Shark fin is a delicacy in many Asian countries. Overexploitation of sharks for shark fin trading has led to a drastic reduction in shark population. To monitor international trade of shark fin products and protect the endangered species from further population decline, we present rapid, user-friendly and sensitive diagnostic loop-mediated isothermal amplification (LAMP) and effective polymerase chain reaction (PCR) assays for all twelve CITES-listed shark species. Species-specific LAMP and PCR primers were designed based on cytochrome oxidase I (COI) and NADH2 regions. Our LAMP and PCR assays have been tested on 291 samples from 93 shark and related species. Target shark species could be differentiated from non-target species within three hours from DNA extraction to LAMP assay. The LAMP assay reported here is a simple and robust solution for on-site detection of CITES-listed shark species with shark fin products.
Dalbergia L.f. is a pantropical genus consisting of 269 species of trees, shrubs, and woody lianas. This genus is listed in CITES Appendices because of illegal logging and trafficking driven by the high economic value of its heartwood. Some species are also used medicinally. Species identification of Dalbergia timber and herbs is challenging but essential for CITES implementation. Molecular methods had been developed for some timber species, mostly from Madagascar and Southeast Asia, but medicinal species in south China were usually not included in those studies. Here, we sequenced and assembled the chloroplast genomes of five Dalbergia species native to Hong Kong, four of which are medicinal plants. Our aim is to find potential genetic markers for the identification of medicinal Dalbergia species based on divergence hotspots detected in chloroplast genomes after comparative and phylogenetic analysis. Dalbergia chloroplast genomes displayed the typical quadripartite structure, with the 50 kb inversion found in most Papilionoideae lineages. Their sizes and gene content are well conserved. Phylogenetic tree of Dalbergia chloroplast genomes showed an overall topology similar to that of ITS sequences. Four divergence hotspots (trnL(UAA)-trnT(UGU), ndhG-ndhI, ycf1a and ycf1b) were identified and candidate markers for identification of several Dalbergia species were suggested.
Asparagus species are widely used for medicinal, horticultural, and culinary purposes. Complete chloroplast DNA (cpDNA) genomes of three Asparagus specimens collected in Hong Kong—A. aethiopicus, A. densiflorus ‘Myers’, and A. cochinchinensis—were de novo assembled using Illumina sequencing. Their sizes ranged from 157,069 to 157,319 bp, with a total guanine–cytosine content of 37.5%. Structurally, a large single copy (84,598–85,350 bp) and a small single copy (18,677–18,685 bp) were separated by a pair of inverted repeats (26,518–26,573 bp). In total, 136 genes were annotated for A. aethiopicus and A. densiflorus ‘Myers’; these included 90 mRNA, 38 tRNA, and 8 rRNA genes. Further, 132 genes, including 87 mRNA, 37 tRNA, and 8 rRNA genes, were annotated for A. cochinchinensis. For comparative and phylogenetic analysis, we included NCBI data for four congenerics, A. setaceus, A. racemosus, A. schoberioides, and A. officinalis. The gene content, order, and genome structure were relatively conserved among the genomes studied. There were similarities in simple sequence repeats in terms of repeat type, sequence complementarity, and cpDNA partition distribution. A. densiflorus ‘Myers’ had distinctive long sequence repeats in terms of their quantity, type, and length-interval frequency. Divergence hotspots, with nucleotide diversity (Pi) ≥ 0.015, were identified in five genomic regions: accD-psaI, ccsA, trnS-trnG, ycf1, and ndhC-trnV. Here, we summarise the historical changes in the generic subdivision of Asparagus. Our phylogenetic analysis, which also elucidates the nomenclatural complexity of A. aethiopicus and A. densiflorus ‘Myers’, further supports their close phylogenetic relationship. The findings are consistent with prior generic subdivisions, except for the placement of A. racemosus, which requires further study. These de novo assembled cpDNA genomes contribute valuable genomic resources and help to elucidate Asparagus taxonomy.
Authentication of medicinal materials by deoxyribonucleic acid (DNA) technology is gaining popularity. In 2010, our team has created Medicinal Materials DNA Barcode Database (MMDBD) version 1.0 to provide an interactive database for documenting DNA barcode sequences of medicinal materials. This database now contains DNA barcode sequences of medicinal materials listed in the Chinese Pharmacopoeia, Dietary Supplements Compendium and Herbal Medicine Compendium of the US Pharmacopoeia and selected adulterants. The data archive is regularly updated and currently it stores 62 011 DNA sequences of 2111 medicinal materials. Our team has recently completed the major improvement on the interfaces and incorporated essential bioinformatics tools to facilitate the authentication work. MMDBD version 1.5 contains detailed information of each medicinal material including their material names, medical part, pharmacopeia information, biological classification in rank of family and status on the Convention on International Trade in Endangered Species of Wild Fauna and Flora and the International Union for Conservation of Nature’s Red List of Threatened Species, if any. DNA sequences can be retrieved by search in Latin scientific name, Chinese name, family name, material name, medical part and simplified Chinese character stroke. A `BLAST’-based engine for searching DNA sequences is included in the MMDBD version 1.5. Since primer design is a key step in DNA barcoding authentication, we have integrated the `Clustal Omega alignment tool’ and `Primer3’ in the form of web interface. These new tools facilitate multiple sequence comparison and the design of primers for amplification of a target DNA barcode region, allowing DNA barcoding authentication.
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