The novel developed microsatellite markers revealed potential hybridization among Cymbidium species and the interspecies sub-division of C. goeringii and C. ensifolium.

BACKGROUND: Orchids (Cymbidium spp.) exhibit significant variations in floral morphology, pollinator relations, and ecological habitats. Due to their exceptional economic and ornamental value, Cymbidium spp. have been commercially cultivated for centuries. SSR markers are extensively used genetic tools for biology identification and population genetics analysis. RESULT: In this study, nine polymorphic EST-SSR loci were isolated from Cymbidium goeringii using RNA-Seq technology. All nine SSR loci showed transferability in seven other congeneric species, including 51 cultivars. The novel SSR markers detected inter-species gene flow among the Cymbidium species and intra-species sub-division of C. goeringii and C. ensifolium, as revealed by neighborhood-joining and Structure clustering analyses. CONCLUSION: In this study, we developed nine microsatellites using RNA-Seq technology. These SSR markers aided in detecting potential gene flow among Cymbidium species and identified the intra-species sub-division of C. goeringii and C. ensifolium.

Molecular quantification for differentiation of fresh and dried Jinqian Baihua She.

Freshly-used crude drugs have unique functions and advantages in TCM practice of treating diseases. Jinlong Capsule is a patent traditional Chinese medicine product effective for treatment of hepatocarcinoma, and fresh Jinqian Baihua She (JBS, the body of juvenile Bungarus multicinctus) is one of its important ingredients. The emergence of counterfeit fresh JBS, often identified as dried JBS with almost identical appearance, poses a difficult problem in the quality control of the product. Herein we report a molecular quantification-based method for differentiation of fresh and dried JBS by determining the copy number of a specific DNA marker in the samples. Using species-specific primers and TaqMan probes, we established a real-time quantitative PCR system for amplification of a fragment in the 658-bp cytochrome oxidase subunit I (COI) region from JBS specimens. The amplicon copy number in the muscle tissues ranged from 1.14 × 107 to 4.83 × 107 copies/mg in fresh JBS samples, as compared with 1.13 × 105-8.91 × 106 copies/mg in dried JBS samples. Based upon Fisher discriminant analysis, we used 1.27 × 107 copies/mg as the cut-off value for differentiating fresh and dried JBS, which was validated in the single-blinded validation test of fresh and dried JBS samples. This qPCR system may provide an efficient means for accurate identification of fresh JBS to improve the quality control of the medicinal product.

Development of a Competitive Time-Resolved Fluoroimmunoassay for Paclitaxel.

INTRODUCTION: Paclitaxel (Tax) is a diterpene alkaloid isolated from Taxus species and has proved clinically effective in treating a number of malignancies. Current quantitative analytical methods for Tax such as high-performance liquid chromatography (HPLC) often involve complicated sample preparation procedures with low recovery rates. OBJECTIVE: To establish a rapid and sensitive time-resolved fluoroimmunoassay (TRFIA) for measuring Tax in Taxus materials with convenient sample preparation and a high recovery rate. METHODS: Rabbit anti-mouse IgG was coated onto a 96-well microplate, which was then incubated with standard solutions of Tax and anti-Tax monoclonal antibody 3A3. A Eu3+ -labelled conjugate of Tax and human serum albumin was used as the tracer. The luminescent system was enhanced with a solution containing 2-naphthoyltrifluoroacetone. RESULTS: The established TRFIA showed a linear response within the Tax concentration range of 3.2 to 80 ng/mL, with a limit of detection of 1.4 ng/mL. The intra- and inter-assay coefficient of variation of the assay was 9.6% and 9.7%, respectively, with an average recovery rate from spiked samples of 108.5%. Tax contents in Taxus samples were determined using both the established TRFIA system and a previously established enzyme-linked immunosorbent (ELISA), and the results of two assays were well correlated. CONCLUSION: This TRFIA system shows a high sensitivity, precision and accuracy for detection of Tax. This assay, which is convenient and less time-consuming, allows rapid analysis of Tax and provides another option for Tax measurement for quality control of Taxus materials and products. Copyright © 2017 John Wiley & Sons, Ltd.

[Identification of Original Species of Fish Maw by DNA Barcoding].

Objective: To identify the original species of fish maw sold in Guangzhou market by DNA barcoding technology. Methods: Mitochondrial cytochrome C subunit I (CO I) gene fragment of eleven fish maw samples were amplified and sequenced with the self-designed primers. UPGMA phylogenetic tree were constructed for clustering analysis. The species origin of each sample was identified with the identification engine provided in the Barcode of Life Data Systems (BOLD). Results: The self-designed primers were effective in fish maw CO I amplification and sequencing, with success rates both of 100%. BOLD identification and UPGMA clustering analysis indicated the fish maw samples were derived from five fish species of three families. Conclusion: DNA barcoding combined with BOLD identification system can accurately identify the species origin of commercial fish maw.

Advances in research of the artificial cultivation of Ophiocordyceps sinensis in China.

Ophiocordyceps sinensis (syn. Cordyceps sinensis), a traditional Chinese medicine called DongChongXiaCao (DCXC) in Chinese, is well known and has been used in Asia countries since the fifteenth century, and it contains some valuable medicinal component defined by modern pharmacological science. DCXC only appears at high altitudes on the Qinghai-Tibetan Plateau. Consequently, it is difficult to find and harvest. Because of its rarity and medicinal value, DCXC has always been one of the most expensive medicines known. As the price of DCXC has risen in recent years, thousands of migrants have entered into the various grasslands to search for them in season, which makes ecological environments of the grassland more fragile. In order to relieve the environmental pressures and protect this valuable resource, the artificial cultivation of DCXC involving two aspects of the genus Hepialus and the fungi of the host larvae should be employed and applied at the first available time point. In this article, the reproduction of moth larvae of the genus Hepialus is first described, which includes their ecological characteristics and the methods of artificial feeding. Second, the generation and isolation method of the fungi from DCXC are subsequently summarized, and then the mechanism of fungal spores to attack the moth larvae are restated. Finally, the basic model of artificial cultivation of DCXC is introduced; meanwhile, the potential application of modern biotechnology to the artificial cultivation is analyzed in prospect. This review article will not only expand people's knowledge regarding the artificial cultivation of DCXC, but also hopefully provide an informative reference for the development of this valuable resource and the environmental protection of alpine meadows.

Permanent genetic resources added to Molecular Ecology Resources Database 1 August 2012 - 30 September 2012.

This article documents the addition of 83 microsatellite marker loci and 96 pairs of single-nucleotide polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Bembidion lampros, Inimicus japonicus, Lymnaea stagnalis, Panopea abbreviata, Pentadesma butyracea, Sycoscapter hirticola and Thanatephorus cucumeris (anamorph: Rhizoctonia solani). These loci were cross-tested on the following species: Pentadesma grandifolia and Pentadesma reyndersii. This article also documents the addition of 96 sequencing primer pairs and 88 allele-specific primers or probes for Plutella xylostella.

Permanent genetic resources added to molecular ecology resources database 1 April 2012 - 31 May 2012.

This article documents the addition of 123 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Brenthis ino, Cichla orinocensis, Cichla temensis, Epinephelus striatus, Gobio gobio, Liocarcinus depurator, Macrolophus pygmaeus, Monilinia vaccinii-corymbosi, Pelochelys cantorii, Philotrypesis josephi, Romanogobio vladykovi, Takydromus luyeanus and Takydromus viridipunctatus. These loci were cross-tested on the following species: Cichla intermedia, Cichla ocellaris, Cichla pinima, Epinephelus acanthistius, Gobio carpathicus, Gobio obtusirostris, Gobio sp. 1, Gobio volgensis, Macrolophus costalis, Macrolophus melanotoma, Macrolophus pygmaeus, Romanogobio albipinnatus, Romanogobio banaticus, Romanogobio belingi, Romanogobio kesslerii, Romanogobio parvus, Romanogobio pentatrichus, Romanogobio uranoscopus, Takydromus formosanus, Takydromus hsuehshanesis and Takydromus stejnegeri.

Development of microsatellite loci for Blastophaga javana (Agaonidae), the pollinating wasp of Ficus hirta (Moraceae).

PREMISE OF THE STUDY: To investigate genetic structure of the pollinator and, indirectly, gene flow in its plant host, Blastophaga javana microsatellite primers were developed. METHODS AND RESULTS: Ten polymorphic microsatellite loci were developed using the Fast Isolation by AFLP of Sequences Containing (FIASCO) repeats protocol. Numbers of alleles per locus ranged from 2 to 19, with observed and expected heterozygosities ranging from 0.000 to 0.800 and from 0.000 to 0.925, respectively. CONCLUSIONS: These markers may be useful for further investigation of population genetics of Blastophaga javana and other congeneric species.