[Evaluation of genetic diversity and population structure of Bletilla striata based on SRAP markers].

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.

Different evolutionary processes in shaping the genetic composition of Dendrobium nobile in southwest China.

Knowledge of population genetic structure and intrapopulation genetic variation is important for understanding population dynamics and evolutionary processes. Dendrobium nobile is an endangered traditional Chinese tonic medicine. In order to analyze the population differentiation and genetic diversity in D. nobile and propose proper conservation measurements, we genotyped 102 individual plants from 7 natural populations distributed across southwest China at 9 microsatellite loci. Seven pairs of genomic SSR primers were newly designed, and two pairs were chosen from the EST-SSRs. According to the results, the genetic process of D. nobile on Hainan Island and the Chinese mainland might be affected by different evolutionary processes, the genetic drift caused by founder effect has played an important role in shaping the genetic constitution of the Island population. The population of D. nobile in Hainan Island was highly differentiated and displayed low levels of genetic diversity. For the conservation management plans of D. nobile, we propose that individuals in Hainan Island with rare alleles need to be conserved with top priority, and those individuals with rare alleles and the most common alleles also should be concerned. The seven new microsatellite loci may be informative for further evaluation and conservation of the genetic diversity of D. nobile.

[DNA barcoding research and its application on medicinal plants of Bletilla H. G. Reichenbach].

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.

[Sequence analysis of LEAFY homologous gene from Dendrobium moniliforme and application for identification of medicinal Dendrobium].

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.

Genomic Sequence and Transcriptome Analysis of the Medicinal Fungus Keithomyces neogunnii.

The filamentous fungus Keithomyces neogunnii can infect the larvae of Lepidoptera (Hepialus sp.) and form an insect-fungi complex, which is utilized as an important traditional Chinese medicine. As a valuable medicinal fungus, K. neogunnii produces diverse bioactive substances (e.g., polysaccharide, vitamins, cordycepic acid, and adenosine) under cultivation conditions. Herein, we report the first high-quality genome of the K. neogunnii single-spore isolate Cg7.2a using single-molecule real-time sequencing technology in combination with Illumina sequencing. The assembled genome was 32.6 Mb in size, containing 8,641 predicted genes and having a GC content of 52.16%. RNA sequencing analysis revealed the maximum number of differentially expressed genes in the fungus during the stroma formation stage compared with those during the mycelium stage. These data are valuable to enhance our understanding of the biology, development, evolution, and physiological metabolism of K. neogunnii.

[Evaluation of genetic diversity of Dendrobium officinale wild populations based on RAMP markers].

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.

Further mapping of quantitative trait loci for female sterility in wheat (Triticum aestivum L.).

Epistasis underlying fertility plays an important role in crop breeding. Although a new female sterile mutant in wheat, XND126, has been identified and a major quantitative trait locus (QTL), taf1, for the female sterility has been mapped, the genetic architecture of the female sterility needs to be further addressed. To identify the interaction involving the gene(s) controlling the female sterility, an investigation was carried out for the seed setting ratio in an F2 population derived from the cross between XND126 and Gaocheng 8901. Among 1250 simple sequence repeat (SSR) primer pairs in the whole genome, a total of 21 markers, obtained by recessive class approach, along with other ten tightly linked markers on reference maps in wheat, were used to survey 243 F2 individuals. As a result, 28 markers were mapped into five genetic linkage groups. The performance for female sterility for each F2 individual was evaluated simultaneously at the Urumqi and Huai'an experimental stations in 2006-2007. The two phenotypic datasets along with marker information were jointly analysed in the detection of QTL using penalized maximum likelihood approach. A total of six QTLs, including two main-effect QTLs, three epistatic QTLs and one environmental interaction and accounting for 0.67-24.55% of the total phenotypic variance, were identified. All estimated effects accounted for 53.26% of the total phenotypic variation. The taf1 detected in previous study was also located on the same marker interval on chromosome 2DS. These results enrich our understanding of the genetic basis of the female sterility.

[Development of microsatellites of Dendrobium officinale and its application in purity identification of germplasm].

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.

Efficient mapping of a female sterile gene in wheat (Triticum aestivum L.).

Studies on inheritance of fertility are of great importance in wheat breeding. Although substantial progress has been achieved in molecular characterization of male sterility and fertility restoration recently, little effort has been devoted to female sterility. To identify the gene(s) controlling female sterility in wheat efficiently, an investigation was conducted for the seed setting ratio using a set of F2 populations derived from the cross between a female sterile line XND126 and an elite cultivar Gaocheng 8901. Bulked segregation analysis (BSA) method and recessive class approach were adopted to screen for SSR markers potentially linked to female fertility gene loci in 2005. Out of 1080 SSRs in wheat genome, eight markers on chromosome 2D showed a clear difference between two disparate bulks and small recombination frequency values, suggesting a strong linkage signal to the sterility gene. Based on the candidate linked markers, partial linkage maps were constructed with Mapmaker 3.0 (EXP) instead of whole genome maps, and quantitative trait locus (QTL) mapping was implemented with software QTLNetwork 2.0. A major gene locus designated as taf1, was located on chromosome 2DS. The above result was confirmed by the analysis for 2007 data, and taf1 was identified on the same chromosome 2DS with a confidence interval of 2.4 cM, which could explain 44.99% of phenotypic variation. These results provided fundamental information for fine mapping studies and laid the groundwork for wheat fertility genetic studies.

Phylogeography of the endangered orchid Dendrobium moniliforme in East Asia inferred from chloroplast DNA sequences.

The aim of the current study was to elucidate the phylogeographic history of Dendrobium moniliforme, an endangered orchid species, based on two chloroplast DNA (cpDNA) markers (trnC-petN and trnE-trnT). One hundred and thirty-five samples were collected from 18 natural populations of D. moniliforme covering the entire range of the Sino-Japanese Floristic Region (SJFR) of East Asia. A total of 35 distinct cpDNA haplotypes were identified in these populations, of which 23 haplotypes were each present in only one sample and thus restricted to a single population. The significantly larger NST value (0.586) than GST (0.328) (p < 0.05) demonstrated the presence of strong phylogeographic structure. Phylogenetic analyses indicated that all haplotypes were clustered into two lineages. The genetic diversity of D. moniliforme was high at the species level, reflected in its haplotype diversity (Hd=0.8862), nucleotide diversity (Pi=0.00361), total genetic diversity (HT=0.9011), and significant differentiation (ΦST=0.5482). Based on mismatch distribution analysis and neutrality tests, population expansion was evident in all sampled populations and also in all populations sampled in mainland China. Three refuge areas were identified, one each in southwestern China, central-southeastern China, and the CKJ (Taiwan, Japan and Korea) Islands. The results supported the hypothesis that glacial refugia were maintained on different spatial-temporal scales in the SJFR during the last glacial maximum or earlier cold periods, suggesting that Quaternary refugial isolation promoted allopatric speciation of D. moniliforme in East Asia.

Iteration expansion and regional evolution: phylogeography of Dendrobium officinale and four related taxa in southern China.

The genus Dendrobium was used as a case study to elucidate the evolutionary history of Orchidaceae in the Sino-Japanese Floristic Region (SJFR) and Southeast Asia region. These evolutionary histories remain largely unknown, including the temporal and spatial distribution of the evolutionary events. The present study used nuclear and plastid DNA to determine the phylogeography of Dendrobium officinale and four closely related taxa. Plastid DNA haplotype and nuclear data were shown to be discordant, suggesting reticulate evolution drove the species' diversification. Rapid radiation and genetic drift appeared to drive the evolution of D. tosaense and D. flexicaule, whereas introgression or hybridization might have been involved in the evolution of D. scoriarum and D. shixingense. The phylogeographical structure of D. officinale revealed that core natural distribution regions might have served as its glacial refuges. In recent years, human disturbances caused its artificial migration and population extinction. The five taxa may have originated from the Nanling Mountains and the Yungui Plateau and then migrated northward or eastward. After the initial iteration expansion, D. officinale populations appeared to experience the regional evolutionary patterns in different regions and follow the sequential or rapid decline in gene exchange.

Adverse Effects of Hydroalcoholic Extracts and the Major Components in the Stems of Impatiens balsamina L. on Caenorhabditis elegans.

Impatiens balsamina L. (Balsaminaceae), an annual herb found throughout China, has been extensively used in traditional Chinese medicine (TCM). However, our knowledge regarding the adverse effects of I. balsamina in vivo is very limited. In this present study, the nematode Caenorhabditis elegans model was employed to fully assess the adverse effects of hydroalcoholic (EtOH 55%) extracts of I. balsamina stems (HAEIBS) in vivo. After exposure to 10 mg/mL HAEIBS, the major organism-level endpoints of C. elegans of percent survival, frequency of head thrash and body bends, and reproduction had decreased by 24%, 30%, and 25%, respectively. The lifespan of C. elegans was also greatly reduced after HAEIBS exposure compared to the controls. The active compounds in HAEIBS were separated using high speed countercurrent chromatograph (HSCCC) and characterized by high performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR). Two compounds, lawsone and 2-methoxy-1,4-naphthoquinone (MNQ), and their adverse effects were then more thoroughly detailed in this study. It was found that lawsone is the major toxin in HAEIBS with a higher toxicity than MNQ in terms of negative impact on C. elegans mortality, locomotion, reproduction, and lifespan. Our data also suggests that the C. elegans model may be useful for assessing the possible toxicity of other Chinese medicines, plant extracts, and/or compounds.

[The mixed major gene plus polygenes inheritance for female fertility in wheat (Triticum aestivum L.)].

Three sets of data for the P1, P2, F1, and F2 populations derived from three crosses between the normal fertility wheat (Triticum aestivum L.) cultivars with different ecotypes and the female sterile line (XND126) were used to investigate the inheritance of female fertility in wheat using mixed major gene plus polygenes inheritance model in 2005 and 2006. The results from the joint segregation analysis of the four generations showed that female fertility in wheat is controlled by two major genes plus polygenes, and the interaction between the two major genes is also detected.

Comparative chloroplast genomes of photosynthetic orchids: insights into evolution of the Orchidaceae and development of molecular markers for phylogenetic applications.

The orchid family Orchidaceae is one of the largest angiosperm families, including many species of important economic value. While chloroplast genomes are very informative for systematics and species identification, there is very limited information available on chloroplast genomes in the Orchidaceae. Here, we report the complete chloroplast genomes of the medicinal plant Dendrobium officinale and the ornamental orchid Cypripedium macranthos, demonstrating their gene content and order and potential RNA editing sites. The chloroplast genomes of the above two species and five known photosynthetic orchids showed similarities in structure as well as gene order and content, but differences in the organization of the inverted repeat/small single-copy junction and ndh genes. The organization of the inverted repeat/small single-copy junctions in the chloroplast genomes of these orchids was classified into four types; we propose that inverted repeats flanking the small single-copy region underwent expansion or contraction among Orchidaceae. The AT-rich regions of the ycf1 gene in orchids could be linked to the recombination of inverted repeat/small single-copy junctions. Relative species in orchids displayed similar patterns of variation in ndh gene contents. Furthermore, fifteen highly divergent protein-coding genes were identified, which are useful for phylogenetic analyses in orchids. To test the efficiency of these genes serving as markers in phylogenetic analyses, coding regions of four genes (accD, ccsA, matK, and ycf1) were used as a case study to construct phylogenetic trees in the subfamily Epidendroideae. High support was obtained for placement of previously unlocated subtribes Collabiinae and Dendrobiinae in the subfamily Epidendroideae. Our findings expand understanding of the diversity of orchid chloroplast genomes and provide a reference for study of the molecular systematics of this family.