, Hpa II, EcoR I, Rsa I, Hha I, Hind III.</p><p><b>RESULT</b>Seven species of Gynostemma and their adulterant could be identified by trnK1f-trnK2r and Rsa.</p><p><b>CONCLUSION</b>PCR - RFLP provides a quick, reliable molecular marker technique for identification of Cynostemma and their adulterant Cayratia japonica.</p>

Phylogeny relationship and molecular identification of ten Huperzia species (Huperziaceae) based on matK gene sequences / 中国中药杂志

<p><b>OBJECTIVE</b>To study the phylogeny relationship and molecular identification of 10 species from Huperzia (Huperziaceae) based on matK gene sequences data.</p><p><b>METHOD</b>Total DNA of nine species from Huperzia was extracted; matK gene sequence was amplified by PCR. PCR product was directly sequenced after purification.</p><p><b>RESULT</b>The chloroplast matK gene nucleotide sequences from 9 species of Huperzia species were sequenced. The matK gene nucleotide sequences length was 1 589 bp. Analysis with Huperzia lucidula matK gene nucleotide sequences (download from GenBank) and taking Lycopodiella cernua as outgroup, Maximum Parsimony, Neighbor-Joining analyses and genetic distances were conducted using MEGA 3.1 software. 35 variable sites and 35 parsimony informative sites have been found. Pairwise genetic distances among 10 species of Huperzia was 1.59% - 0.25%.</p><p><b>CONCLUSION</b>The results were consistent with the taxonomy in morphological of Huperzia. But H. longipetiolata and H. serrata were resolved into in different clade. There are 19 different sites of matK gene sequences between H. longipetiolata and H. serrata, the genetic distances is 0.121%. It is suggested H. longipetiolata should be as an independent species.</p>

Genetic analysis of some pepper [capsicum annuum L.] genotypes using three DNA-based markers

RAPD-PCR RFLP of the 18S r-RNA gene and chloroplast DNA polymorphisms were employed to estimate the genetic similarity among five different genotypes of pepper [Capsicum ammum L.]. RAPD analysis was performed using ten random primers. The genetic similarity was estimated as band sharing [BS] for each primer between the genotypes [B 1, B2, K9, AC and F[1], of the last two]. The results showed the highest genetic similarity of 90.0% between genotypes B2 and AC. This high numeric similarity suggested a common lineage or a very little genetic variation existing between these two genotypes. The lowest genetic similarity of 74.0% was detected between genotypes B1 and F[1]. Two pairs of primers were used to amplify 18 S-RNA gene and chloroplast DNA. Both the restriction fragment patterns of the 18S r-RNA gene using four restriction enzymes viz. BamH I, EcoR I, Hind III and Hinf I; and the patterns of chloroplast DNA did not reveal any polymorphism. The obtained data demonstrated the usefulness of molecular analyses in the detection of genetic relationships and evaluating the relative effectiveness of the different types of DNA-based markers in revealing variation among Egyptian and foreign pepper genotypes

Cucumber chloroplast trnL(CAA) gene: nucleotide sequence and in vivo expression analysis in etiolated cucumber seedlings treated with benzyladenine and light.

The nucleotide sequence of a 714 bp BamHI-EcoRI fragment of cucumber chloroplast DNA was determined. The fragment contained a gene for tRNA(Leu) together with its flanking regions. The trnL(CAA) gene sequence is about 99% in similarity to broad bean, cauliflower, maize, spinach and tobacco corresponding genes. The relative expression level of the gene was determined by Northern (tRNA) gel blot and Northern (total cellular RNA) slot-blot analyses using the trnL gene probe in 6-day old etiolated cucumber seedlings and the seedlings that had been kept in the dark (dark-grown), treated with benzyladenine (BA) and kept in the dark (BA-treated dark-grown), illuminated (light-grown), and treated with BA and illuminated (BA-treated light-grown), for additional 4, 8 or 12 hr. The trnL transcripts and tRNA(Leu) levels in BA-treated dark-grown seedlings were 5 and 3 times higher, respectively after 4 hr BA treatment, while in the BA treated light-grown seedlings the level of trnL transcripts was only 3 times higher and had no detectable effect on mature tRNA(Leu) when compared to the time-4 hr dark-grown seedlings. However, the level of mature tRNA(Leu) did not show marked changes in the light-grown seedlings, whereas the level of trnL transcripts increases 3 times after 8 hr illumination of dark-grown seedlings. These data indicate that both light and cytokinin can signal changes in plastid tRNA gene expression. The possible regulatory mechanisms for such changes are discussed.