The Complete Chloroplast Genomes of Nine Smilacaceae Species from Hong Kong: Inferring Infra- and Inter-Familial Phylogeny.

The Smilacaceae is a cosmopolitan family consisting of 200-370 described species. The family includes two widely accepted genera, namely Smilax and Heterosmilax. Among them, the taxonomical status of Heterosmilax has been continuously challenged. Seven Smilax and two Heterosmilax species can be found in Hong Kong, with most of them having medicinal importance. This study aims to revisit the infra-familial and inter-familial relationships of the Smilacaceae using complete chloroplast genomes. The chloroplast genomes of the nine Smilacaceae species from Hong Kong were assembled and annotated, which had sizes of 157,885 bp to 159,007 bp; each of them was identically annotated for 132 genes, including 86 protein-coding genes, 38 transfer RNA genes, and 8 ribosomal RNA genes. The generic status of Heterosmilax was not supported because it was nested within the Smilax clade in the phylogenetic trees, echoing previous molecular and morphological studies. We suggest delimitating the genus Heterosmilax as a section under the genus Smilax. The results of phylogenomic analysis support the monophyly of Smilacaceae and the exclusion of Ripogonum from the family. This study contributes to the systematics and taxonomy of monocotyledons, authentication of medicinal Smilacaceae, and conservation of plant diversity.

Characterization and phylogenetic analysis of the complete chloroplast genome of Emilia sonchifolia (L.) DC.

Emilia sonchifolia is a herb with antioxidant, anti-inflammatory, antitumor, and wound healing properties. The complete chloroplast genome (cp genome) of the genus Emilia was sequenced for the first time. The cp genome of E. sonchifolia is 151,474 bp in length. It contained a large single-copy (LSC) region (84,004 bp), and small single-copy (SSC) region (17,980 bp), and two inverted repeats (IRs, 24,745 bp). Phylogenetic analysis of 24 species was conducted. E. sonchifolia was found to be closely related to Pericallis hybrida and Dendrosenecio spp. The sequenced cp genome would be useful to understand the phylogeny and genomic studies of the genus Emilia.

Comparison of DNA extraction methods on CITES-listed timber species and application in species authentication of commercial products using DNA barcoding.

Quality and quantity of DNA extracted from wood is important for molecular identification of wood species, which can serve for conservation of wood species and law enforcement to combat illegal wood trading. Rosewood (Dalbergia and Pterocarpus) and agarwood (Aquilaria) are the most commonly found hardwood in timber seizure incidents. To monitor international trade of timber and commercial wood products and to protect these endangered wood species from further population decline, in this study, we have compared three extraction protocols for DNA extraction from 12 samples of rosewood and agarwood timber logs, and later applied the best DNA extraction protocol on 10 commercial wood products claimed to be rosewood and agarwood. We also demonstrated the applicability of DNA mini-barcoding with multi-loci combination with reference library for identifying the species of timber and commercial wood products. We found that a silica column-based method with guanidine thiocyanate-containing binding buffer served the best in DNA extraction from different parts of wood in all three genera with good quality and quantity. Single barcode region ITS2 or multi-loci combinations including ITS2 barcode region generally provide better discriminatory power for species identification for both rosewood and agarwood. All 10 products were identified to species-level using multi-loci combination. In terms of accuracy in labelling, 80% of them were labelled correctly. Our work has shown the feasibility of extracting good quality of DNA from authentic wood samples and processed wood products and identifying them to species level based on DNA barcoding technology.

Characterization and phylogenetic analysis of the complete chloroplast genome of Cyanthillium cinereum (L.) H. Rob.

Cyanthillium cinereum is a member of the tribe Vernonieae from the family Compositae. The tribe was traditionally placed in the subfamily Cichorioideae, but is recently proposed to be placed in its own subfamily Vernonioideae. The complete chloroplast genome (cp genome) of the genus Cyanthillium is sequenced for the first time. The cp genome of C. cinereum is 152,750 bp in length. It contained a large single copy (LSC) region (83,871 bp), and small single copy (SSC) region (18,487 bp), and two inverted repeats (IRs, 25,196 bp). Phylogenetic analysis of 20 species was conducted. C. cinereum and Gymnanthemum amygdalinum which are members of tribe Vernonieae nested outside of the monophyletic clade formed by members of subfamily Cichorioideae. The findings would be useful to understand the phylogeny of the genus Cyanthillium and the subfamily Vernonioideae.

The complete chloroplast genome of Iris speculatrix Hance, a rare and endangered plant native to Hong Kong.

Iris speculatrix is a rare and endangered plant first discovered in and native to Hong Kong. The whole chloroplast genome of I. speculatrix is 152,368 bp in length. It contained a large single copy region (82,003 bp), a small single copy region (17,941 bp), and two inverted repeats (26,212 bp). Phylogenetic analysis of 17 species of Iridaceae was conducted. I. speculatrix was found to be sister to a group of 12 Iris species, including I. setosa, I. lacteal, and I. uniflora. The sequenced chloroplast whole genome would be useful to understand the phylogeny and to conservation of I. speculatrix.

Comparative Analysis of Chloroplast Genomes of Dalbergia Species for Identification and Phylogenetic Analysis.

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.

Complete chloroplast genomes of Asparagus aethiopicus L., A. densiflorus (Kunth) Jessop 'Myers', and A. cochinchinensis (Lour.) Merr.: Comparative and phylogenetic analysis with congenerics.

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.

A specific and bioactive polysaccharide marker for Cordyceps.

Cordyceps is one of the most expensive and widely used functional foods. But the authenticity is still a concern due to the lack of appropriate markers. By targeting polysaccharides, this study aimed to develop a specific, and bioactive marker for Cordyceps. Firstly, the results of screening tests of 250 samples by examining both genetic markers and polysaccharide profile showed that a unique polysaccharide fraction (named CCP) was particular to the caterpillar parts. Its potential as a marker was further demonstrated by its ability to induce NO and cytokine production in RAW 264.7 cells. CCP was characterized to be an α-1,4-glucan with a branch at C-6 by the conventional structure analyzing and de novo oligosaccharides sequencing. The content of CCP was closely correlated to the traditional classification criteria. Generally, CCP was a marker that simultaneously enables qualitative and quantitative analysis of Cordyceps.

Assessing the reliability of medicinal Dendrobium sequences in GenBank for botanical species identification.

DNA-based method is a promising tool in species identification and is widely used in various fields. DNA barcoding method has already been included in different pharmacopoeias for identification of medicinal materials or botanicals. Accuracy and validity of DNA-based methods rely on the accuracy and taxonomic reliability of the DNA sequences in the database to be compared against. Here we evaluated the annotation quality and taxonomic reliability of selected barcode loci (rbcL, matK, psbA-trnH, trnL-trnF and ITS) of 41 medicinal Dendrobium species downloaded from GenBank. Annotations of most accessions are incomplete. Only 53.06% of the 2041 accessions downloaded contain a reference to a voucher specimen. Only 31.60% and 4.8% of the entries are annotated with country of origin and collector or assessor, respectively. Taxonomic reliability of the sequences was evaluated by a Megablast search based on similarity to sequences submitted by other research groups. A small number of sequences (211, 7.14%) was regarded as highly doubted. Moreover, 10 out of 60 complete chloroplast genomes contain highly doubted sequences. Our findings suggest that sequences of GenBank should be used with caution for species-level identification. The scientific community should provide more important information regarding identity and traceability of the sample when they deposit sequences to public databases.

Rapid detection of CITES-listed shark fin species by loop-mediated isothermal amplification assay with potential for field use.

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.

A rapid sample-to-answer analytical detection of genetically modified papaya using loop-mediated isothermal amplification assay on lab-on-a-disc for field use.

With genetically modified (GM) food circulating on the market, a rapid transgenic food screening method is needed to protect consumer rights. The on-site screening efficiency of GM food testing is low. We report rapid sample-to-answer detection of GM papayas with loop-mediated isothermal amplification (LAMP) and a compact, portable, integrated microfluidic platform using microfluidic lab-on-a-disc (LOAD). GM samples were differentiated from non-GM papaya, based on the detection of a specific GM (P-35S (Cauliflower mosaic virus 35S promoter)) and non-GM DNA marker (papain) in 15 min. The detection limits for DNA and juice from papaya were 10 pg/µL and 0.02 µL, respectively. Our LOAD platform is a simple and robust solution for GM screening, which is anticipated to be a foundation for on-site testing of transgenic food.

Medicinal Materials DNA Barcode Database (MMDBD) version 1.5-one-stop solution for storage, BLAST, alignment and primer design.

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.