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dna metabarcoding uncovers fungal communities in Zingiberis Rhizoma

Chinese Herbal Medicines 2024年第4期16卷 679-685页

作者： Chune Fan Yanan Xu Yufeng Li Meihua Yang Jianping Han Xiaohui Pang Institute of Medicinal Plant Development Chinese Academy of Medical Sciences&Peking Union Medical CollegeBeijing 100193China

Objective: Zingiberis Rhizoma(ZR, Ganjiang in Chinese), also known as dried ginger, is a popular spice and medicinal herb that has been used for several thousand years. However, ZR is easily contaminated by fungi and mycotoxin under suitable conditions, and might be hazardous to the health and safety of consumers, thus concerns about the herb's safety have been raised. The aim of this study was to investigate the fungal community and the effects of collection areas and processing methods on the fungal community in ***: A total of 18 ZR samples were collected from four provinces of China, and the samples were divided into four groups based on collecting sites. Meanwhile, the samples collected in Sichuan Province, China were divided into three groups based on the processing methods. We employed the Illumina Mi Seq PE300 platform and targeted the internal transcribed spacer 2(ITS2) sequences to investigate fungal contamination in ZR samples, and the difference in fungal community among the groups of different collection sites and processing ***: All 18 samples were contaminated with fungi. Ascomycota was the dominant phyla, accounting for 34.46%-100% of the fungal reads. At the genus level, Candida, Diutina, and Aspergillus were the most dominant genera, with relative abundances of 0–98.37%, 0–99.82%, and 0–79.08%, ***, four potential toxigenic fungi and seven human pathogens were found. Furthermore, differences in the community composition of ZR samples from four collecting sites and three processing methods were ***: dna metabarcoding provides a novel insight into fungal community diversity in ZR samples,providing references to ensure the sustainable utilization and quality research of ZR.

关键词： dna metabarcoding processing methods spices toxigenic fungi Zingiberis Rhizoma

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dna metabarcoding analysis of fungal community on surface of four root herbs

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Chinese Herbal Medicines 2024年第1期16卷 143-150页

作者： Yujie Dao Jingsheng Yu Meihua Yang Jianping Han Chune Fan Xiaohui Pang Institute of Medicinal Plant Development Chinese Academy of Medical Sciences&Peking Union Medical CollegeBeijing 100193China

Objective: Angelicae Sinensis Radix(ASR, Danggui in Chinese), Cistanches Herba(CH, Roucongrong in Chinese), Ginseng Radix et Rhizoma(PG, Renshen in Chinese), and Panacis Quinquefolii Radix(PQ,Xiyangshen in Chinese), widely used as medicine and dietary supplement around the world, are susceptible to fungal and mycotoxin contamination. In this study, we aim to analyze their fungal community by dna ***: A total of 12 root samples were collected from three main production areas in China. The samples were divided into four groups based on herb species, including ASR, CH, PG, and PQ groups. The fungal community on the surface of four root groups was investigated through dna metabarcoding via targeting the internal transcribed spacer 2 region(ITS2).Results: All the 12 samples were detected with fungal contamination. Rhizopus(13.04%-74.03%),Aspergillus(1.76%-23.92%), and Fusarium(0.26%-15.27%) were the predominant genera. Ten important fungi were identified at the species level, including two potential toxigenic fungi(Penicillium citrinum and P. oxalicum) and eight human pathogenic fungi(Alternaria infectoria, Candida sake, Hyphopichia burtonii, Malassezia globosa, M. restricta, Rhizopus arrhizus, Rhodotorula mucilaginosa, and Ochroconis tshawytschae). Fungal community in ASR and CH groups was significantly different from other groups,while fungal community in PG and PQ groups was relatively ***: dna metabarcoding revealed the fungal community in four important root herbs. This study provided an important reference for preventing root herbs against fungal and mycotoxin contamination.

关键词： Angelicae Sinensis Radix Cistanches Herba dna metabarcoding fungi Ginseng Radix et Rhizoma ITS2 Panacis Quinquefolii Radix root herbs

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Seasonal variations in the plant diet of the Chinese Monal revealed by fecal dna metabarcoding analysis

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Avian Research 2022年第2期13卷 208-215页

作者： Bin Wang Yun Li Guiquan Zhang Jian Yang Cao Deng Haoyu Hu Long Zhang Xiaoqin Xu Caiquan Zhou Key Laboratory of Southwest China Wildlife Resources Conservation(Ministry of Education) China West Normal UniversityNanchong637002China Institute of Ecology China West Normal UniversityNanchong637002China Key Laboratory of State Forestry and Grassl and Administration on Conservation Biology of Rare Animalsin the Giant Panda National Park(China Conservation and Research Center for the Giant Panda) Dujiangyan611830China Key Laboratory of Animal Disease and Human Health of Sichuan Province College of Veterinary MedicineSichuan Agricultural UniversityChengdu611130China Institute of Chinese Materia Medica China Academy of Chinese Medical SciencesBeijing100700China

The Chinese Monal(Lophophorus thuysii)is an alpine-obligate galliform species of global conservation *** has been listed as a first class protected wildlife species in China,requiring conservation actions during the 14 th Five-Year Plan ***,the diet composition of Chinese Monal and its seasonal variations have rarely been studied,constraining the effective conservation of the ***,we investigated the plant diet composition of the Chinese Monal and its seasonal variations using a dna metabarcoding approach on fecal *** collected 190 fecal samples of the Chinese Monals from the central Qionglai Mountains located in China,and analyzed the plant diet of this species using a dna metabarcoding *** profiling of higher plants in the fecal samples was performed using the second internal transcribed spacer(ITS2)*** analyses,including rarefaction curves,nonmetric multidimensional scaling(NMDS)and permutational multivariate analysis of variance(PERMANOVA),were used to explore the seasonal variations in diet *** Chinese Monal foraged a wide range of plant recipes composed of 35 families and 83 genera throughout the year,with Brassicaceae,Apiaceae,and Poaceae as the dominant families,and Cardamine as the dominant *** species consumed plants from 62 genera from 28 families during the breeding season(n=81)and 66 genera from 31 families during the non-breeding season(n=109).Further,the plant diet composition significantly varied between the breeding and non-breeding seasons,especially for the frequency of occurrence and relative read abundances at genus *** study analyzed the plant diet of the Chinese Monal at a high resolution for the first time,and the results revealed that the seasonal variations in its plant diet composition was adapted to plant phenology and foraging *** species,a previously confirmed important food resource for the Chinese Monal,were not detected in any fe

关键词： Chinese monal Conservation management Diet seasonality dna metabarcoding Endangered species

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Multilocus dna metabarcoding diet analyses of small cetaceans:a case study on highly vulnerable humpback dolphins and finless porpoises from the Pearl River Estuary,China

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Integrative Zoology 2023年第1期18卷 183-198页

作者： Xiyang ZHANG Dingyu LUO Ri-Qing YU Yuping WU School of Marine Sciences Zhuhai Key Laboratory of Marine Bioresources and EnvironmentGuangdong Provincial Key Laboratory of Marine Resources and Coastal EngineeringPearl River Estuary Marine Ecosystem Research StationMinistry of EducationSun Yat-sen UniversitySouthern Marine Science and Engineering Guangdong LaboratoryZhuhaiChina Department of Biology Center for EnvironmentBiodiversity and ConservationThe University of Texas at TylerTylerTexasUSA

Accurate diet identiﬁcation of top predators is crucial to fully understand their ecological *** to terrestrial animals,gathering dietary information from cetaceans is notoriously difﬁ***,we applied a multilocus metabarcoding approach to investigate the diet of vulnerable Indo-Paciﬁc humpback dolphins and Indo-Paciﬁcﬁnless porpoises from the Pearl River Estuary(PRE),*** analyses identiﬁed 21 preyﬁsh species from the 42 humpback dolphin stomachs,as well as 10 species ofﬁsh and 1 species of cephalopod from the 13ﬁnless porpoise *** of the taxa were assigned to the species level,highlighting that the multimarker approach could facilitate species identiﬁ*** of the prey species were small-and medium-sizedﬁshes that primarily fed on *** calculated similarity index revealed a moderated dietary overlap between the 2 cetaceans,presumably due to the feeding of the 2 predators in association withﬁshing vessels in the PRE.A more diverse diet was observed in humpback dolphins in the closedﬁshing season compared to theﬁshing season,implying the inﬂuence on the dolphin diet due to the availability of commercialﬁshery ***,according to the results of species rarefaction curves,ourﬁndings on the feeding habits of the 2 cetaceans are still limited by insufﬁcient sample size and therefore should be interpreted with *** study represents aﬁrst attempt to apply the multilocus dna metabarcoding technique in the diet analysis of small cetaceans,although more efforts are needed to improve this type of analysis.

关键词： diet species analyses dna metabarcoding Indo-Paciﬁcﬁnless porpoise Indo-Paciﬁc humpback dolphin Pearl River Estuary

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Biomonitoring for traditional herbal medicinal products using dna metabarcoding and single molecule, real-time sequencing

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Acta Pharmaceutica Sinica B 2018年第3期8卷 488-497页

作者： Tianyi Xin Zhichao Xu Jing Jia Christine Leon Songnian Hu Yulin Lin Subramanyam Ragupathy Jingyuan Song Steven G.Newmaster Key Lab of Chinese Medicine Resources Conservation State Administration of Traditional Chinese Medicine of the People's Republic of China Institute of Medicinal Plant Development Peking Union Medical College & Chinese Academy of Medical Sciences Royal Botanic Gardens Beijing Institute of Genomics Chinese Academy of Sciences NHP Research Alliance Biodiversity Institute of Ontario (BIO) University of Guelph

Global concerns have been paid to the potential hazard of traditional herbal medicinal products(THMPs). Substandard and counterfeit THMPs, including traditional Chinese patent medicine, health foods, dietary supplements, etc. are potential threats to public health. Recent marketplace studies using dna barcoding have determined that the current quality control methods are not sufficient for ensuring the presence of authentic herbal ingredients and detection of contaminants/adulterants. An efficient biomonitoring method for THMPs is of great needed. Herein, metabarcoding and single-molecule, realtime(SMRT) sequencing were used to detect the multiple ingredients in Jiuwei Qianghuo Wan(JWQHW), a classical herbal prescription widely used in China for the last 800 years. Reference experimental mixtures and commercial JWQHW products from the marketplace were used to confirm the method. Successful SMRT sequencing results recovered 5416 and 4342 circular-consensus sequencing(CCS) reads belonging to the ITS2 and psb A-trn H regions. The results suggest that with the combination of metabarcoding and SMRT sequencing, it is repeatable, reliable, and sensitive enough to detect species in the THMPs, and the error in SMRT sequencing did not affect the ability to identify multiple prescribed species and several adulterants/contaminants. It has the potential for becoming a valuable tool for the biomonitoring of multi-ingredient THMPs.

关键词： Traditional herbal medic inal products(THMP) Species mixture Authentication dna metabarcoding Single molecule real-time(SMRT) sequencing Circular-consensus sequencing(CCS)

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Feeding ecology and interactions with mammal hosts in a symbiotic genus of birds(Buphagus spp.)in Namibia

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Avian Research 2024年第3期15卷 336-344页

作者： Michael S.Lukubwe Daniel Velarde-Garcéz Fernando Sequeira Susana Lopes Adrian J.F.K.Craig Vanessa A.Mata Department of Zoology and Entomology Rhodes UniversityMakhandaSouth Africa Department of Wildlife Management and Tourism Studies University of NamibiaKatima Mulilo CampusNamibia Departamento de Biologia Faculdade de CiênciasUniversidade do PortoPortoPortugal CIBIO Centro de Investigação em Biodiversidade e Recursos GenéticosInBIO Laboratório AssociadoCampus de VairãoVairãoPortugal BIOPOLIS Program in Genomics Biodiversity and Land PlanningCIBIOCampus de VairãoVairãoPortugal

As the sole obligate symbiotic birds in Africa,oxpeckers offer a unique model for studying symbiotic *** to the multitrophic level they occupy and the context dependent foraging behavior they exhibit,the type of symbiotic relationship can be *** addition to providing a cleaning service to the host by removing ticks,oxpeckers frequently feed on blood,mucus,and saliva,inflicting potential damage on the ***,we used dna metabarcoding on faecal samples to analyze the taxonomic composition of the trophic interactions of the Yellow-billed Oxpecker(Buphagus africanus)and Red-billed Oxpecker(***)in northeastern *** contrast to conventional methods,dna metabarcoding allows for a detailed identification of dietary resources encompassing both mammal hosts and consumed arthropods within the same *** this information,we examined differences in the diet composition between oxpecker species and localities,as well as the co-occurrence between host and arthropod *** findings revealed that oxpeckers predominantly source their diet from mammals,ticks,and flies;however,ticks and flies rarely co-occur in the diet of an *** observed variability among individuals in their feeding ecology,which is strongly correlated with locality and,to a lesser extent,with the mammal *** noted a high degree of mobility between hosts within relatively short periods,with 32%of the samples showing traces of at least two mammal *** study illustrates the dynamic foraging behavior of these specialized symbiotic birds,shedding light on their potential role in pest control services and disease transmission.

关键词： Commensalism Diet dna metabarcoding Oxpeckers Parasitism Trophic ecology

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A single degenerated primer significantly improves COX1 barcoding performance in soil nematode community profiling

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Soil Ecology Letters 2024年第2期6卷 31-44页

作者： Yincai Ren Dorota L. Porazinska Quanhui Ma Shuhan Liu Hongmei Li Xue Qing Department of Plant Pathology Nanjing Agricultural UniversityNanjing210095China Department of Entomology and Nematology University of FloridaGainesvilleFL32611USA Institute of Grassland Science Key Laboratory of Vegetation Ecology of the Ministry of EducationJilin Songnen Grassland Ecosystem National Observation and Research StationNortheast Normal UniversityChangchun130024China

A new COX1 primer for soil nematode metabarcoding was designed,and this primer outperforms other commonly used COX1 primer pairs in species recovery and quantity of PCR products.•The lack of reference database is the main reason that led to the low species recovery in COX1 metabarcoding.•We expanded current NCBI database by adding 51 newly generated COX1 reference *** nematodes play important roles in soil ecosystems and often serve as bioindicators of soil *** identification of soil nematodes is often difficult due to their limited diagnostic characters and high phenotypic *** barcoding and metabarcoding techniques are promising but lack universal primers,especially for mitochondrial COX1 *** this study a degenerated COX1 forward primer COIFGED was *** primer pair(COIFGED/JB5GED)outperforms other four commonly used COX1 primer pairs in species recovery and quantity of polymerase chain reaction(PCR)*** metabarcoding analysis,the reads obtained from the new primer pair had the highest sequencing saturation threshold and amplicon sequence variant(ASV)diversity in comparison to other COX1 as well as 18S rRNA *** annotation of ASVs suggested the new primer pair initially recovered 9 and 6 out of 25 genera from mock communities,respectively,outperformed other COX1 primers,but underperformed the widely used 18S NF1/18Sr2b primers(16 out of 25 genera).By supplementing the COX1 database with our reference sequences,we recovered an additional 6 mock community species bringing the tally closer to that obtained with 18S *** summary,our newly designed COX1 primers significantly improved species recovery and thus can be supplementary or alternative to the conventional 18S metabarcoding.

关键词： degenerated primers dna metabarcoding mitochondrial cytochrome oxidase c subunit I gene phylogeny ribosomal RNA gene soil nematodes

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基于dna条形码技术的中成药五虎散生物组分鉴定研究

基于DNA条形码技术的中成药五虎散生物组分鉴定研究

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作者：穆威杉承德医学院

学位级别：硕士

中成药多为复方制剂,化学成分复杂,原料药材经粉碎加工后鉴定难度增加,现有的传统中药鉴定方法在中成药原料错误投料及真菌杂质等外源污染物的监管方面仍具有一定挑战性。中成药五虎散5种原料药材中,同时包含3个伞形科物种,基于传统方... 详细信息

中成药多为复方制剂,化学成分复杂,原料药材经粉碎加工后鉴定难度增加,现有的传统中药鉴定方法在中成药原料错误投料及真菌杂质等外源污染物的监管方面仍具有一定挑战性。中成药五虎散5种原料药材中,同时包含3个伞形科物种,基于传统方法的鉴定难度增加,同时还包含有毒物种天南星。五虎散投料的准确性问题关系着其临床使用的疗效与安全,积极开展中成药五虎散生物组分鉴定的相关研究刻不容缓。目的:本研究以原粉入药的中成药五虎散为研究对象,基于dna barcoding技术获取5种原料药材及阳性对照药材的参考序列。再通过dna metabarcoding和shotgun metabarcoding两种方法,对中成药五虎散自制样品和市售样品的生物组分进行检测。旨在为中成药五虎散的生物组分鉴定提供不同的策略和技术手段,作为中成药传统鉴定方法的有效补充,形成更全面的生物组分鉴定体系。方法:1.收集五虎散的5种原料药材或基原植物以及阳性对照药材西洋参,首先采用形态学及性状鉴定方法对药材的真伪进行初步鉴定,并采用传统的Sanger测序技术对各药材进行dna条形码分析,经与标准序列进行复查核对,确保药材基原的准确性,获取各药材ITS2、psb A-trn H、mat K和rbc L片段的参考序列。最终基于dna barcoding技术保证五虎散各原料药材及阳性对照药材的准确性并获取参考序列。2.按中国药典五虎散项下的制备方法自制2份五虎散参考样品(HSZY160和HSZY172),其中HSZY172中加入与五虎散中最小入药量等量的西洋参作为阳性对照。从药店收集4份五虎散市售样品(WHS001、WHS002、WHS003和WHS004),使用优化后的dna提取方法提取6份中成药的总dna,基于ITS2片段选择MS2F/MS2R和MS3F/MS3R两对引物进行PCR扩增,利用Illumina Miseq测序平台进行高通量测序。经序列拼接、质控、过滤、去冗余、OTU聚类及BLAST比对等生物信息学分析,最终基于dna metabarcoding技术对中成药五虎散的生物组分进行鉴定。3.以2份五虎散自制样品(HSZY160和HSZY172)和3份五虎散市售样品(WHS001、WHS002和WHS003)为研究对象,使用优化后的dna提取方法提取中成药的总dna,利用Illumina测序平台直接对5个样品的dna进行shotgun测序。原始数据经Trimmonmatic去除接头和低质量序列、MEGAHIT和Meta SPAdes组装序列、Cutadapt软件去除引物,bowtie2短序列Mapping等生物信息学分析,获得ITS2、psb A-trn H、rbc L和mat K序列。将自制样品获得的组装序列与Sanger测序获得的参考序列进行比较以确定组装序列的准确性,利用自制样品考察shotgun metabarcoding的可行性,并将该方法用于市售样品中以验证方法的适用性。最终基于shotgun metabarcoding技术对中成药五虎散生物组分进行分析。结果:1.基于dna barcoding技术五虎散原料药材参考序列获取:在形态学鉴定的基础上,使用dna barcoding技术成功获取了除天南星的psb A-trn H片段和白芷的rbc L片段外,各处方成分及阳性对照药材的ITS2、psb Atrn H、mat K和rbc L片段的序列,经与《中国药典中药材dna条形码标准序列》中相关标准序列复核,表明5种原料药材和西洋参药材均为正品,为保障后续自制样品的准确性奠定了基础,所获序列可作为参考序列使用。2.基于dna metabarcoding技术中成药五虎散生物组分鉴定:经高通量测序两对引物获得的总pair-end数分别为564574和622926条。去除reads数metabarcoding技术中成药五虎散生物组分鉴定:5份样品共获得37.14 G的测序数据,共1421013条paired-end reads。经组装、注释,ITS2、psb A-trn H、mat K和rbc L区域分别获得80、11、9和8个OTU。基于4种dna条形码序列,在2份自制样品中,所有处方成分的组装序列均成功获得,在HSZY172样品中获得到阳性对照西洋参的OTU,且组

关键词：五虎散原料药材 dna条形码 dna metabarcoding shotgun metabarcoding 生物组分物种鉴定

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