The present study aimed to explore the correlation between agronomic traits and quality indexes of Dendrobium nobile and its application value in agricultural breeding. The cultivated strains of D. nobile in Hejiang-Chishui producing areas were extensively collected,and the main agronomic traits and quality indexes were measured. The agronomic traits with significant correlation with quality indexes were screened out by the correlation analysis,and then the parental lines and self-bred F_1 generation plants were furtherverified. Among 96 lines of D. nobile,the content of soluble polysaccharides showed a significant negative correlation with dendrobine( P < 0. 01),and no significant correlation with agronomic traits in stems and leaves. The content of dendrobine exhibited a significant positive correlation with the stem width-thickness ratio( at the largest cross section; P < 0. 01),and no significant correlation with other agronomic traits. Regression analysis further verified the positive correlation between dendrobine content and stem width-thickness ratio( R2> 0. 9). Two lines,JC-10 and JC-35,with significant differences in stem width-thickness ratio were screened out( P <0. 05). The corresponding F1 generation plants by self-pollination both showed that the dendrobine content was higher with greater stem width-thickness ratio( P < 0. 01). The experimental results suggested that within a certain range,the dendrobine content was higher in D. nobile with flatter stem. Therefore,in the breeding of D. nobile,this specific trait could be used for screening plants with high content of quality indexes such as dendrobine.
Agriculture ; Dendrobium/genetics* ; Plant Breeding ; Plant Leaves/genetics* ; Polysaccharides

In this study, the roots, stems and leaves of diploid and autotetraploid Dendrobium huoshanense were used as materials to compare their contents of polysaccharides and alkaloids, and the transcriptome sequencing analysis was carried out. The results showed that the contents of polysaccharides and alkaloids in the roots, stems and leaves of tetraploid were 7.6%, 34.5%, 17.2%, 0.01%, 0.024% and 0.035% higher than those of diploid D. huoshanense, respectively. The contents of active components in different tissues were significantly different. There were 3 687 differentially expressed genes in diploid and tetraploid D. huoshanense, of which 2 346 genes were up-regulated and 1 341 down regulated. Go functional analysis showed that these genes were mainly involved in growth and development, stress resistance and other related functions. KEGG pathway analysis showed that most of the differential genes were concentrated in the processes of carbon metabolism, signal transduction, carbohydrate metabolism, amino acid metabolism and energy metabolism. The differential expression of key genes involved in the metabolism of polysaccharides, terpenes and polyketones, amino acid metabolism, hormone synthesis and signal transduction in diploid and tetraploid plants may be the main reason for the high energy content, the increase of active components and the growth potential of tetraploid plants.
Alkaloids ; Dendrobium/genetics* ; Diploidy ; Plant Roots ; Polysaccharides ; Transcriptome

WRKY transcription factors are novel transcriptional regulatory factors, which play an important role in regulating plant development, metabolism and other physiological processes. In this study, a new Dendrobium officinale WRKY transcription factor, designated as DoWRKY1 was cloned by using RT-PCR and RACE (GenBank Accession No. KF953910). Bioinformatic analysis demonstrated that, the full-length cDNA of DoWRKY1 was 1,704 bp. And DoWRKY1 contained a 1,629 bp open reading frame (ORF) that encoding a peptide of 542 amino acid residues. The putative DoWRKY1 protein contained two conserved WRKY domains and it belonged to the group I WRKY family protein. Yeast one-hybrid experiment showed that DoWRKY1 had transcriptional activation ability in yeast, and it could activate the expression of downstream report genes (His3 and Ade2). Semi-quantitative RT-PCR experiment showed that DoWRKY1 expressed in roots, stems, leaves and protocorm-like bodies. Real-time qRT-PCR proved that DoWRKY1 could be induced by methyl jasmonate (MeJA) and chitosan (Chitosan), and the expression level of this gene can reach the expression peak at 2 h and 1 h, respectively. These results are useful for further determination of the regulation function of this gene in secondary metabolism of D. officinale.
Cloning, Molecular ; Dendrobium ; genetics ; Gene Expression Regulation, Plant ; Plant Proteins ; genetics ; Transcription Factors ; genetics

Isolation of high-quality genomic DNA from dried and processed crude drug is the key for the DNA identification of Dendrobii Caulis. The DNA extract of Dendrobii Caulis was firstly compared using different method to isolate genomic DNA from dried and processed crude drug, including commercial DNA extracted kit and CTAB method. Using modified CTAB method (extracted from large samples), the genomic DNA was successfully isolated from Dendrobii Caulis, including Huangcao and Fengdou. The ITS regions were amplified using the purified DNA as template, and then cloned and sequenced. These ITS sequences were compared with data from Genbank database and our lab, 14 Dendrobium species and five similar species were identified from "Huangcao" and "Huangcao" slice, while six species and three similar species from "Fengdou" according to their sequence similarity. The study demonstrated that the dried Dendrobii Caulis could be identified using DNA molecular method, which could overcome deficiencies and limitations of traditional identification method and has a certain application prospects.
DNA, Intergenic ; genetics ; DNA, Plant ; genetics ; Dendrobium ; classification ; genetics ; Sequence Analysis, DNA

In order to understand the function of GDP-mannose pyrophosphorylase(GMPP) function and its regulation in polysaccharide biosynthesis mechanism in Dendrobium. D. huoshanense was used to clone GMPP gene. GMPP gene expression in D. huoshanense,D. officinale and D. moniliforme was also determined by qPCR. The results showed that the length of D. huoshanense GMPP gene c DNA sequence is 1 867 bp,containing 1 245 bp open reading frame(ORF),encoding 415 amino acids. Phylogenetic tree analysis showed that D. huoshanense,D. officinale and D. moniliforme are closely related with GMPP taken into consideration. Bioinformatics analysis demonstrated that GMPP sequence similarity among the three species reached as high as 99%. qPCR results indicated that GMPP genes was highly expressed in stem of D. huoshanense compared with its leaf,flower and root. According to GMPP gene expression profile in D. huoshanense,D. officinale and D. moniliforme grown in Huoshan area,it was clear that GMPP in D. huoshanense showed the highest expression level. Furthermore,our findings of GMPP gene expression profile will facilitate future researches into its polysaccharide biosynthetic mechanism.
Base Sequence ; Cloning, Molecular ; Dendrobium ; enzymology ; genetics ; Nucleotidyltransferases ; genetics ; Phylogeny ; Plant Proteins ; genetics ; Polysaccharides ; biosynthesis

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.
Chloroplasts ; DNA, Chloroplast ; genetics ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Dendrobium ; classification ; genetics ; Plants, Medicinal ; classification ; genetics

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.
DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Dendrobium ; classification ; genetics ; Genes, Chloroplast ; Genes, Plant ; Introns ; Plants, Medicinal ; classification ; genetics

To identify Herba Dendrobii and its adulterant species on molecular level, the rDNA ITS sequences of 17 species of Herba Dendrobii were studied. Genomic DNA of Dendrobium was extracted using the modified cetyltrimethyl ammonium bromide (CTAB) method. The PCR products of the rDNA ITS sequences of Dendrobium (32 materials) were purified and then sequenced. The characteristic of the sequences and the genetic distance were compared between Bulbophyllum odoratissimum and Dendrobium, Dendrobium interspecies and different populations. Phylogenetic trees were constructed using the UPGMA method by the biology softwares including BioEdit, MEGA4.0 etc. The PCR products were purified and then sequenced. It was built up that the database of rDNA ITS sequences of 17 species of Herba Dendrobii (32 materials). The ITS1 was 228-234 bp, the GC content accounting for 45.7%-53.0%. Its variable sites were 167, accounting for 67.34%. The Parsim-Informative positions were 106, accounting for 42.74%. The ITS2 was 241-247 bp, the GC accounting for 44.8% - 55.7%. The variable sites were 165, accounting for 66.27%. The Parsim-Informative positions were 115, accounting for 46.18%. The genetic distance between B. odoratissimum and Dendrobium was 0.295. The average genetic distance was 0.142 between Dendrobium species, and there were 2-156 variable nucleotides. The average genetic distance between different populations was 0.002, and there were 2-156 variable nucleotides. The genetic distance between B. odoratissimum and Dendrobium was greater than that of Denrobium interspecies. Meanwhile, the genetic distance between Denrobium species was also greater than that of different populations (varieties). The molecular phylogeny tree was constructed on the database of rDNA ITS the sequences of 17 species of Herba Dendrobii using the biology softwares. Then 10 materials on molecular level were authenticated. It is concluded that using of the whole sequences database of 17 species of Herba Dendrobii and heredity analysis softwares, and measuring the sequences of rDNA ITS of the inspected species, can authenticate the Dendrobium on molecular level, and provide basis for molecular authentication.
China ; DNA, Plant ; genetics ; DNA, Ribosomal ; genetics ; DNA, Ribosomal Spacer ; genetics ; Dendrobium ; classification ; genetics ; Molecular Sequence Data ; Phylogeny

Using phenol-sulfuric acid method and hot-dip method of alcohol-soluble extracts, the contents of polysaccharides and alcohol-soluble extracts in 11 F1 generations of Dendrobium officinale were determined. The results showed that the polysaccharides contents in samples collected in May and February were 32.89%-43.07% and 25.77%-35.25%, respectively, while the extracts contents were 2.81%-4.85% and 7.90%-17.40%, respectively. They were significantly different among families. The content of polysaccharides in offspring could be significantly improved by hybridization between parents with low and high polysaccharides contents, and the hybrid vigor was obvious. Cross breeding was an effective way for breeding new varieties with higher polysaccharides contents. Harvest time would significantly affect the contents of polysaccharides and alcohol-soluble extracts. The contents of polysaccharides in families collected in May were higher than those of polysaccharides in families collected in February, but the extracts content had the opposite variation. The extents of quantitative variation of polysaccharides and alcohol-soluble extracts were different among families, and each family had its own rules. It would be significant in giving full play to their role as the excellent varieties and increasing effectiveness by studying on the quantitative accumulation regularity of polysaccharides and alcohol-soluble extracts in superior families (varieties) of D. officinale to determine the best harvesting time.
Breeding ; China ; Dendrobium ; chemistry ; classification ; genetics ; Plant Extracts ; chemistry ; isolation & purification ; Polysaccharides ; chemistry ; isolation & purification

OBJECTIVETo investigate the genetic diversity and structure of Dendrobium huoshanense, a (CT)n enriched microsatellite library was constructed using a magnetic beads enrichment procedure.

METHODThe 3'-biotinylated oligonucleotide probe was used to hybridize with the digested D. huoshanense genomic DNA fragments whose both ends were ligated with adaptors. The hybridized complex was then combined with the streptavidin-coated magnetic beads. The captured microsatellite fragments were eluted, collected and cloned into pMD19-T vector. The recombinant plasmids were transformed into Escherichia coli DH5alpha competent cells. The clones that yielded two or more bands contained microsatellite fractions. Positive clones were screened and sequenced. Thirty pairs of primers were designed and synthesized. Polymorphism at each locus was determined using 24 individuals from a natural population from Huoshan county town in Anhui province.

RESULTTwelve polymorphic microsatellite loci from the microsatellite-enriched genomic library were newly developed across 24 D. huoshanense individuals. In total, 65 alleles were identified, and the number of alleles per locus ranged from 2 to 8. The mean observed and expected heterozygosities were 0.500 and 0.638, respectively. Two loci significantly deviated from Hardy-Weinberg equilibrium (P<0.05), which could be due to the presence of null alleles. Furthermore, three of twelve loci showed significant linkage disequilibrium (P<0.05).

CONCLUSIONThese results suggest that the identified polymorphic microsatellite markers will be useful in population genetic studies of D. huoshanense.