Whole genome sequencing reveals candidate genes involving in PAS resistance in M. Tuberculosis isolated from patients in Thailand.

Para-amino salicylic acid (PAS) was first reported by Lehmann in 1946 and used for tuberculosis treatment. However, due to its adverse effects, it is now used only as a second line anti-tuberculosis drug for treatment of multidrug resistant or extensively drug resistant M. tuberculosis. The structure of PAS is similar to para-amino benzoic acid (pABA), an intermediate metabolite in the folate synthesis pathway. The study has identified mutations in genes in folate pathway and their intergenic regions for their possibilities in responsible for PAS resistance. Genomic DNA from 120 PAS-resistant and 49 PAS-sensitive M. tuberculosis isolated from tuberculosis patients in Thailand were studied by whole genome sequencing. Twelve genes in the folate synthesis pathway were investigated for variants associated with PAS resistance. Fifty-one SNVs were found in nine genes and their intergenic regions (pabC, pabB, folC, ribD, thyX, dfrA, thyA, folK, folP). Functional correlation test confirmed mutations in RibD, ThyX, and ThyA are responsible for PAS resistance. Detection of mutation in thyA, folC, intergenic regions of thyX, ribD, and double deletion of thyA dfrA are proposed for determination of PAS resistant M. tuberculosis.

Unfolding the mitochondrial genome structure of green semilooper (Chrysodeixis acuta Walker): An emerging pest of onion (Allium cepa L.).

Onion is the most important crop challenged by a diverse group of insect pests in the agricultural ecosystem. The green semilooper (Chrysodeixis acuta Walker), a widespread tomato and soybean pest, has lately been described as an emergent onion crop pest in India. C. acuta whole mitochondrial genome was sequenced in this work. The circular genome of C. acuta measured 15,743 base pairs (bp) in length. Thirteen protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, and one control region were found in the 37 sequence elements. With an average 395 bp gene length, the maximum and minimum gene length observed was 1749 bp and 63 bp of nad5 and trnR, respectively. Nine of the thirteen PCGs have (ATN) as a stop codon, while the other four have a single (T) as a stop codon. Except for trnS1, all of the tRNAs were capable of producing a conventional clover leaf structure. Conserved ATAGA motif sequences and poly-T stretch were identified at the start of the control region. Six overlapping areas and 18 intergenic spacer regions were found, with sizes ranged from 1 to 20 bp and 1 to 111 bp correspondingly. Phylogenetically, C. acuta belongs to the Plusiinae subfamily of the Noctuidae superfamily, and is closely linked to Trichoplusia ni species from the same subfamily. In the present study, the emerging onion pest C. acuta has its complete mitochondrial genome sequenced for the first time.

Discovery of a novel mitochondrial DNA molecule associated with tetrad pollen sterility in potato.

BACKGROUND: Tetrad sterility in potato is caused by a specific cytoplasm, called TSCsto, derived from the Mexican wild tetraploid species Solanum stoloniferum. Different S. stoloniferum accessions crossed as females with S. tuberosum resulted in 12 fertile hybrids and 27 sterile hybrids exhibiting tetrad sterility. RESULTS: Whole-mitochondrial-genome sequencing was performed for two fertile hybrids and three hybrids exhibiting tetrad sterility. Two to seven contigs, with the total assembly lengths ranging from 462,716 to 535,375 bp, were assembled for each hybrid. Unlike for the reference mitochondrial genome (cv. Désirée), two different recombinant-type contigs (RC-I and RC-II) were identified. RC-I featured by the rpl5-ψrps14 gene joined to the nad6 gene, generating a novel intergenic region. Using a PCR marker (P-3), we found that this intergenic region occurred exclusively in interspecific hybrids exhibiting tetrad sterility and in their parental S. stoloniferum accessions. A part of this intergenic sequence was expressed in the pollen. From a large survey in which P-3 was applied to 129 accessions of 27 mostly Mexican wild species, RC-I was found in diploid S. verrucosum and polyploid species. From eight accessions of S. verrucosum used as females, 92 interspecific hybrids were generated, in which only those carrying RC-I exhibited tetrad sterility. CONCLUSIONS: RC-I was clearly associated with tetrad sterility, and the RC-I-specific intergenic region likely contains a causal factor of tetrad sterility.

Development of a DNA barcode library of plants in the Thai Herbal Pharmacopoeia and Monographs for authentication of herbal products.

Traditional herbal medicine has long been practiced as a method of health care in many countries worldwide. The usage of herbal products has been increasing and is expected to continue to do so in the future. However, admixture and adulteration are concerns regarding the quality of herbal medicine, including its safety and efficacy. We aimed to develop a reference DNA barcode library of plants listed in the Thai Herbal Pharmacopoeia (THP) and Monographs of Selected Thai Materia Medica (TMM) (n = 101 plant species) using four core barcode regions, namely, the ITS2, matK, rbcL and trnH-psbA intergenic spacer regions, for authentication of the plant origin of raw materials and herbal products. Checking sequences from samples obtained from local markets and the Thai Food and Drug Administration (Thai FDA) against our digital reference DNA barcode system revealed the authenticity of eighteen out of twenty tested samples as claimed on their labels. Two samples, no. 3 and 13, were not Cyanthillium cinereum (L.) H.Rob. and Pueraria candollei Wall. ex Benth. as claimed, respectively. They were recognized as Emilia sonchifolia (L.) DC. and Butea superba (Roxb.), respectively. Hence, it is important for the Thai FDA or regulatory agencies to immediately initiate strict enforcement for the development of pharmacopoeial standards as well as revisions or modifications of available regulatory guidelines and to implement close monitoring for the quality control of herbal products in terms of authentication before they enter the herbal market. The centralized digital reference DNA barcode database developed here could play a very important role in monitoring or checking the authenticity of medicinal plants.

Identification of Valeriana fauriei and other Eurasian medicinal valerian by DNA polymorphisms in psbA-trnH intergenic spacer sequences in chloroplast DNA.

In order to differentiate among Valeriana fauriei Briq. and other Eurasian medicinal valerian (V. dioica L., V. hardwickii Wall., V. jatamansi Jones, and V. officinalis L.), we attempted to establish DNA markers. DNA sequences for the psbA-trnH intergenic spacer region of chloroplast DNA (psbA-trnH) and 18S ribosomal RNA, internal transcribed spacer 1 (ITS1), 5.8S ribosomal RNA, internal transcribed spacer 2 (ITS2), and 28S ribosomal RNA of nuclear DNA in V. fauriei and other Eurasian medicinal valerian were compared. Using partial sequences of psbA-trnH (nucleotide positions 1-75 from the 5' end of the intergenic spacer region), V. fauriei and other Eurasian medicinal valerian could be correctly identified to the species level. In addition, the partial sequences of psbA-trnH in V. fauriei contained five different haplotypes, and it was possible to distinguish the origins of valerian from Japan and Eurasia (China and Korea). On the other hand, individuals had heterogeneous sequences of ITS1 and ITS2, making it impossible to use direct sequencing and DNA markers of ITS1 and ITS2 to distinguish species and origins of V. fauriei and other Eurasian medicinal valerian.

Species diversity of Pleosporalean taxa associated with Camellia sinensis (L.) Kuntze in Taiwan.

Pleosporales species are important plant pathogens, saprobes, and endophytes on a wide range of economically important plant hosts. The classification of Pleosporales has undergone various modifications in recent years due to the addition of many families described from multiple habitats with a high level of morphological deviation. Numerous asexual genera have been described in Pleosporales that can be either hyphomyceteous or coelomycetous. Phoma- or coniothyrium-like species are common and have been revealed as polyphyletic in the order Pleosporales and linked with several sexual genera. A total of 31 pleosporalean strains were isolated in different regions of Taiwan between 2017 and 2018 from the leaves of Camellia sinensis plants with symptoms of leaf spot disease. These strains were evaluated morphologically and genotypically using multi-locus sequence analyses of the ITS, LSU, SSU, rpb2, tef1 and tub2 genes. The results demonstrated the affiliation of these strains with the various families in Pleosporales and revealed the presence of one new genus (Neoshiraia) and eight new species (Alloconiothyrium camelliae, Amorocoelophoma camelliae, Leucaenicola camelliae, L. taiwanensis, Neoshiraia camelliae, N. taiwanensis, Paraconiothyrium camelliae and Paraphaeosphaeria camelliae). Furthermore, to the best of our understanding, Didymella segeticola, Ectophoma pomi and Roussoella mexican were reported for the first time from C. sinensis in Taiwan.

Identification of Tibetan medicine Dida based on DNA barcoding.

The purpose of this study was to test the ability of DNA barcoding to identify the herbal raw trade of Tibetan medicine Dida in China. A reference database for plant-material DNA barcodes was successfully constructed and used to identify 36 commercially samples of Dida collected from Southwest China. The ITS sequence was amplified from these samples and the efficiency of the PCR amplification of ITS was 100%. The DNA sequencing results revealed that 3 samples (8.3%) were authenticated as Swertia chirayita, 2 sequences (5.6%) were authenticated as Swertia mussotii, 3 sequences (8.3%) were authenticated as Swertia ciliata, as recorded in the Tibetan Pharmacopeia. The other samples were authenticated as adulterants and all of them originated from common plants belonging to Saxifraga, Swertia and Halenia. This result indicates Dida pieces that are available in the market have complex origins and may indicate a potential safety issue and DNA barcoding is a convenient tool for market supervision.

Design and validation of a real-time PCR technique for assessing the level of inclusion of fungus- and yeast-based additives in feeds.

A reliable method for quantification of non-viable microbe-based nutritional and zootechnical additives introduced into feed is essential in order to ensure regulatory compliance, feed safety and product authenticity in industrial applications. In the present work, we developed a novel real-time quantitative polymerase chain reaction (qPCR) -based analysis protocol for monitoring two microbial additives in feed matrices. To evaluate the applicability of the method, pelleted wheat- and maize-based broiler chicken diets containing a non-viable phytase-producing strain of Aspergillus niger produced in solid state fermentation (150 or 300 g/t) and a non-viable selenium-enriched Saccharomyces cerevisiae (100 or 200 g/t) as model feed ingredients, were manufactured and subjected to analysis. Power analysis of the qPCR results indicated that 2 to 6 replicate feed samples were required to distinguish the product doses applied, which confirms that the microbial DNA was efficiently recovered and that potential PCR inhibitors present in the feed material were successfully removed in DNA extraction. The analysis concept described here was shown to be an accurate and sensitive tool for monitoring the inclusion levels of non-viable, unculturable microbial supplements in animal diets.

Characterizing fungal communities in medicinal and edible Cassiae Semen using high-throughput sequencing.

Cassiae Semen (CS) has been widely used as roasted tea and traditional Chinese medicine for decades. However, CS is easily contaminated by fungi and mycotoxins during pre-harvest and post-harvest process, thus posing a potential threat to consumer health. In this study, we used the Illumina MiSeq PE300 platform and targeted the internal transcribed spacer 2 sequences to survey the occurrence of fungi in raw and roasted CS samples. Results showed the fungal contamination in all 12 test samples. Ascomycota was the prevailing fungus at the phylum level, with the relative abundance of 66.50%-99.42%. At the genus level, Aspergillus, Cladosporium, and Penicillium were the most dominant genera, accounting for 0.66%-85.51%, 0.20%-29.11%, and 0.11%-32.92% of the fungal reads, respectively. A total of 68 species were identified, among which six potential toxigenic fungi belonging to Aspergillus, Penicillium, Candida, and Schizophyllum genera were detected. Moreover, differences in fungal communities were observed in raw and roasted CS samples. In conclusion, amplicon sequencing is feasible for analyzing fungal communities in CS samples, which provides a new approach to investigate the fungal contamination in edible-medicinal herb, thereby ensuring food safety and drug efficacy.

The combination of ITS2 and psbA-trnH region is powerful DNA barcode markers for authentication of medicinal Terminalia plants from Thailand.

The dried fruits of Terminalia plant (Combretaceae) called "Samo" have been used as herbal medicine in Thai traditional medicine. Four "Samo" crude drugs, namely, Samo thai, Samo thed, Samo dee-ngu, and Samo phiphek, are used as the main ingredients in Triphala and Trisamo recipes. Their commercial products are available in processed and powdered form, but are difficult to authenticate by conventional methods. In this study, we aimed to discriminate species of genus Terminalia for the identification of their crude drugs by a DNA barcoding technique. A total of 208 closely related nucleotide sequences were obtained from nine Terminalia species collected from Thailand and the DDBJ/EMBL/GenBank database. An effective DNA barcode marker was selected from six DNA loci (matK, rbcL, psbA-trnH, ITS, ITS1, and ITS2) and their two-locus combination. All sequences were analyzed by three major methods: (1) BLAST search; (2) the genetic divergence method using Kimura 2-parameter (K2P) distance matrices; and (3) tree topology analysis based on the neighbor-joining method. Comparison of the six candidate DNA loci indicated that ITS identified Terminalia with 100% accuracy at the species and genus levels in the BLAST1 method. ITS2 showed the highest K2P variability. The data from the single markers and the two-locus combinations revealed that only the two-locus combinations, namely, the combinations of rbcL, ITS, ITS1, and ITS2 with psbA-trnH, clearly discriminated all the species. From the results of DNA sequence analysis and the three methods, ITS2 is recommended for the identification of Terminalia species to supplement psbA-trnH.

Evaluation of white Kwao Krua (Pueraria candollei Grah. ex Benth.) products sold in Thailand by molecular, chemical, and microscopic analyses.

The tuberous roots of Pueraria candollei Grah. ex Benth. (Fabaceae), commonly known as white Kwao Krua, are used to relieve menopausal symptoms in Thai traditional medicine because they contain phytoestrogens. Black and red Kwao Krua crude drugs exist as well, but they have different botanical origins and pharmacological activities. There is a high demand for white Kwao Krua products, but because of the limited availability of the plant material, it is suspected that the adulteration and misidentification of white Kwao Krua crude drugs and products occur. In this study, we authenticated white Kwao Krua products collected from Thai herbal markets by molecular, chemical, and microscopic analyses. The nucleotide sequences in the internal transcribed spacer (ITS) and trnH-psbA regions of 23 samples of authentic P. candollei were analyzed, and both regions were found to have intraspecific DNA polymorphisms. Based on the single nucleotide polymorphisms in the ITS1 region, species-specific primer sets of P. candollei were designed to authenticate white Kwao Krua and differentiate it from red and black Kwao Krua. Only the PCR products of KWP02 were not amplified by the primer sets. Isoflavonoid contents and microscopic features were used to support the results of molecular analysis to clarify the botanical origin of white Kwao Krua. Molecular, chemical and microscopic methods confirmed that all the Thai Kwao Krua products examined in this study contained authentic "white Kwao Krua" as claimed on their labels.

Differentiation, chemical profiles and quality evaluation of five medicinal Stephania species (Menispermaceae) through integrated DNA barcoding, HPLC-QTOF-MS/MS and UHPLC-DAD.

Stephania species is one of the alkaloid-rich genus of the family Menispermaceae. Most plants of the genus Stephania possess medicinal value, whose main components are alkaloids. However, the non-medical species are often mistakenly used as herbs because of the difficulty in identification of the species. A systematic method which involved the combination of DNA barcoding, HPLC-QTOF-MS/MS and UHPLC was established for differentiation, chemical profiles and quality evaluation of medicinal Stephania species. Firstly, twenty batches of Stephania species samples were classified into five Stephania species by DNA barcoding. Secondly, 114 alkaloids including 22 tetrahydroprotoberberines, 13 protoberberines, 27 aporphines, 13 benzylisoquinolines, 12 hasubanans, 3 morphines and 24 other alkaloids were clearly or tentatively identified. Thirdly, thirteen representative components were simultaneously detected by UHPLC-DAD to characterize the differences of chemical compositions among five Stephania species. In conclusion, this method was comprehensive and effective for identification, chemical profiles and quality evaluation of medicinal Stephania species. It will provide a basis for holistic quality evaluation of medicinal Stephania species.

Dual and divergent transcriptional impact of IS1548 insertion upstream of the peptidoglycan biosynthesis gene murB of Streptococcus agalactiae.

Fourteen different insertion sequences belonging to seven families were identified in the genome of Streptococcus agalactiae. Among them, IS1548, a mobile element of the ISAs1 family, was linked to clonal complex (CC) 19 strains associated with neonatal meningitis and endocarditis. IS1548 impacts S. agalactiae in two reported ways: i) inactivation of virulence genes by insertion in an open reading frame (e.g. hylB or cpsD), ii) positive modulation of the expression of a downstream gene by insertion in an intergenic region (e.g. lmb). We previously identified an unknown integration site of IS1548 in the intergenic region between the folK and the murB genes involved in folate and peptidoglycan biosynthesis, respectively. In this work, we analyzed the prevalence of IS1548 in a large collection of nine hundred and eleven S. agalactiae strains. IS1548 positive strains belong to twenty-nine different sequence types and to ten CCs. The majority of them were, however, clustered within sequence type 19 and sequence type 22, belonging to CC19 and CC22, respectively. In contrast, IS1548 targets the folK-murB intergenic region exclusively in CC19 strains. We evaluated the impact of the insertion of IS1548 on the expression of murB by locating transcriptional promoters influencing its expression in the presence or absence of IS1548 and by comparative ß-galactosidase transcriptional fusion assays. We found that in the absence of IS1548, genes involved in folate biosynthesis are co-transcribed with murB. As it was postulated that a folic acid mediated reaction may be involved in cell wall synthesis, this co-transcription could be necessary to synchronize these two processes. The insertion of IS1548 in the folK-murB intergenic region disrupt this co-transcription. Interestingly, we located a promoter at the right end of IS1548 that is able to initiate additional transcripts of murB. The insertion of IS1548 in this region has thus a dual and divergent impact on the expression of murB. By comparative ß-galactosidase transcriptional fusion assays, we showed that, consequently, the overall impact of the insertion of IS1548 results in a minor decrease of murB gene transcription. This study provides new insights into gene expression effects mediated by IS1548 in S. agalactiae.

The ribosomal intergenic spacer (IGS) in the potato and tobacco cyst nematodes, Globodera pallida, G. rostochiensis and G. tabacum.

The potato cyst nematodes Globodera pallida and G. rostochiensis (PCN), and tobacco cyst nematode (TCN), G. tabacum, are the most important parasitic nematodes of potato and tobacco worldwide. Ribosomal DNA provides useful molecular data for diagnostics, the study of polymorphisms and for evolutionary research in eukaryotic organisms including nematodes. Here we present data on the structure and organization of a rarely studied part of the intergenic spacer (IGS) region of the PCN and TCN genome of cyst nematodes. This region has shown potential for diagnostic purposes and population studies in other organisms including nematodes. In nematodes, the ribosomal RNA gene cluster comprises three genes: 5.8S, 18S and 28S rRNA, which are separated by spacer regions: the intergenic spacer (IGS), non-transcribed spacer (NTS), externally transcribed spacer (EST) and the internally transcribed spacer (ITS). The intergenic spacer (IGS) region consists of an external transcribed spacer (ETS) and a non-transcribed spacer (NTS) which is located between the 28S of one repeat and the 18S gene of the next repeat within the rRNA genes cluster. In this study, the first flanking portion of the IGS was amplified, cloned and sequenced from PCN and TCN. Primers were then designed to amplify the whole IGS sequence. PCR amplification of IGS from G. tabacum, G. pallida, and G. rostochiensis yielded respectively: a single amplicon of 3 kb, three amplicons sized 2.5, 2.6 and 2.9 kb, and two amplicons sized 2.8 and 2.9 kb. Results showed that Globodera spp. has more than one variant copy of the IGS, with both long and short repetitive DNA elements. An approximately 400 bp long region without any internal repetitive elements, were identified in a position between the two repetitive regions suggesting that there is a 5S gene in the IGS of these species.

Detecting Schisandrae Chinensis Fructus and Its Chinese Patent Medicines with a Nucleotide Signature.

Schisandrae Chinensis Fructus (Wuweizi) is often adulterated with Schisandrae Sphenantherae Fructus (Nanwuweizi) in the herbal market. This adulteration is a threat to clinical treatment and safety. In this study, we aimed to develop a nucleotide signature for the identification of Wuweizi and its Chinese patent medicines based on the mini-DNA barcoding technique. We collected 49 samples to obtain internal transcribed spacer 2 (ITS2) sequences and developed a 26-bp nucleotide signature (5'-CGCTTTGCGACGCTCCCCTCCCTCCC-3') on the basis of a single nucleotide polymorphism (SNP) site within the ITS2 region that is unique to Wuweizi. Then, using the nucleotide signature, we investigated 27 batches of commercial crude drug samples labeled as Wuweizi and eight batches of Chinese patent medicines containing Wuweizi. Results showed that eight commercial crude drug samples were adulterants and one of the Chinese patent medicines contained adulterants. The nucleotide signature can serve as an effective tool for identifying Wuweizi and its Chinese patent medicines and can thus be used to ensure clinical drug safety.

Full-Length Multi-Barcoding: DNA Barcoding from Single Ingredient to Complex Mixtures.

DNA barcoding has been used for decades, although it has mostly been applied to somesingle-species. Traditional Chinese medicine (TCM), which is mainly used in the form ofcombination-one type of the multi-species, identification is crucial for clinical usage.Next-generation Sequencing (NGS) has been used to address this authentication issue for the pastfew years, but conventional NGS technology is hampered in application due to its short sequencingreads and systematic errors. Here, a novel method, Full-length multi-barcoding (FLMB) vialong-read sequencing, is employed for the identification of biological compositions in herbalcompound formulas in adequate and well controlled studies. By directly sequencing the full-lengthamplicons of ITS2 and psbA-trnH through single-molecule real-time (SMRT) technology, thebiological composition of a classical prescription Sheng-Mai-San (SMS) was analyzed. At the sametime, clone-dependent Sanger sequencing was carried out as a parallel control. Further, anotherformula-Sanwei-Jili-San (SJS)-was analyzed with genes of ITS2 and CO1. All the ingredients inthe samples of SMS and SJS were successfully authenticated at the species level, and 11 exogenousspecies were also checked, some of which were considered as common contaminations in theseproducts. Methodology analysis demonstrated that this method was sensitive, accurate andreliable. FLMB, a superior but feasible approach for the identification of biological complexmixture, was established and elucidated, which shows perfect interpretation for DNA barcodingthat could lead its application in multi-species mixtures.

DNA barcoding detects floral origin of Indian honey samples.

The unique medicinal and nutritional properties of honey are determined by its chemical composition. To evaluate the quality of honey, it is essential to study the surrounding vegetation where honeybees forage. In this study we used conventional melissopalynological and DNA barcoding techniques to determine the floral source of honey samples collected from different districts of the state of Mizoram, India. Pollen grains were isolated and genomic DNA was extracted from the honey samples. PCR amplification was carried out using universal barcode candidates ITS2 and rbcL to identify the plant species. Furthermore, TA cloning was carried out to screen the PCR amplicon libraries to identify the presence of multiple plant species. Results from both the melissopalynological and DNA barcoding analyses identified almost exactly the same 22 species, suggesting that both methods are suitable for analysis. However, DNA barcoding is easier and widely practiced. Hence, it can be concluded that DNA barcoding is a useful tool in determining the medicinal and commercial value of honey.

Sequencing and Comparative Analysis of the Chloroplast Genome of Angelica polymorpha and the Development of a Novel Indel Marker for Species Identification.

The genus Angelica (Apiaceae) comprises valuable herbal medicines. In this study, we determined the complete chloroplast (CP) genome sequence of A. polymorpha and compared it with that of Ligusticum officinale (GenBank accession no. NC039760). The CP genomes of A. polymorpha and L. officinale were 148,430 and 147,127 bp in length, respectively, with 37.6% GC content. Both CP genomes harbored 113 unique functional genes, including 79 protein-coding, four rRNA, and 30 tRNA genes. Comparative analysis of the two CP genomes revealed conserved genome structure, gene content, and gene order. However, highly variable regions, sufficient to distinguish between A. polymorpha and L. officinale, were identified in hypothetical chloroplast open reading frame1 (ycf1) and ycf2 genic regions. Nucleotide diversity (Pi) analysis indicated that ycf4⁻chloroplast envelope membrane protein (cemA) intergenic region was highly variable between the two species. Phylogenetic analysis revealed that A. polymorpha and L. officinale were well clustered at family Apiaceae. The ycf4-cemA intergenic region in A. polymorpha carried a 418 bp deletion compared with L. officinale. This region was used for the development of a novel indel marker, LYCE, which successfully discriminated between A. polymorpha and L. officinale accessions. Our results provide important taxonomic and phylogenetic information on herbal medicines and facilitate their authentication using the indel marker.

Cultivar differentiation of Citri Reticulatae Pericarpium by a combination of hierarchical three-step filtering metabolomics analysis, DNA barcoding and electronic nose.

The traditional Chinese medicine Citri Reticulatae Pericarpium (CRP) was mainly originated from the dried pericarp of Citrus reticulata 'Chachi' (Crc), Citrus reticulata 'Dahongpao' (Crd), Citrus reticulata 'Unshiu' (Cru) and Citrus reticulata 'Tangerina' (Crt) in China. Since these four cultivars have great similarities in morphology, reliable methods to differentiate CRP cultivars have rarely been reported. To discriminate the differences of these CRP cultivars, herein an efficient and reliable method by combining metabolomics, DNA barcoding and electronic nose was first established. The hierarchical three-step filtering metabolomics analysis based on liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) indicated that 9 species-specific chemical markers including 6 flavanone glycosides and 3 polymethoxyflavones could be considered as marker metabolites for discrimination of the geoherb Crc from other cultivars. A total of 19 single nucleotide polymorphism (SNP) sites were found in nuclear internal transcribed spacer 2 (ITS2) of CRP, and three stable SNP sites (33, 128 and 174) in the ITS2 region can distinguish the four CRP cultivars. The electronic nose coupled with chemometrics could also be used to effectively distinguish Crc from other CRP cultivars. Therefore, our results indicated that the integrated method will be an effective strategy for discrimination of similar herbal medicines.

Phylogenetic Comparison between Italian and Worldwide Hericium Species (Agaricomycetes).

A broad literature concerns the genus Hericium, mainly regarding the medicinal properties of H. erinaceus. Congeneric species of H. erinaceus have been poorly investigated. We collected basidiomata of H. alpestre, H. coralloides and H. erinaceus in Italy and isolated the corresponding mycelia in pure culture. Analysis of the respective internal transcribed spacer regions confirmed the morphological identification of the strains. Internal transcribed spacer sequences from the Italian strains were phylogenetically compared along with 64 other sequences available from Gen-Bank, the CBS Strain Database, and the European Nucleotide Archive (ENA) for the same Hericium . Geographic origin and host plant species were cross-checked using the above data banks. Bayesian phylogenetic analysis produced a phylogram that permitted good discrimination among Hericium species. It provides an updated phylogeny within the genus Hericium and a better understanding of affinity among the species analyzed. The main Hericium clade includes the following: the H. erinaceus group and the H. alpestre/H. coralloides group, where the two species cluster separately. This study also allowed us to differentiate the H. erinaceus group on a biogeographical basis. The phylogenetic comparison further confirms the importance of a joint morphological-molecular approach to avoid misidentification and to guarantee the quality of strains for further chemical and medicinal characterization.