THE CHINESE UNIVERSITY OF HONG KONG

Li Dak Sum Yip Yio ChinR & D Centre

for Chinese Medicine

Shiu-Ying Hu HerbariumSchool of Life Sciences

Organized by: Supported by:

Chung Chi College,CUHK

Institute for Control of Chinese Traditional Medicine and Ethnic Medicine, National Institutes for Food and Drug Control,

China

2nd International Conference on DNA technology for authentication, quality control

and conservation of herbal material

LI DAK SUM YIP YIO CHIN R & D CENTRE FOR CHINESE MEDICINE

2 - 5 December, 2018

Faculty of Science,CUHK

香港中文大學李達三葉耀珍中醫藥研究發展中心LI DAK SUM YIP YIO CHIN R & D CENTRE FOR CHINESE MEDICINE

THE CHINESE UNIVERSITY OF HONG KONG

FOREWORD

Welcome to the 2nd International Conference on DNA Technology for Authentication, QualityControl and Conservation of Herbal Material (ICDNA2018). The first conference was held in2016.

As a premier conference in the field, ICDNA2018 provides a forum for reporting anddiscussing the latest developments in the research and application of DNA technology onmolecular authentication, quality control and conservation of herbal material. Thisconference aims at gathering global leaders of the field to discuss: 1) the current trend inDNA authentication, including but not limited to metabarcoding, genome skimming andchloroplast sequencing, for the raw material and the products; 2) the use of DNA technologyfor understanding biodiversity, evolution and pharmacological properties of medicinal herbs;3) the use of appropriate DNA technologies for standardization, authentication and qualityassessment of medicinal herbs; 4) the use of omics for quality control and efficacy study. Weanticipate that this conference will further stimulate research and development in this field.

We are pleased that many well-known researchers and collaborators are present. We hopethat more collaboration will be fostered. In recent years, DNA technology has beenincreasingly accepted by different regulatory agencies and we thank these agencies forsupporting the conference and sending staff to attend. We hope the conference will enhancethe use of DNA technologies in regulation.

Finally, we welcome you all to Hong Kong. We hope participants will have a fruitful time andyou will also take some spare time to enjoy Hong Kong.

Pang Chui ShawConference ConvenorICDNA 2018 @ CUHK

香港新界沙田香港中文大學 TEL 電 話: (852) 3943 1363 WEBSITE 網址: rdccm.cuhk.edu.hkCUHK, Shatin, N.T., HKSAR FAX 圖文傅真: (852) 2603 5646 E-MAIL 電郵: rdccm@cuhk.edu.hk

Organizing Committee

Chairperson:

Prof. Pang Chui Shaw Li Dak Sum Yip Yio Chin R & D Centre for Chinese Medicine,

School of Life Sciences, Institute of Chinese Medicine, The Chinese University of Hong Kong

Members:

Dr. Stanley Chun Kai CheungLi Dak Sum Yip Yio Chin R & D Centre for Chinese Medicine,

The Chinese University of Hong Kong

Prof. Clara Bik San Lau Institute of Chinese Medicine,

The Chinese University of Hong Kong

Dr. David Tai Wai Lau Shiu-Ying Hu Herbarium, School of Life Sciences,

The Chinese University of Hong Kong

Mr. Kenneth Chi Fai Leung School of Life Sciences,

The Chinese University of Hong Kong

Prof. Karl Wah Keung TsimCentre for Chinese Medicine R & D and Division of Life Science,

Hong Kong University of Science and Technology

Miss Mavis Hong Yu YikLi Dak Sum Yip Yio Chin R & D Centre for Chinese Medicine,

The Chinese University of Hong Kong

Conference Venue: Ballroom II-III, 2/F, Courtyard by Marriott Hong Kong Shatin, 1 On Ping Street, Sha Tin, Hong Kong香港沙田石門安平街1號萬怡酒店2樓宴會廳II-III

Venue: Ballroom II-III, 2/F, Courtyard by Marriott Hong Kong Shatin14:00 – 17:00 Registration

18:30 – 21:30

Welcome Dinner (Invited & Registered Guests)Venue:

F Zone, Shop E-F, 11/F, Kings Wing Plaza 1, Shek Mun, Sha Tin石門京瑞廣場一期11樓E-F號舖放 Zone

Enquiries:

Dr. Stanley Cheung Miss Mavis YikScientific Officer Project CoordinatorLi Dak Sum Yip Yio Chin R & D Centre Li Dak Sum Yip Yio Chin R & D Centrefor Chinese Medicine for Chinese Medicine Tel: (852) 9289 7939 Tel: (852) 6209 3118Email: stanley@cuhk.edu.hk Email: mavisyik@cuhk.edu.hk

PostersArea

ConferenceRoom

MoMo Café (Lunch)

Registration Area

2 December, 2018 (Sunday)

Day 1

Venue: Ballroom II-III, 2/F, Courtyard by Marriott Hong Kong Shatin08:30 - 09:00 Registration & Set Up Poster

09:00 - 09:30

Opening Ceremony & Group Photo

Dr. Edwin Lok-Kin TsuiAssistant Director (Traditional Chinese Medicine), Department of Health, HKSAR

Prof. Fanny Mui-Ching CheungPro-Vice-Chancellor, The Chinese University of Hong Kong

Prof. Pang-Chui Shaw Director, Li Dak Sum Yip Yio Chin R & D Centre for Chinese Medicine, The Chinese University of Hong Kong

Chairperson: Prof. Zhi-Xiu Lin

09:30 - 10:00Dr. Caroline HowardNational Institute for Biological Standards and Control (NIBSC), UKTitle: The Application of DNA Barcoding in the British Pharmacopoeia (BP)

10:00 – 10:30

Mr. Ka-Lok WongDepartment of Health, Hong Kong, ChinaTitle: The Establishment and Work Progress of Government Chinese Medicines Testing Institute

10:30 – 11:15 Tea Break & Set Up of Posters

11:15 – 11:45Prof. Monique Simmonds Royal Botanic Gardens, Kew, UKTitle: Authentication of Plants Entering the Trade: From Genes to Isotopes

11:45 - 12:15

Dr. Yohei Sasaki Kanazawa University, JapanTitle: Phylogenetic Relationships Based on DNA Information and Application for the Species Identification

12:15 - 12:30

Dr. Kannika ThongkhaoChulalongkorn University, ThailandTitle: Combined DNA Barcoding and HPTLC for Differentiation of the Poisonous Urobotrya Siamensis from the Edible Melientha Suavis and SauropusAndrogynus for Food Safety

12:30 - 14:00Lunch

Venue : MoMo Café, Courtyard by Marriott Hong Kong Shatin

3 December, 2018 (Monday)

Theme: DNA Barcodes --- Development and Application.

Day 2

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Chairperson: Prof. Tae-Jin Yang

14:00 - 14:30

Prof. Lian-Ming GaoKunming Institute of Botany, Chinese Academy of Sciences, ChinaTitle: Using Comprehensive DNA Barcode Reference Library and Super-barcodes to Identify Yews (Taxus) Worldwide

14:30 - 15:00Prof. Suchada SukrongChulalongkorn University, ThailandTitle: DNA Barcoding in Drug Discovery: From Bench to Bedside

15:00 - 15:15

Ms. Xin-Dan LiuJinan University, ChinaTitle: Identification and Spatiotemporal Regulation of the Patchoulol Synthase Gene Responsible for Sesquiterpene Biosynthesis in Different Pogostemoncablin (Blanco) Benth. Cultivars

15:15 - 16:00 Tea Break & Poster Presentation (Odd Number Posters)

16:00 - 16:15

Ms. Mahima KarthikeyanBharathiar University, IndiaTitle: Species Delimitation of Ficus krishnae C.DC. from Ficus benghalensis L. Complex Based on DNA Barcoding and HP-TLC

16:15 - 16:30

Dr. Senthilkumar UmapathySRM Institute of Science and Technology, IndiaTitle: Phylogenetic Signal of Steroidal and Glyco-alkaloids in Genus SolanumBased on DNA Barcodes and HPTLC Fingerprints

16:30 - 16:45

Dr. Yat-Tung LoThe Chinese University of Hong Kong, Hong Kong, ChinaTitle: Way to Reveal Unknown Species Identity in Processed Materials -Development of Adaptor Ligation-mediated PCR Technology with DNA Barcoding

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Venue: Ballroom II-III, 2/F, Courtyard by Marriott Hong Kong Shatin09:00 - 09:30 Registration

Chairperson: Prof. Clara Bik-San Lau

09:30 - 10:00

Prof. Kelvin Kam-Chuen Chan Liverpool John Moores University, UK & Western Sydney University, AustraliaTitle: The Role of DNA Technology on Pharmacopoeia Standards of Chinese Materia Medica

10:00 - 10:30

Mr. Edwin van Huis Naturalis Biodiversity Center, NetherlandsTitle: A DNA-mark for All Plant Species in the Amazon: A Species Information System for Science, the Environment and Society

10:30 - 11:15 Tea Break & Poster Presentation (Even Number Posters)

11:15 - 11:45

Dr. Steven NewmasterUniversity of Guelph, CanadaTitle: Guidelines for Assembling Biological Reference Materials (RM) and Validation of Methods for Molecular Diagnostic Identification of Botanicals

11:45 - 12:05

Prof. Chang LiuInstitute of Medicinal Plant Development, Chinese Academy of Medical Science, ChinaTitle: Zhen-Tracking: A Block-Chain Based Tracking System Based on Genetic Fingerprints

12:05 - 12:25Ms. Zhi-Yun YangKunming Institute of Botany, Chinese Academy of Sciences, ChinaTitle: A Rapid and Accurate Identification Method for Opium Poppy Seedings

12:25 - 14:00Lunch

Venue : MoMo Café, Courtyard by Marriott Hong Kong Shatin

4 December, 2018 (Tuesday)

Theme: New DNA Technology.

Day 3

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Chairperson: Prof. Chun-Kwok Wong

14:00 - 14:30

Prof. Jiang Xu (Representing Prof. Shi-Lin Chen)Institute of Chinese Materia Medica, Chinese Academy of Chinese MedicalSciences, ChinaTitle: The Development of One-step NGS TCM Barcoding Machine

14:30 - 15:00

Dr. Zheng-Fei LuHerbalife Nutrition, USATitle: DNA Technologies for Botanical Ingredient Authentication: A Quality Control Perspective

15:00 - 15:15

Mr. Hyun-Seung ParkSeoul National University, KoreaTitle: Chloroplast and Mitochondrial Genome Flux Makes Confusion for Plant DNA Barcoding

15:15 - 16:00 Tea Break & Poster Presentation

16:00 - 16:20Dr. Ramalingam Sathishkumar Bharathiar University, IndiaTitle: Biological Authentication of Natural Indian Honey Samples

16:20 - 16:50Prof. Simon Quanbin HanHong Kong Baptist University, Hong Kong, ChinaTitle: A Polysaccharide Marker for Authentication of Cordyceps

16:50 - 17:05

Ms. Grace Wing-Chiu ButThe Chinese University of Hong Kong, Hong Kong, ChinaTitle: A Rapid Sample-to-answer Analytical Detection of Genetically ModifiedPapaya Using Loop-mediated Isothermal Amplification Assay on Lab-on-a-discfor Field Use

18:00 - 21:00

Gala Dinner (Invited & Registered Guests)Venue:

Ballroom V2&3, ClubONE Riviera, 55-57 Tai Chung Kiu Road, Sha Tin, Hong Kong香港沙田大涌橋路55-57號會所1號水中天宴會廳 V2&3

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Venue: Ballroom II-III, 2/F, Courtyard by Marriott Hong Kong Shatin09:00 - 09:30 Registration

Chairperson: Prof. Suchada Sukrong

09:30 - 10:00

Prof. Tae-Jin YangSeoul National University, KoreaTitle: Genome Sequence of Panax ginseng and Genomics-based DNA Barcoding of Important Medicinal Plants in Korea

10:00 - 10:30

Prof. Parani MadasamySRM University, IndiaTitle: PlantBarcodes: A Plant DNA Barcode Database for Species Identification and Herbal Material Authentication

10:30 - 11:00 Tea Break & Dismount of Posters

11:00 - 11:20Prof. Jerome Ho-Lam HuiThe Chinese University of Hong Kong, Hong Kong, ChinaTitle: Population Genomics of Incense Trees Aquilaria sinensis in Hong Kong

11:20 – 11:40

Dr. Chun-Xia ZengKunming Institute of Botany, Chinese Academy of Sciences, ChinaTitle: Developing a DNA Barcoding System for Plant Materials with Low Quality Genomic DNAs Using Next-Generation Sequencing

11:40 - 11:55

Mr. Sae-Hyun LeeSeoul National University, KoreaTitle: Inter- and Intra- chloroplast Genome Diversity and Classification of Germplasm in Cynanchum Species

11:55 - 12:10

Dr. Karen Hoi-Yan WuThe Chinese University of Hong Kong, Hong Kong, ChinaTitle: Medicinal Materials DNA Barcode Database (MMDBD) v1.5 – One-stop Platform for Storage, BLAST, Alignment & Primer Design

12:10 - 12:30

Closing Ceremony

Prof. Pang-Chui ShawDirector, Li Dak Sum Yip Yio Chin R & D Centre for Chinese Medicine, School of Life Sciences, Institute of Chinese Medicine, CUHK

12:30 - 14:00Lunch

Venue : MoMo Café, Courtyard by Marriott Hong Kong Shatin

5 December, 2018 (Wednesday)

Theme: Genome and Database.

Day 4

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Visit & Tour (14:00 - 17:00)

Gathering Time: 14:00 [Pre-registration is required.]Gathering Location: Lobby @ Courtyard by Marriott Hong Kong Shatin.

1.

Department of Health: Government Chinese Medicines Testing Institute (GCMTI)Time: 14:30 – 16:00Address: 7/F, Biotech Centre 2, No.11 Science Park West Avenue, Phase 2, Science Park, ShatinQuota: 20

2.

Agriculture, Fisheries and Conservation Department: Endangered Species Resource Centre (ESRC) and HerbariumTime: 14:30 – 16:30Address: 6/F, Cheung Sha Wan Government Offices, 303 Cheung Sha Wan Road, Cheung Sha WanQuota: 25

Opening Ceremony - Invited Guest

Edwin Lok Kin TsuiAssistant Director (Traditional Chinese Medicine), Department of Health, HKSAR, ChinaEmail: edwintsui@dh.gov.hk

Biography

Dr. Edwin Lok Kin Tsui is the Assistant Director (Traditional Chinese Medicine) of theDepartment of Health. He oversees the implementation of the Chinese MedicineOrdinance and its subsidiary legislations, the promotion of safe and efficacious use ofChinese medicine and practice, the conducting of Chinese medicine related public healthand health promotion activities, and the operation of the Government Chinese MedicinesTesting Institute which has commenced operation since March 2017.

He is the Director of the World Health Organization (WHO) Collaborating Centre forTraditional Medicine, and is also serving as focal point of Hong Kong SAR of theInternational Regulatory Cooperation for Herbal Medicines under the WHO.

Prior to this, he had been responsible for and contributed in various fields in public healthprotection and promotion including port health measures, food safety, contingencyplanning, surveillance, investigations and control of public health crisis. He had assistedthe WHO in 2011 and 2014 to develop WHO strategic documents on International HealthRegulations and border quarantine measures. In 2016, he had been invited to be WHOTemporary Advisor and provided professional advice in development of traditionalmedicines in the Western Pacific regions of the WHO.

Abstracts of Invited Speakers

The Application of DNA Barcoding in the British Pharmacopoeia (BP)

Caroline HowardNational Institute for Biological Standards and Control (NIBSC), UKEmail: Caroline.Howard@nibsc.org

Biography

Dr. Caroline Howard studied at De Montfort University, Leicester UK, attaining a 1st class(Hons) degree in Biology, on the Biotechnology streamand then a PhD in ‘DNAauthentication of Herbal Medicines’ in 2007 working with Professor Slater. She continuedin this research group for 2-3 years after this, funded by industry and grants. In 2014, shewas recruited by the Medicines and Healthcare products Regulatory Agency (MHRA) towork for the British Pharmacopoeia and investigate the use of DNA methods foridentification of Herbal Drugs. The scope of the group was expanded, and she is now thePrincipal Scientist of the BP Herbal Laboratory, managing a team which is responsible forthe analysis of herbal drugs for the purpose of monograph publication, including DNAidentification, HPTLC and physical testing. She remains research active, currently co-supervising PhD students with collaborators from De Montfort University, the BotanicGardens Kew, the University of Copenhagen and others.

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Abstract

Caroline Howard

National Institute for Biological Standards and Control (NIBSC), Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG UK

Corresponding Author Email: Caroline.Howard@nibsc.org

The BP-NIBSC Herbals Laboratory is focusing on monograph development for herbalmedicines with the combination of three distinct specialties: Phytochemical, Physicaltesting and Molecular expertise.

Combining the expertise of the British Pharmacopoeia, NIBSC, and a group of externalexperts from Kew Gardens, industry and academia, the lab is optimised to house a varietyof monograph tests, to advance the safety of herbal medicines worldwide.Physical tests, such as foreign matter testing, ash and water content analysis, are utilisedalongside the DNA barcoding and High Performance Thin Layer Chromatography (HPTLC)to investigate and assess a variety of herbal medicines. TCMs, Ayurvedic and Europeanmedicines including Bei Sha Shen, Holy Basil, and Clivers are among some of themonographs developed.

DNA barcoding is routinely utilised to identify the plant species of the starting materialused in the entire lab workflow, which in-turn supports the development of HPTLCchemical fingerprints and the quality parameters set by the physical testing. The necessityto include DNA barcoding in the work flow of the laboratory led to the decision to publishthe methodologies used in the BP Appendix, along with the target specific referencesequence for each herbal drug in the Supplementary Chapter. The development of thesemethods, along with the BP Nucleic Acid Reference Material (BPNARM) will be described.This unique laboratory setup allows for the exchange of information and expertisetargeted to any herbal species. The accumulation of all the information positively affectsmonograph development, with each area working in harmony to produce a unified result.

The Establishment and Work Progress of the Government Chinese Medicines Testing Institute

Ka-Lok WongGovernment Chinese Medicines Testing Institute, Department of Health, HKSAR, ChinaEmail: dnaanalyst@dh.gov.hk

Biography

Mr. K. L. Wong is currently DNA Analyst in the Government Chinese Medicines TestingInstitute, Department of Health, Hong Kong SAR, PR China. He obtained his M.Phil degreewith Professor P.C. SHAW at Department of Biochemistry, The Chinese University of HongKong (CUHK). Under supervision of Prof SHAW, he then started R&D of Chinese medicines(CM) at CUHK focusing on: (1) molecular authentication of Chinese medica materia(CMM); (2) Establishing CMM DNA barcode database; (3) determining the properties andfunctions of proteins in CM. In 2016, he joined Department of Health. Following this, hewas assigned to establish DNA Laboratory of Government Chinese Medicines TestingInstitute. The current role is to implement research project on developing DNA testingmethod for CMM.

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Abstract

Ka-Lok Wong

Government Chinese Medicines Testing Institute, 7/F of Biotech Centre 2, No. 11 Science Park West Avenue, Phase 2, Science Park, Shatin

Corresponding Author Email: dnaanalyst@dh.gov.hk

Government Chinese Medicines Testing Institute (GCMTI), managed by the Department ofHealth, specialises in the testing of, and scientific research on, Chinese medicines with aview to setting internationally recognised reference standards for the safety, quality andtesting methods of Chinese medicines (CM).

In view that it will take a few years to build the permanent testing institute and there arepressing demands from the Chinese medicine and testing sectors to start the operation ofthe institute as early as possible, a testing institute with reduced manpower and scope forinterim operation commenced operation in its temporary site located in Hong KongScience Park in March 2017.

Committed to promoting the integration of multi-disciplinary approaches for Chinesemedicines identification, Chinese Medicines Herbarium and internationally recognisedlaboratories, including Macroscopic & Microscopic Laboratory, Chemistry Laboratory, andDNA laboratory, have been set up in GCMTI to embark on scientific research on testingmethods. Special design requirements and measures are taken in DNA Laboratory in orderto produce reliable and traceable results. Physically separated laboratory rooms aredesignated for pre-PCR and post-PCR processing procedures, and unidirectional workflowfrom pre-amplified areas to post-amplified areas is adopted.

At present, GCMTI is carrying out 6 research projects on developing testing methodstailored for the Chinese medicine and testing sectors in Hong Kong. Among them, DNAtest methods for the identification of CM with quality control plan is being developed.Besides, a CM DNA barcode library will be established to enhance the traceability in CM.Through the use of DNA technologies and other approaches, GCMTI aims to ensuringefficacy and safety of Chinese medicines. The methods and standards developed byGCMTI will be transferred for the use of the public and related sectors with a view tostrengthening the capability for quality control and identification of Chinese medicines.

Authentication of Plants Entering the Trade: From Genes to Isotopes

Monique S.J. Simmonds Royal Botanic Gardens, Kew, UKEmail: M.Simmonds@kew.org

Biography

Prof. Monique S.J. Simmonds is Deputy Director of Science at the Royal Botanic Gardens,Kew. She has 30 years’ experience of research into different aspects of plant and fungalchemistry with an emphasis on the economic value of natural products. More recentlyshe has increased her research on methods that can be used to authenticate plants beingtraded as well as those being used by local communities. This has included chemical andDNA characters. She has co-authored patent for compounds in the area of malaria, cancer,pest control, cosmetics and have been involved in getting food, medicinal and cosmeticproducts to market. In 2016 in collaboration with scientists at the University ofCambridge she set up Polypharmakos a company that aims to develop new antibiotics andinnate immune modulators derived from plant and fungal compounds. She is also veryinterested in researching the efficacy of traditional based medicines, with an emphasis onplants used in traditional Chinese medicine as well as UK herbal remedies. Her researchillustrates how Kew’s diverse and validated unique range of collections as well as theexpertise of its scientists and horticultural staff can help address economical importantissues associated with plant/fungal use.

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Abstract

Monique S.J. Simmonds

Royal Botanic Gardens, Kew, Richmond, Surrey TW 9 3AB, UK

Corresponding Author E-mail : M.Simmonds@kew.org

This talk will provide an overview of some of the current authentication projects beingundertaken at Kew that require different DNA based techniques to resolve identificationof material entering the trade. This includes the challenges of looking at bulk material foradulterants as well as developing methods that can differentiate between closely relatedspecies. Challenges are often associated with the robustness of the different methods.The techniques are highly relevant for a range of industries dealing with plants for thefood, timber, medicinal and cosmetic sectors.

The use of DNA based techniques are placed in the context of trying to deal with not onlyissues with identification but also quality and province. The methods are in some casesavailable but data are not always available on material being traded so there is a real needfor verified collections to build up robust DNA based databases. The talk will be illustratedby examples from the different trade sectors and thus highlight the need for greater linksbetween regulators, academics and industry. These interactions need to be encouraged ifrobust and simple methods to monitor the sustainability and providence of plants are tobe monitored.

Phylogenetic Relationships Based on DNA Information and Application for the Species Identification

Yohei SasakiKanazawa University, JapanEmail: sasaki@p.kanazawa-u.ac.jp

Biography

Dr. Yohei Sasaki received his Ph.D. in Pharmaceutical Sciences from the Institute of NaturalMedicine, University of Toyama, Japan in 2003, following as to which he worked as aresearcher in the Medicinal Plant Garden at Hoshi University, Tokyo, Japan, from 2003 to2010. Currently he holds the position of Associate Professor at the laboratory ofPharmacognosy in the Faculty of Pharmaceutical Sciences at Kanazawa University, Japan,where he also is the Director of the Medicinal Plant Garden owned by the aforementioned University.

His research topics include the following;(1) Development of Japanese-produced herbal medicine: In Japan, most of the herbs andingredients used in the making of traditional medicine (Kampo) are imported from foreigncountries, especially from China. Methods to establish how to cultivate, process andproduce Japanese medicines are performed. As with Ayurvedic medicine (Indiantraditional medicine), the original plants are difficult to be grown in Japan due to theclimate differentiation. He ventures to find the possible substitution and alternativeswithin the plants found in Japan. This is also includes to be the principle of Ayurveda.(2) Quality evaluation of herbal medicine: To examine the quality of herbal medicine, heestablished the methods for analysis such as morphological, chemical, molecularbiological technique. He is one of the first person to apply the LAMP (Loop-MediatedIsothermal Amplification) method to plants, i.e. Curcuma longa (2007), and Panax ginseng(2008).

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Abstract

Yohei Sasaki

Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan

Corresponding Author Email: sasaki@p.kanazawa-u.ac.jp

The technique of DNA analysis of plant have become common. Consequently, the DNAalignment of dried or processed drugs such as herbal medicines as well as fresh plants canbe analyzed without special technique. This information is used for phylogeneticrelationships between taxonomically closely related plants and authentication of theoriginal plants. The presence of specific DNA alignment among the individuals respectivelymakes this research feasible. The DNA barcoding system is mainly based on mutationsfound in rbcL and matK regions which are rapidly spreading. However, the barcoding isapplied only for the DNA mutations. Presumably, If we do not spot the difference in thoseregions of the plant individually, the DNA barcoding therefore becomes inapplicable. Theexamination of DNA mutations is easier between the plant species than between thesubspecies, variety, or form. Additionally, it is also difficult to find the DNA mutationsbetween intraspecific plant individuals.

Although DNA barcoding is also used for phylogenetic analysis of medicinal plant andauthentication, it is not bound to other species for analysis. Taking into consideration thePaeonia lactiflora plant, there are medicinal type and ornamental type in Paeony and boththe types are identified as the same plant. There almost has been no difference in rbcL,matK, and ITS between medicinal and ornamental type of Paeony. Nevertheless, we haveattempted to identify another technique, i.e., using the Next Generation Sequencer. Today,I would like to introduce the progress about our trials.

Using Comprehensive DNA Barcode Reference Library and Super-Barcodes to Identify Yews (Taxus) Worldwide

Lian-Ming GaoKunming Institute of Botany, Chinese Academy of Sciences, ChinaEmail: gaolm@mail.kib.ac.cn

Biography

Dr. Lian-Ming GAO is a professor of Kunming Institute of Botany, Chinese Academy ofScience (CAS), and deputy director of CAS Key Laboratory for Plant Diversity andBiogeography of East Asia. He obtained his first degree (BS) in Biology from HebeiUniversity in 1996, followed by a PhD in botany at Kunming Institute Botany, CAS in 2002.His major research interests include plant phylogenomics, population genetics,phylogeography, speciation and adaptive evolution, and DNA barcoding on some plantgroups, especially on Taxus and Rhododendron. From last few years, he also started toconduct the community phylogeny and assembly mechanism based on elevationalgradient transect and a 25 ha subalpine forest dynamics plot with evolutionaryperspective. He obtained several grants including the general program and internationalprogram of National Natural Science Foundation of China (NSFC), and basic special projectof the Ministry of Science and Technology of the People’s Republic of China. He haspublished over 90 papers in peer-reviewed journals, such as PNAS, Molecular Ecology,New Phytologist, Diversity and Distributions, Molecular Ecology Resources, and ScientificReport and so on.

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Abstract

Lian-Ming Gao1*, Jie Liu1, De-Zhu Li2

1. Key Laboratory for Plant Diversity and Biogeography of East Asia , Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China2. Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China

Corresponding Author Email: gaolm@mail.kib.ac.cn

Yews are important medicinal plants due to containing anti-cancer drug taxol. However,the accurate identification of target species and population origin is challenging. Reliableidentification can be achieved via a comprehensive DNA barcode reference library,accompanied by precise distribution data. We construct a comprehensive DNA barcodereference library, and generate distribution maps using species distribution modeling(SDM), for all 15 Taxus species worldwide. We find that trnL-trnF is the ideal barcode forTaxus which could distinguish all Taxus species, when considering the species-specific sites,and in combination with ITS identify hybrids. Taxonomical, geographical and geneticdimensions of sampling strategy were all found to affect the comprehensiveness of theresulting DNA barcode library. Maps from SDM showed that most species had allopatricdistributions, except three in the Sino-Himalayan region. Using the barcode library anddistribution map data, two unknown forensic samples were identified to species (and inone case, population) level, and another was determined as a putative interspecific hybrid.This integrated species identification system for Taxus can be used for bio-surveillance,conservation management and to monitor and prosecute illegal trade. Similaridentification systems are recommended for other IUCN- and -CITES listed taxa. We alsosequenced plastomes from two to three individuals for each of all the 16 recognized yewspecies. We demonstrate that entire plastomes can be used to successfully discriminateall Taxus species with 100% support, suggesting that they are useful as super-barcodes forspecies identification. We also propose that accD and rrn16-rrn23 are promising specialbarcodes to discriminate yew species. Our newly developed Taxus plastomic sequencesprovide a resource for super-barcodes and conservation genetics of several endangeredyews.

The Development of One-step NGS TCM Barcoding Machine

Shi-Lin ChenInstitute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, ChinaEmail: slchen@icmm.ac.cn

Biography

Professor Shilin Chen, the Director of the Institute of Chinese Materia Medica, ChinaAcademy of Chinese Medical Sciences, now also works as the Director of the WorldHealth Organization Collaborating Center for Traditional Medicine, Academician ofInternational Academy of Sciences for Europe and Asia, Vice President of the Consortiumfor Globalization of Chinese Medicine, Visiting Professor of Tokyo University of Pharmacyand Life Sciences, Japan and University of Macau, China, Team Leader of ChangjiangScholars and Innovation Team, Ministry of Education(MOE), Co-Editor-in-Chief of PharmCrops. He was trained at Royal Botanic Garden, Kews, U.K. and used to be visitingprofessor of Hong Kong Polytechnic University, China. He has firstly in the worldestablished the DNA Barcoding identification system for Chinese herbal medicine species,and he has written some related works, including Standard DNA barcodes of ChineseMateria Medica in Chinese Pharmacopoeia. He has so far published totally more than 400academic works, including 200 SCI papers on famous academic journals, such as NatureCommun, Biotech Adv and PNAS. His academic works have been cited more than 10,000times, H-index: 50. He has been honored the Wujieping Award for Medical Innovation, theStar of Nobel Prize and Agilent Thought Leadership Award, etc.

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Abstract

XU Jiang1, Liao Baosheng1, Fang Xiao2,Zou Jing2,Zheng Xiasheng1, Yin Xianmei1, Liang Xinming2, Jiang Hui2, Chen Shilin1*

1. Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China2. MGI, Shengzhen, China

Corresponding Author Email: slchen@icmm.ac.cn

Traditional herbal medicines, such as plant- and fungi-based remedies, have beenused formore than 5,000 years. However, the genetic background, the agricultural traits, and themedicinal quality of most traditional herbs are poorly understood. With rapid advances inhigh throughput sequencing technologies and greatly reduced costs, a new disciplinecalled “herbgenomics” has emerged. Researchers are now systematically categorizingmedicinal herbs by sequencing, assembling, and annotating their genomes, and byanalyzing their genes’ functions. Genomic information, together with transcriptomic,proteomic, and metabolomic data, can therefore be used to predict secondary metabolitebiosynthetic pathways and their regulation, triggering a revolution in discovery-basedresearch aiming to understand the genetics and biology of herbs. Herbgenomics providesan effective platform to support the chemical and biological analyses of complex herbalproducts that may contain more than one active component. Therefore, it is now beingapplied to many areas of herbrelated biological research to help understand the quality oftraditional medicines and for molecular herb identification through the establishment ofan herbal gene bank. Based on this database, collaborating with MGI, we launched a one-step NGS DNA Barcoding machine, both for single herb identification, or for unprocessedherb powder identification. Functional herbal genomics can contribute to model herbresearch platforms, geoherbal research, genomics-assisted herb breeding, and herbalsynthetic biology, all of which are important for securing the sourcing of TCMs and theiractive compounds in the future.

A DNA-mark for All Plant Species in the Amazon: A Species Information System for Science, The Environment and Society

Edwin van HuisNaturalis Biodiversity Centre, NetherlandsEmail: edwin.vanhuis@naturalis.nl

Biography

Mr. Edwin J.F.B. van Huis (1958) is a Master of Science in Business Administration from theErasmus University Rotterdam, and a Master of Science in Biology from the University ofFlorida (USA) and the University of Utrecht. He started his professional career working forthe Ministry of Culture (1986 – 1992). He then became the managing Director of theRijksmuseum in Amsterdam and after that the General Director of the NetherlandInstitute for Sound and Vision. In 2011 he became General Director of NaturalisBiodiversity Centre in Leiden. Naturalis is the National Natural History Museum of theNetherlands, a museum and research institute with more that 800 staff. It has a one ofthe largest collections in the world, 42 million plants, animals and minerals.

IC18-I7

Abstract

Edwin van Huis

Naturalis Biodiversity Centre, PO box 9517, 2300 RA Leiden, The Netherlands

Corresponding Author Email: edwin.vanhuis@naturalis.nl

We propose a system to genetically identify all plant species in the Amazon, starting withtrees. A species information system, such as this, is critical to rev our efforts safeguardingthis extremely valuable natural resource and unlocking the potential of this biome as asource of knowledge and a resource for the bio-based economy. Naturalis BiodiversityCenter in the Netherlands, the National Institute for Amazonian Research (INPA) in Brazil,the Natural History Museum of Denmark and the National History Museum of the UnitedKingdom, have taken this initiative that will include the world’s major natural-historymuseums, the collections in the Amazonian countries and the larger scientific community.Together we have the knowledge, the skills, the experience and the collections to makethis ambitious project a success.

Guidelines for Assembling Biological Reference Materials (RM) and Validation of Methods for Molecular Diagnostic Identification of Botanicals

Steven NewmasterUniversity of Guelph, CanadaEmail: snewmast@uoguelph.ca

Biography

Dr. Steven Newmaster has been a botany & genomics professor at the University ofGuelph, Canada for 18 years. He conducts research on genomics, medicinal plants andmolecular diagnostic identification systems with more than 120 publications and $20million in R&D funding. Currently he is conducting leading international research onmolecular diagnostic tools for NHP authentication, genomic diversity in plants, medicinalplants and natural health products. His R&D has resulted in the development of policy onbiodiversity, and International trade of herbal products including product authenticationcertification within the food and natural product industry within Canada, USA, Europe andAsia. He advises on the authentication of herbal products to regulators and is developingnew QA/QC industry standards for testing natural ingredient authenticity. He is one of thefounders for the world’s first international certification program (TRU-ID), which hasprocessed over 30 million natural ingredients in the USA, Canada, and E.U.

IC18-I8

Abstract

Steven Newmaster

NHP Research Alliance, College of Biological Sciences, University of Guelph, Canada

Corresponding Author Email: snewmast@uoguelph.ca

Increasing world population and global loss of biodiversity have resulted in considerabledemands for botanical ingredients. This situation is intensified by the world-wide increaseof 12-15% annually in the consumption of natural health products. Consequently, theadulteration of natural health products (NHPs) is frequently in the news, which concernsconsumers and brand owners who seek quality health products. The NHP ResearchAlliance at the University of Guelph is seeking collaboration with industry, researchers andother NHP stakeholders in research projects and the assemblage of a comprehensivebiological Reference Material (RM) genomic DNA-based libraries for natural botanicalingredients. Validation of this library includes a database with taxonomic herbariumvouchers of known provenance, genome scans and is validated using analytical chemistry(NMR) as chemical fingerprints. We present here a snap shot of current research projectsat the NHP Research Alliance including DNA testing of botanical extracts and validationmethods for the use of onsite molecular diagnostic tools for quick screening of targetspecies ingredients and adulterants.

Telling Plant Species Apart: Techniques and Applications

Pete M. HollingsworthRoyal Botanic Garden Edinburgh, UKEmail: p.hollingsworth@rbge.org.uk

Biography

Prof. Pete Hollingsworth is Director of Science and Deputy Keeper at the Royal BotanicGarden Edinburgh, an Honorary Professor of the Kunming Institute of Botany, a VisitingProfessor of the University of Edinburgh, and a Visiting Professor of the University ofJohannesburg. His research focuses on understanding and conserving plant biodiversity.He has played a major role in the International Barcode of Life Project aiming to build aunified DNA-based index of life on earth. He also has a strong interest in linking scientificresearch to practical conservation outcomes, and this includes the science ofreintroductions and habitat restoration, understanding pest and pathogen threats to thenatural environment, wildlife forensics, strategies for the conservation of genetic diversity,and large-scale analyses of threats to biodiversity. He also works closely with conservationcharities and is currently the Chair of the Board of Trustees of the BumblebeeConservation Trust.

IC18-I9

Abstract

Peter M. Hollingsworth*; Michelle van der Bank**

*Royal Botanic Garden Edinburgh, 20a Inverleith Row, Edinburgh, UK, EH3 5LRs

**African Centre for DNA Barcoding (ACDB), Department of Botany and Plant Biotechnology, University of Johannesburg, PO BOX 524, Auckland Park, 2006 South Africa

Corresponding Author E-mail : p.hollingsworth@rbge.org.uk

Telling plant species apart is often difficult, especially if the material is sub-optimal orprocessed. This is a particular challenge for the identification of herbal products in trade.In this talk we will present some case-study examples using standard DNA barcodingapproaches to identify the plant components of traditional medicines. We will secondlyexplore complementary and/or extended approaches to DNA barcoding, with anassessment of what is required to increase resolving power. Finally, we will consider howwhat we know, and what we don’t know, about the nature of plant species, requiresconsideration in the ongoing search for the ‘perfect’ rapid, cheap, reliable, high-resolutionplant identification system.

Genome Sequence of Panax ginseng and Genomics-based DNA Barcoding of Important Medicinal Plants in Korea

Tae-Jin YangSeoul National University, KoreaEmail: tjyang@snu.ac.kr

Biography

Prof. Tae-Jin Yang is in the department of plant science at Seoul National University since2006. Before, he worked as researcher in rural development administration, Korea, for 10years. He studied and got master and Ph.D. degree at Seoul National University. He didresearch as postdoctoral researcher at Clemson University and Arizona UniversityGenomics institute for 4 years. His research interests are on the plant genomics, evolution,molecular breeding and NGS-based high-throughput plastid genome sequencing andphylogenomics. He involved in genome sequencing of rice, Brassica crops and nowfocusing on genome sequencing and evolution of Panax ginseng. He developed novelNGS-based pipeline for high throughput complete sequencing of plastid and nuclearribosomal DNA, simultaneously, which is useful for phylogenomics. Recently, he isdeveloping unique DNA markers using the complete sequences of CP genomes andribosomal DNA and chemical markers to authenticate various medicinal plants utilized forfunctional foods or natural medicines which is supported from Ministry of Food and DrugSafety, Korea. Up to now, he published more than 186 SCI papers. Recent years, hepublishes 20-30 papers every year about plant genomics era including DNA barcoding.

IC18-I10

Abstract

Tae-Jin Yang

Department of Plant Science and Plant Genomics and Breeding Institute, College of Agriculture and Life Sciences, Seoul National University, Seoul, 151-921, Korea

Corresponding Author Email: tjyang@snu.ac.kr

There are approximately 300,000 plant species in the world, of which more than 100,000species are somehow worthy as natural products. However, these functional plants stillremain in the state of undeveloped resources which is not suitable for agriculturalproduction. Because the medicinal plant species are so diverse, the production scale ofeach species is small or depends on natural gathering. Breeding to develop superiorcultivars for medicinal plants have not been established or are in the initial stage. In thispresentation, I will introduce the ginseng genome sequence which unveiled the genomicarchitecture of tetraploid P. ginseng by de novo genome assembly, representing 2.98 Gbpwith 59,352 annotated genes. We found two whole genome duplications and continentalmigration which diversify Panax species. We also obtained the complete chloroplastgenomes and 45S nuclear ribosomal DNA sequences from seven Panax species, andrelated genus. We identified 1,128 SNPs in coding sequences (CDSs), distributed among72 of the 79 protein-coding genes in the genomes of the seven Panax species anddeveloped 18 SNP markers from the chloroplast CDS regions that included two or threespecies-specific markers for each species and can be used to authenticate all the sevenPanax species from the others, thereby furthering efforts to protect the ginseng industryfrom economically motivated adulteration. Also, I will briefly introduce DNA barcodingmethods based on plant genomic research and the authentication technology for rawmaterials or processed products for diverse medicinal plants. Finally, I am going torepresent a case which show inadequate application of DNA markers which ignore thegenetic diversity of the originating plants and genome flux. I like to suggest the necessityto make a golden standard for the authentication of plant species using multiple DNAmarkers.

PlantBarcodes: A Plant DNA Barcode Database for Species Identification and Herbal Material Authentication

Madasamy Parani SRM University, IndiaEmail: parani.m@ktr.srmuniv.ac.iin

Biography

Prof. Madasamy Parani has completed his undergraduate degree in Agriculture andPostgraduate degree in Plant Breeding and Genetics from Tamil Nadu AgriculturalUniversity, India. He joined as Research Assistant in M. S. Swaminathan ResearchFoundation, Chennai, India in 1993 and grown up to Scientist in 2002. During this period,he completed his PhD from Anna University, Chennai. Then he worked as Post DoctoralFellow in University of Florida, Florida, USA and Research Assistant Professor in Universityof Toledo, Ohio, USA between the year 2002 and 2006. He joined SRM University, Chennai,India as Assistant Professor in 2006, and currently he is heading the Department ofGenetic Engineering. He is the General Secretary of the Indian Youth Science CongressAssociation since 2010. He has been a visiting scientist to University of Heidelberg(Germany), University of Bologna (Italy), and International Rice Research Institute(Philippines). He has been a visiting professor to University of Guelph (Canada) and TexasA&M University (USA). He has received Prof. Uma Kanth Sinha Memorial Award for YoungScientist from Indian Science Congress (2002) and India DBT-Cutting Edge ResearchEnhancement and Scientific Training Award (2012). He has 50 publications and 2 patentsto his credit. His areas of research include genomics, gene editing and DNA barcoding.

IC18-I11

Abstract

Madasamy Parani*, Durairaj Sherlin, Durga Jayabalan, Pandian E, Booma V

Genomics Laboratory, Department of Genetic Engineering, SRM Institute of Science and Technology, Kattankulathur, Tamil Nadu, India, 603202

Corresponding Author E-mail : parani.m@ktr.srmuniv.ac.iin

DNA barcoding is an effective tool for species identification using any part of the plants inany form. As a result, it is a very much useful tool for authentication of herbal material,which often do not possess intact morphological characters. Here, we reportdevelopment of “PlantBarcodes”, a web-based DNA barcode database, exclusively forplants, which can be used for species identification, and authentication of herbal materialas well. PlantBarcodes is an open development user-friendly database and server, whichcan be used freely. Species identification in PlantBarcodes can be done using rbcL, matK,trnH-psbA, and ITS2 DNA barcode markers. It uses BLAST algorithm for similarity search,however, provides the ability to make use of the indels in calculating identity/divergence.It ranks the species in the increasing order of divergence including indels. For thematching species, the DNA barcode information including chromatogram files areprovided. It also provides the taxonomic description of the species along with the imagesof live specimen and scanned images of herbarium. Users can provide feedback if theyfound any discrepancy in species description or images. It also offers the users to deposittheir data for the purpose of archival and publication. PlantBarcodes is a useful resourceto the researchers as well as all the stakeholders of herbal trade. PlantBarcodes can beaccessed at www.plantbarcodes.org.

Abstracts of Oral Presentations

Biological Authentication of Natural Indian Honey Samples

Mohanasundaram Saravanan and Ramalingam Sathishkumar*

Plant Genetic Engineering Laboratory, Department of Biotechnology, Bharathiar University, Coimbatore, Tamil Nadu, India.

Presenting Author Email: rsathish@buc.edu.inCorresponding Author E-mail: rsathish@buc.edu.in

Abstract

Honey is a natural product highly devoured due its known health benefits, determined bylocal flora and honeybees foraging habit. Honey show vast variation in its composition basedon its geographical location. Hence, determining flora and its origin is essential toauthenticate the natural honey samples. Towards this conventional melissopalynology andmore recent DNA barcoding techniques were used to evaluate the 47 natural honey samplescollected from different districts of Mizoram, India, one of the apiculture hubs. Inmelissopalynology, first pollen grains were isolated and observed under microscope todetermine the plant composition. For DNA barcoding, genomic DNA was extracted from theisolated pollen present in honey samples. PCR amplification was carried out using universalbarcode candidates ITS2 and rbcL to detect the plant species. Further, to detect multipleplant species, TA cloning was carried out to screen the PCR amplicon libraries. Resultsidentified 22 species, which were almost same in melissopalynology and DNA barcodingmethods that comprised native and horticultural plants. Honey samples exhibited aprevalence of pollen types from variety of floral sources. The major taxa were Combretumindicum, Nicotiana tabacum, Cucumis sativus, Amaranthus tricolor and Mikania species, asbee colonies make an independent choice among the species available. It can be concludedthat, melissopalynology / DNA barcoding can be used as the potential tools to know thesource and location of the natural honey. Thus, outcome of this study confirms thatbiological authentication of natural honey will help the food regulators to determine itsmedicinal and commercial value.

IC18-A7

Species Delimitation of Ficus krishnae C.DC. from Ficusbenghalensis L. Complex Based on DNA Barcoding and HP-TLC

Mahima K1, J.V Sudhakar2 and Sathishkumar Ramalingam*1

1, *: Plant Genetic Engineering Laboratory, Department of Biotechnology, BharathiarUniversity, Coimbatore- 641 046 Tamil Nadu. India.

2: Department of Botany, Dr. S.R.K Govt. Arts College, Pondicherry University, UT of Puducherry, Tamil Nadu, India- 533 464, Tamil Nadu. India.

Presenting Author Email: mahimakarthikeyan863@gmail.comCorresponding Author E-mail: rsathish@buc.edu.in

Abstract

In recent times DNA barcoding has been established as a universal tool for speciesdiscrimination using short genetic sequence among plant and animal species. In the presentinvestigation, DNA barcoding was carried out on important closely allied Ficus species, Ficuskrishnae and Ficus benghalensis from India to resolve its taxonomical identity, as they areconsidered synonym by taxonomists. In contrast to the morphological characters, the DNAsequence analysis of universal barcode regions like nuclear DNA (ITS2) and chloroplast DNA(trnH-psbA) for the two species revealed substantial variations in sequence, clearly provingthe disparity between the species at molecular level. ITS2 and trnH-psbA in terms of speciesresolution show 100% species discrimination. Our results based on phylogenetic analysis ofthe concatenated dataset strongly support the view that Ficus krishnae be considered as adistinct species; not a variety of Ficus benghalensis. To confirm the DNA barcoding results,we have performed HP- TLC profiling of Ficus krishnae and Ficus benghalensis, whichshowed significant difference in the alkaloids, flavonoids and terpenoids profiles. From thisstudy, it’s clear that both DNA barcodes and HP-TLC profiling is useful to delineate theclosely related species.

IC18-A10

DNA Technologies for Botanical Ingredient Authentication: A Quality Control Perspective

Zhengfei Lu

Herbalife Nutrition, Suite 650, 990 West 190th Street, Torrance, CA 90502.

Presenting Author Email: yanjunzh@herbalife.comCorresponding Author E-mail: yanjunzh@herbalife.com

Abstract

Current good manufacturing practices (cGMPs) require dietary supplement manufacturersto conduct at least one appropriate test to verify the identity of any component to be usedin their products. Identification and authentication of botanical ingredients presentchallenges due to profile complexity and damage to DNA during processing. Because of thespecificity of DNA sequences, DNA-based methods are valuable contributions in the toolboxof botanical authentication, which previously contained only morphological and chemicalmethods. Depending on the circumstances, DNA technologies may be the most viable toolto precisely define the target botanical in supply chain management, as well as tracingmaterial identity throughout the manufacturing processes and to detect potentialadulterants in finished products. Herbalife leads the dietary supplement industry in DNA-based botanical authentication. Here, we present an example on how different DNAtechnologies are applied to trace and qualify botanical ingredients from starting botanicalparts before manufacturing to finished products delivered to the consumer.

IC18-A16

Developing a DNA Barcoding System for Plant Materials with Low Quality Genomic DNAs Using Next-generation Sequencing

Chun-Xia Zeng1, Peter M. Hollingsworth2, Jing Yang1, Zheng-Shan He1, Zhi-Rong Zhang1, De-Zhu Li1*, Jun-Bo Yang1*

1. Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan 650201, China.

2. Royal Botanic Garden Edinburgh, 20A Inverleith Row, Edinburgh, EH3 5LR UK.

Presenting Author Email: zengcx@mail.kib.ac.cnCorresponding Author E-mail: dzl@mail.kib.ac.cn, jbyang@mail.kib.ac.cn

Abstract

“Next-generation” massive parallel sequencing or NGS technology fits in well with thefragmented nature of herbarium-derived DNAs. Furthermore, sequencing libraries for thehistorical specimens using only 500 pg of DNA input in each library have been generatedsuccessfully. High-copy number compartments such as plastomes and rDNA can beobtained on a routine basis. As a practical test of routine recovery of plastiomic and rDNAsequences from herbarium specimens or highly processed plant material, in forms of leaves,roots, seeds, and even processed powders which lack key morphological diagnosticcharacters, we sequenced 80 species of such samples. Paired-end reads were generated,successfully yielding plastomic assemblies for 73 species and nuclear rDNAs for 62 species,respectively. In addition, the standard DNA barcodes, rbcL from 74 out of 80 samples andmatK from 75 out of 80 samples, were recovered and were subjected to a blast searchagainst NCBI database. In all cases, the closest match to the sequence was from the correctgenus (55/75) and species (20/75). This study confirmed that NGS was shown to be aneffective and cost-efficient way to authenticate herbarium-derived or highly processedtraditional Chinese medicine (TCM) plant materials. Besides plastid sequences,mitochondrial genes, nuclear genes, and microsatellite (SSR) loci would be the furtherexplored in resulting sequences and could be developed for species identification.

IC18-A21

A Rapid and Accurate Identification Method for Opium Poppy Seedings

Zhi-Yun Yang, Ting-Shuang Yi, Jie Cai, Hong Wang*, Jun-Bo Yang*

Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan 650201, China.

Presenting Author Email: zhangzhiyun@mail.kib.ac.cnCorresponding Author E-mail: wanghong@mail.kib.ac.cn, jbyang@mail.kib.ac.cn

Abstract

Papaver somniferum L., commonly known as opium poppy is a famous medical plant used asopioid analgesics, and is widely planted and illegally traded as a hard drug. To find a rapidand efficient way of drug ban, it is urgently needed to establish a molecular identificationsystem to identify poppy seeds and seedlings. Taking the advantages of next-generationsequencing (NGS) technologies, we skimmed the Papaver somniferum genome with ca. 2 Gpraw data, and developed 254 genomic SSR sites. After verification in three individuals fromPapaver somniferous and other two morphologically similar species by gel electrophoresis,121 SSR sites were chosen for further screening in nine individual of Papaver somniferousand the other 52 individuals which are from 8 species Papaver and Roemeria refracta, aclose relative of poppy.

We designed four SSR primer pairs (PSS5, PSS20, PSS70, and PS120) and successfullyamplified unique SSR segments in Papaver somniferum with negative results in all othersampled species. After pairwise matching, PSS20 and PS120, generating clear strips of ca.300 bp and 100 bp respectively, were chosen for further multiplex PCR. AtpB200, which canproduce ca. 200 bp strip in most angiosperm species, was added as an internal controlprimer pair. We established a multiplex PCR proto- col to get three segments of different sizeat the same time. The optimal proportion of PSS20: PS120: atpB200 is 8:10:2.5. The 17 μlPCR reaction system comprises 10μl 2× Taq Mix, 4μl ddH2O, 2μl mixed primers, and 1μl DNA.The optimized PCR condition is listed as follows: 95°C for 4 min, then 35 cycles of 94°C for 20sec, 55°C for 20 sec, and 72°C for 20 sec, followed by 72°C for 4 min. PCR products weredetected by agarose gel (4%) electrophoresis. Taking all three bands (100 bp, 200bp, 300bp)together into consideration, we can effectively identify Papaver somniferum samples. Onlyone band of 200 bp represents other species and absence of 200 bp band indicates PCRfailure. Our newly-developed method can identify opium poppy accurately in six hours.

IC18-A23

Identification and Spatiotemporal Regulation of the Patchoulol Synthase Gene Responsible for Sesquiterpene Biosynthesis in Different PogostemonCablin (Blanco) Benth. Cultivars

Xindan Liu1, Ying Zhang1,*, Menghua Wu2, Hui Cao2

1. College of Pharmacy, Jinan University, Guangzhou, China.2. Research Center for Traditional Chinese Medicine of Lingnan, Jinan University,

Guangzhou, China.

Presenting Author Email: 627983246@qq.comCorresponding Author E-mail: zhangying@jnu.edu.cn

Abstract

Pogostemon cablin (patchouli) is an aromatic medicinal plant which accumulates largeamounts of essential oils. The tertiary sesquiterpene alcohol patchoulol is one of the majorbioactive constituents. The profile of phytochemical volatiles in various patchouli cultivarsand their importance as aroma compounds have been studied in detail, but much is stilllacking in our understanding of the genetic regulation of their production. Here, we reporton the identification and spatiotemporal regulation of PTS, the patchoulol synthase-encoding gene, involved in sesquiterpene aroma synthesis. The recombinant PTS wasidentified as patchoulol synthase by liquid chromatography-mass spectrometry (LC-MS) andwestern blotting. Real-time PCR analyses indicated that the optimum harvest season(reflecting the appropriate developmental stage of the plant) of the HN-1, HN-2, SP-1, YN-1,YN-2, and GY cultivars was in November, whereas the best time to harvest the SP-2 cultivarwas in August. In addition, phytochemical assay revealed that the patchoulol concentrationsin stem were positively associated with those in leaf. This work will not only be beneficial tothe patchouli resources conservation but also provide a basis for the full development andutilization of patchouli resources.

IC18-A31

Zhen-Tracking: A Block-Chain Based Tracking System Based on Genetic Fingerprints

Chang Liu1, Weiwei Fang2, Feng Xue2

1. Institute of medicinal Plant Development, Chinese Academy of Medical Science, 151 Malinawa North Road 151, Beijing, China.

2. School of Computer and Information Technology, Beijing Jiaotong University, 3 Shang Yuan Cun, Haidian District, Beijing, China.

Presenting Author Email: cliu6688@yahoo.comCorresponding Author E-mail:

Abstract

The Chinese Food and Drug Administration has issued the guidance for constructing trackingsystems to ensure the quality and safety of drugs. However, the classical tracking system hasdifficulty for wide application due to the lack of trust among the participants. Recently, theblock chain technology has been used in many areas due to its decentralized, distributedand immutable characteristics. Here, we constructed a tracking system based on geneticfingerprints of medicinal plants and the block-chain technology, called “Zhen-Tracking”,using the Python programming language. The system includes a mobile client and a block-chain based data storage system. The client app can be used by everyone, for example,those involved in the production, sale and consumption of the products by scanning the QRcode containing the genetic information of the products to be tracked. For the producers,they provide the genetic fingerprints of the plant materials to construct a QR code, whoseinformation will be stored on the chain. For the sale persons, they scan the code to store therelevant circulation information on the block chain. For the consumers, they can scan the QRcode to obtain the information about the sources and circulation paths of the products. TheZhen-Tracking system have several advantages such as large capacity, high degree of security,free of centralized management and etc. In a decentralized global environment, the systemwill be invaluable for ensuring of the safety and efficacies of products of health food andtraditional medicines.

IC18-A40

DNA Barcoding in Drug Discovery: From Bench to Bedside

Suchada Sukrong*

1. Research Unit of DNA barcoding of Thai Medicinal Plants, Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand.

Presenting Author Email: suchada.su@chula.ac.thCorresponding Author E-mail: suchada.su@chula.ac.th

Abstract

Camptothecin (CPT), a plant-derived compound, is being used as an anticancer. CPT is anatural tryptophan-derived quinoline alkaloid exhibiting anticancer activity due to its abilityto inhibit topoisomerase I (TopI) enzyme involved in DNA topology. There are a number ofplants reported to contain CPT including some Ophiorrhiza spp.. The production of CPT andits derivatives in the genus Ophiorrhiza found in Thailand were explored as a process of drugdiscovery. Two DNA barcode regions, chloroplast matK and the targeted nuclear TopI gene,were utilized to determine the species within Ophiorrhiza. The phylogenetic tree revealed amajor clade of Ophiorrhiza taxa correlated with production of CPT and its derivatives. Itsuggested that the genetic makeup tends to affect the qualitative and quantitativeproperties of a phytochemical such as CPT. Consequently, matK and TopI gene could be usedfor the prediction of CPT production ability of any members of Ophiorrhiza. Additionally, themutation points in the TopI amino acid sequences also supported the nucleotidephylogenetic tree on the prediction of CPT-producing ability. Interestingly, the substitution atposition 722 (Asn722Ser) is identical to that found in CPT-resistance human cancer cells. Theother mutations have not been found to date in CPT-resistance human cancer cells. Weproposed that unique amino acid substitutions in TopI of CPT-producing plants could beused as amino acid markers toward recognition of the point mutations in CPT-resistancecancer patients in the future. Besides, comparison of the TopI mutation in producing andnonproducing plants may be exploited for designing new drugs to prevent resistance incancer cells and combat drug resistance in cancer patients.

IC18-A52

Combined DNA Barcoding and HPTLC for Differentiation of the Poisonous Urobotrya Samensis from the Edible Melientha Suavis and Sauropus Androgynus for Food Safety

Kannika Thongkhao1,2, Thatree Padungchareon3, Suchada Sukrong1,2*

1. Research Unit of DNA barcoding of Thai Medicinal Plants, Chulalongkorn University Drug and Health Product Innovation Promotion Center, Chulalongkorn University, Bangkok, Thailand.

2. Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, 3Rangsit University, Pathumthani, Thailand.

Presenting Author Email: kannika.thongkhao@gmail.comCorresponding Author E-mail: suchada.su@chula.ac.th

Abstract

Urobotrya siamensis Hiepko (Phak Wan Mao) was a cause of death in Southeast Asiacountries due to the confusing with Melientha suavis Pierre (Phak Wan Pa). Both plantsbelong to Opiliaceae family and their leaves look very similar. M. suavis is now popularserved as vegetable for dishes and production of this species tends to increase according tothe demand of the market. However, Sauropus androgynous (Phak Wan Ban), edible plant inthe Phyllanthaceae, also shares the same Thai vernacular name with U. siamensis and M.suavis. Leaves of S. androgynus have been reported to have nutritive value. Hence, the aimof this study is to differentiate the poisonous U. siamensis from the edible species, S.androgynus and M. suavis, using the DNA barcoding couple with HPTLC techniques. CoreDNA barcode regions, rbcLa, matK, ITS and psbA-trnH, were amplified and sequenced.Polymorphic sites were detected from rbcLa gene. Indels were observed in psbA-trnH regionof the U. siamensis and led to size different of the PCR products. The chemical profiles of thetoxic and edible species showed unique patterns. This study is the first report that combinedDNA barcoding and chemical profile for differentiation of poisonous plant from the edibleones. The successful of this study will support us the knowledge for developing a routinedetection tool to detect the poisonous U. siamensis contamination for food safety.

IC18-A59

Chloroplast and Mitochondrial Genome Flux Makes Confusion for Plant DNA Barcoding

Hyun-Seung Park, Sae Hyun Lee, Jae-Hyeon Jeon, Jee Young Park and Tae-Jin Yang*

Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea.

Presenting Author Email: fgenesis@snu.ac.krCorresponding Author E-mail: tjyang@snu.ac.kr

Abstract

Food adulteration, also known as economically motivated adulteration (EMA), of herbalproducts is a severe threat both to human health and to herb-based industries. AlthoughDNA barcoding is a promising approach for preventing EMA, major unaddressedshortcomings of barcoding techniques have caused substantial interruption of the herbalindustry, including recent cases in Korea and the USA. Here, our de novo assembly of theplastid and mitochondrial genomes of the Cynanchum wilfordii and C. auriculatum in Korearevealed that their organellar genomes contain a substantial proportion of mitochondrialsequences of plastid origin (MTPTs). A broad survey of MTPTs in more than 80 plantsincluding model and non-model species found dynamic and lineage-specific plastid–mitochondrial genome flux that has diversified the mitochondrial genome structure. Further,we demonstrate a DNA marker paradox derived from co-amplification of MTPTs can causemis-authentication of herbal products. We propose a guideline of using multiple targetmarkers for EMA regulation to protect both human health and the integrity of the herbalsupplement and functional food industry.

Acknowledgement: This work was carried out with the support of the Cooperative ResearchProgram for Agriculture Science & Technology Development (grant number, PJ013238) RuralDevelopment Administration, Republic of Korea.

IC18-A61

Phylogenetic Signal of Steroidal and Glyco-alkaloids in Genus Solanum Based on DNA Barcodes and HPTLC Fingerprints

U. Senthilkumar1,2*, N. Kasthuri Bai2, R. Pandian2, K. Ilango2, M. Parani1

1. Centre for DNA Barcoding, Department of Genetic Engineering, School of Bioengineering, SRM Institute of Science and Technology, Kattankulathur, Chennai-603 203, India.

2. Interdisciplinary Institute of Indian System of Medicine (IIISM), SRM Institute of Science and Technology, Kattankulathur, Chennai- 603 203, India.

Presenting Author Email: sensonsam85@gmail.comCorresponding Author E-mail: sensonsam85@gmail.com

Abstract

Plants have evolved with high diversity of alkaloids either for their defenses (allelochemicals)or survival, which leads to evolution of specialized metabolites and its natural derivatives.The steroidal alkaloids and glycol-alkaloids are such ones. Phylogenetic approach offers apredictive approach on selection of plants based on the evolutionary relatedness that leadsto discovery of novel drugs. However, the analysis of combined data from both phylogenyand phytochemical investigations are poorly established. In this study, we have created thephylogenetic framework for Solanum (Solanaceae), which is an important genus bothmedicinally and commercially. Maximum likelihood and Bayesian analyses were carried outusing both nuclear (ITS2) and plastid (ndhF, trnL-trnF) DNA sequences for 25 species fromIndia. We have investigated the steroidal-alkaloides and glyco-alkaloids i.e. α-Solanine,Solasodine, Solanidine from the leaves and berries based on High Performance Thin LayerChromatography (HPTLC) and found significant phylogenetic signal at interspecific level. Inthis study, we have also have mapped the metabolites to derive the phylogeneticrelationships between crop and non-crop species based on the presence and absence dataas well as the percentage of the metabolites (quantified) present. In conclusion, the studyreveals that the phylogenetic signals suggest in selection of non-poisonous wild Solanumspecies and to avoid the excessive intake of crops (edible Solanum) based on the content ofsteroidal alkaloids that are known to be poisonous at higher dosage.

IC18-A62

The Role of DNA Technology in Setting Pharmacopoeia Standards of Chinese Materia Medica

Kelvin Chan

School of Pharmacy & Biomolecular Sciences, Liverpool John Moores University, Liverpool L3 3AF, UK and National Institute of Chinese Medicine, Western Sydney University, NSW 2560, Australia.

Presenting Author Email: k.k.chan@ljmu.ac.ukCorresponding Author E-mail: k.k.chan@ljmu.ac.uk

Abstract

Traditional Chinese medicine (TCM) has its values, in healthcare and medical treatments ofdiseases where western medicine has difficulties to cope, as understood by most Chinesecommunities and some open-minded people in other regions worldwide [1, 2]. Chinaoccupies majority of the world market share in exporting crude medicinal plant parts asChinese materia medica (CMM) and supplies other TCM knowhows overseas [3]. Yet veryfew registered TCM products as drugs or functional supplements gain access to theregulatory systems abroad.

Problems and difficulties are recognised by manufacturers in coping with regulatoryrequirements regarding the quality, safety and efficacy (QSE) of herbal medicinal productscontaining various chemical entities in the finished products; as only few bioactivecomponents are known [4]. Therefore, the good practices in every aspect from initialmedicinal plant materials to final products with assured QSE are the key roles to providetraceability of the starting materials used for producing the final product for registrationsubmission. Pharmacopoeia standard of starting materials for research and manufacturing isthe minimum requirement to comply with the regulatory guidelines for registration purpose[4].

The introduction of DNA technology for QC tracking CMM’s origin has added value on theidentity and traceability but the chemical profile is key for clinical efficacy [5]. It will bebeneficial for identification of CMM when chemical and other tests fail. This lecture willfocus on areas that DNA technology will be applied in monitoring quality of medicinal plants.

References1. J Ethnopharmacology, 2012; 140: 455-642.2. World Journal of TCM, 2016; 2(4): 6-28.3. Helmut Kaiser Consultancy HKC22, Traditional Chinese Medicine: Market, Development,

Products and Companies, 2010-2015-2025>. www.hkc22.com4. BMC Chinese Medicine, 2009, 4: 18.5. Biotechnology Advances, 2014, 32: 1237-1244.

IC18-A63

A Polysaccharide Marker for Authentication of Cordyceps

Li-Shuang Zhou1, Karen Wu2, Miao-Miao Chen1, Grace But2, Xin Wen1, Hau-Yee Fung1, Pang-Chu Shaw2,*, Quan-Bin Han1,*

1. School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China.2. School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China.

Presenting Author Email: simonhan@hkbu.edu.hkCorresponding Author E-mail: pcshaw@cuhk.edu.hk; simonhan@hkbu.edu.hk

Abstract

Chemical marker based methods have been popularly used in quality control of Chinesemedicines, however, all these chemical markers are small molecules, and the quality controlof many saccharide-dominant Chinese medicines remains a challenge without properchemical markers. Cordyceps sinensis (CS) is right such an example. Conventional methods,like DNA barcoding and microscopic authentication, involve time-consuming, complicated,and high-cost operations. More importantly, they all fail to provide quantitative qualityevaluation.

Our preliminary studies have found a unique polysaccharide CSP which only exists in thecaterpillar part of C. sinensis, but not in samples of other Cordyceps species we have. Wehypothesize that CSP might be a proper chemical marker to be used in the qualityassessment of CS. This project aims to verify CSP’s specificity to CS by large-scale sampleanalysis via comparative study of chemical/DNA analysis. The success of CSP to be used aschemical marker in quality control of CS will help identify a new quality control parameterfor Chinese medicines.

IC18-A66

Inter- and Intra- Chloroplast Genome Diversity and Classification of Germplasm in Cynanchum Species

Sae Hyun Lee, Hong-Seob Yu, Ho Jun Joh, Hyun-Seung Park, Jee Young Park, Tae-Jin Yang *

Department of Plant Science and Plant Genomics and Breeding Institute, College of Agriculture and Life Sciences, Seoul National University, Seoul, 08826, Korea.

Presenting Author Email: whyskyisgray@naver.comCorresponding Author E-mail: tjyang@snu.ac.kr

Abstract

Cynanchum wilfordii has long been regarded as a medicinal plant in Korea. Because of itsvarious pharmacological effects, especially for menopause, the annual production hasincreased continuously. However, morphological similarity between C. auriculatum, and C.wilfordii may cause Economically Motivated Adulteration (EMA) in the market. Therefore, itis necessary to develop authentication methods to distinguish two species. Most of themolecular markers are designed from chloroplast genome sequences for the identificationof plant species. For Cynanchum species, several markers have been developed for speciesidentification, but they often fail to do so which might be caused by intra-species variationsfrom natural diversity. Therefore, to develop more stable authentication markers to protectEMA issue, we assembled complete chloroplast genome and 45s rDNA of four C. wilfordiiusing NGS data with de novo assembly of low coverage WGS sequence. By comparativeanalysis, we identified six SNPs and five InDels in the chloroplast genome of four C. wilfordiiaccessions. Four SNPs were identified in the intergenic region and two SNP was found in theexon region. We also identified five InDels, two in the intergenic region and three in theexon region. Markers that can verify inter- and intra- chloroplast genome diversity in twoCynanchum species were developed and applied to breeding line germplasms collected fromall over Korea. As a result, the germplasms were grouped into thirteen types. This researchwas supported by “Cooperative Research Program for Agriculture Science & TechnologyDevelopment (Project No. PJ013238)”, Rural Development Administration, Republic of Korea.

IC18-A71

Medicinal Materials DNA Barcode Database (MMDBD) v1.5 – One-stop Platform for Storage, BLAST, Alignment & Primer Design

HY Wu1,2, TH Wong1,3, Grace WC But2, Stacey SK Tsang3, David TW Lau3 and, PC Shaw1,2

1. Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong, China.2. LDS YYC R & D Centre for Chinese Medicine, The Chinese University of Hong Kong, Hong

Kong, China.3. SY Hu Herbarium, School of Life Sciences, The Chinese University of Hong Kong, Hong

Kong, China.

Presenting Author Email: karenwhy@cuhk.edu.hkCorresponding Author E-mail: pcshaw@cuhk.edu.hk

Abstract

Authentication of medicinal materials by DNA technology is gaining popularity in herbalindustry. In 2010, our team has created Medicinal Materials DNA Barcode Database(MMDBD) version 1.0, an interactive database of DNA barcode sequences of medicinalmaterials. MMDBD now contains DNA barcode sequences of medicinal materials listed inthe Chinese Pharmacopoeia, Dietary Supplements Compendium and Herbal MedicineCompendium of the US Pharmacopoeia and selected adulterants. The data archive isregularly updated and currently it stores 65 520 DNA sequences of 2225 medicinal materials.A major improvement has been carried out on the interfaces and essential bioinformaticstools were incorporated to facilitate the authentication work.

MMDBD version 1.5 contains detailed information of each medicinal material, including thematerial name, medical part, pharmacopeia information, biological classification in rank offamily and its status on the Convention on International Trade in Endangered Species of WildFauna and Flora and the International Union for Conservation of Nature’s Red List ofThreatened Species, if any. User can retrieve DNA sequences by search in Latin scientificname, Chinese name, family name, material name, medical part and simplified Chinesecharacter stroke. A `BLAST’-based engine is included in MMDBD v1.5 to allow searching DNAsequence based on similarity. In addition, we have integrated the `Clustal Omega alignmenttool’ and `Primer3’ in the form of web interface for easy primer design. These new toolsfacilitate multiple sequence comparison and the design of primers for amplification of atarget DNA barcode region, allowing DNA barcoding authentication.

IC18-A78

A Rapid Sample-to-answer Analytical Detection of Genetically Modified Papaya Using Loop-mediated Isothermal Amplification Assay on Lab-on-a-discfor Field Use

Jacky FC Loo1,2 *, Grace WC But1 *, HC Kwok3, PM Lau1, SK Kong1, HP Ho2, PC Shaw1,4

1. School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China.2. Department of Biomedical Engineering, The Chinese University of Hong Kong, Hong

Kong, China.3. Department of Electronic Engineering, The Chinese University of Hong Kong, Hong Kong,

China.4. Li Dak Sum Yip Yio Chin R & D Centre for Chinese Medicine and Institute of Chinese

Medicine, The Chinese University of Hong Kong, Hong Kong, China.

Presenting Author Email: gracebut@link.cuhk.edu.hkCorresponding Author E-mail: pcshaw@cuhk.edu.hk

Abstract

With genetically modified (GM) food circulating on the market, a rapid transgenic foodscreening method is needed to protect consumer rights. The on-site screening efficiency ofGM food testing is low. We report rapid sample-to-answer detection of GM papayas withloop-mediated isothermal amplification (LAMP) and a compact, portable, integratedmicrofluidic platform using microfluidic lab-on-a-disc (LOAD). GM samples weredifferentiated 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. Thedetection limits for DNA and juice from papaya were 10 pg/μL and 0.02 μL, respectively. OurLOAD platform is a simple and robust solution for GM screening, which is anticipated to be afoundation for on-site testing of transgenic food.

IC18-A79

Way to Reveal Unknown Species Identity in Processed Materials - Development of Adaptor Ligation-mediated PCR Technology with DNA Barcoding

Yat-Tung Lo and Pang-Chui Shaw*

1. Li Dak Sum Yip Yio Chin R & D Centre for Chinese Medicine, State Key Laboratory of Research on Bioactivities and Clinical Applications of Medicinal Plants (CUHK) and School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China.

Presenting Author Email: ytlo@cuhk.edu.hkCorresponding Author E-mail: pcshaw@cuhk.edu.hk

Abstract

DNA barcoding is a commonly used laboratory technique for molecular authentication ofherbal materials. Universal primers are used to amplify segment of DNA with severalhundred base-pair in size before it is sequenced for species identification. However, suchPCR amplification in processed products is difficult because of severe DNA fragmentation.The use of “DNA mini-barcode” to amplify smaller DNA fragments is often limited to certainherbal species. To maximize the usefulness of currently available universal barcodingprimers, an adaptor ligation-mediated PCR protocol was derived to amplify sets of targetDNA fragments isolated from herbal products. The specially designed adaptor withasymmetric strands and terminal modification avoids amplification of non-target DNAsequences. DNA extracted from medicinal granules of Angelica sinensis, Hedyotis diffusa andPanax notoginseng were ligated with the adaptors and amplified by only one side of theuniversal barcode primers plus an adaptor primer to obtain partial ITS2 or psbA-trnHsequences. Results showed that various length of DNA fragments within the target DNAregions were amplified and could be used to identify the concerned species. When thistechnique is combined with high-throughput sequencing technologies, the species identityand sample quality of unknown herbal products can be quickly revealed. The adaptorligation-mediated PCR is therefore a promising universal method for species identification inprocessed herbal products.

IC18-A80

Population Genomics of Incense Trees Aquilariasinensis in Hong Kong

Sean Tsz Sum Law1, Wenyan Nong1, Annette Wong1, Zhe Qu1, Ho Yin Yip1, Nianhe Xia2, Lee Man Chu1, David T.W. Lau1, Jerome H.L. Hui1

1. School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China.2. Chinese Academy of Sciences, Guangzhou, China.

Presenting Author Email: jeromehui@cuhk.edu.hkCorresponding Author E-mail: jeromehui@cuhk.edu.hk

Abstract

The Incense tree Aquilaria sinensis (Lour.) Spreng, belongs to the plant familyThymelaeaceae, and is native/endemic to the southern China including Hong Kong. Indeed,it is commonly believed that Hong Kong derives its name from this evergreen tree (i.e.Incense/Fragrant Harbour). A special feature that distinguishes the Incense tree from theother plants is that they can produce agarwood or Chen Xiang, whereas the trading of whichused to be an important industry in Hong Kong. Owing to the decline in its distribution dueto habitat loss, Incense tree is currently classified under the “List of Wild Plant Under StateProtection (Category II)” in mainland China, and is defined as the vulnerable species in theIUCN Red List of Threatened Species in CITES. Surprisingly, those that are planted in HongKong have not been affected from these natural threats. In other words, the Incense trees A.sinensis in Hong Kong represent an important conservation target for China and the world.Here, we have sequenced genomes of A. sinensis collected in different places in Hong Kong,in light to understand its population structure as well as potentially implement thisknowledge practically on its conservation.

IC18-A84

Abstracts of Poster Presentations

Biological Authentication of Natural Indian Honey Samples

Mohanasundaram Saravanan and Ramalingam Sathishkumar*

Plant Genetic Engineering Laboratory, Department of Biotechnology, Bharathiar University, Coimbatore, Tamil Nadu, India.

Presenting Author Email: rsathish@buc.edu.inCorresponding Author E-mail: rsathish@buc.edu.in

Abstract

Honey is a natural product highly devoured due its known health benefits, determined bylocal flora and honeybees foraging habit. Honey show vast variation in its composition basedon its geographical location. Hence, determining flora and its origin is essential toauthenticate the natural honey samples. Towards this conventional melissopalynology andmore recent DNA barcoding techniques were used to evaluate the 47 natural honey samplescollected from different districts of Mizoram, India, one of the apiculture hubs. Inmelissopalynology, first pollen grains were isolated and observed under microscope todetermine the plant composition. For DNA barcoding, genomic DNA was extracted from theisolated pollen present in honey samples. PCR amplification was carried out using universalbarcode candidates ITS2 and rbcL to detect the plant species. Further, to detect multipleplant species, TA cloning was carried out to screen the PCR amplicon libraries. Resultsidentified 22 species, which were almost same in melissopalynology and DNA barcodingmethods that comprised native and horticultural plants. Honey samples exhibited aprevalence of pollen types from variety of floral sources. The major taxa were Combretumindicum, Nicotiana tabacum, Cucumis sativus, Amaranthus tricolor and Mikania species, asbee colonies make an independent choice among the species available. It can be concludedthat, melissopalynology / DNA barcoding can be used as the potential tools to know thesource and location of the natural honey. Thus, outcome of this study confirms thatbiological authentication of natural honey will help the food regulators to determine itsmedicinal and commercial value.

IC18-A7

Computational Identification of miRNAs from Allium cepa L Predicts its Therapeutic Potential

Dhivya Selvaraj* and Sathishkumar Ramalingam

Plant Genetic Engineering Laboratory, Department of Biotechnology, Bharathiar University, Coimbatore - 641046, India.

Presenting Author Email: bioinfodhivya@gmail.comCorresponding Author E-mail: rsathish@buc.edu.in

Abstract

Advancement of massive parallel sequencing facilitates a quick execution of genomicsequencing in therapeutics. Genome sequence is an important tool for evaluating unusualdisorders and therapeutic targets. MicroRNAs (miRNAs) are a class of approximately 22nucleotides single-stranded non-coding RNA molecules that plays an essential role in bothgene expression and silencing. It has been reported that miRNAs might be one of the hiddenbioactive ingredients in medicinal plants. Allium cepa L (Liliaceae) is rich in nutrients, henceit is used to boost immunity, to treat cancer and other disorders like cholesterol, diabetesetc. Flavour precursors like cysteine and glutathione, essential processes such as sulphuruptake by plants, redox homeostasis and xenobiotic detoxification. In this study, we proposeA. cepa miRNAs might be involved in the pharmacological activities. As nucleotidesequences of miRNAs are highly conserved among the plants by homology alignment it canbe identified. EST sequences of Allium cepa L (Onion) were searched against known miRNAsequences using BLASTN tool and potential miRNA were identified from 20225 ESTs and10725 of GSS (Genome survey sequence) sequences. Conserved and novel miRNAs wereidentified from Allium cepa and potential human genes have been predicted usingTargetScan, miRanda, PITA, and RNAhybrid methods. Furthermore, for understanding ofmiRNA-based regulation of bioactive ingredients in Allium cepa L, the genes involved interpenoids, lectins and viscotoxins biosynthesis have been characterized, and theircorresponding regulatory miRNAs have been predicted. The outcome of this study mightfacilitate bioengineering research for the production of pharmacologically activecomponents.

IC18-A9

meatIDNGS: A Method for Species Detection of Mixture of Commonly Used Animal Sample

Mei Jiang, Chang Liu*

Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, PekingUnion Medical College, Beijing 100193, China.

Presenting Author Email: mjiang0502@163.comCorresponding Author E-mail: cliu6688@yahoo.com

Abstract

The DNA barcoding technology was used to identify the species source ofbiological material. However, the commonly used PCR-based approach can only detect thespecific specie. We aimed to develop a method that are able to identify biologicalingredients from multiple unknown species. Here we developed a software package(meatIDNGS) for species identification of animal mixture using the next generationsequencing (NGS) technology. The software package mainly includes a database and twoprograms. The database consists of the mitochondrial genome of 18 commonly used animalspecies: Anas platyrhynchos, Bos taurus, Camelus bactrianus, Canis lupus familiaris, CervusNippon hortulorum, Equus asinus, Equus caballus, Gallus gallus, Mus musculus, Mustelaputorius voucher, Myocastor coypus, Neovison vison, Oryctolagus cuniculus, Ovis aries,Nyctereutes procyonoides, Rattus norvegicus, Sus scrofa domesticus and Vulpes vulpes. Then,we used the NGS data of mixed meat with 18 species for qualitative testing and used theNGS data containing the mixtures of pork and chicken in different proportions forquantitative testing. And, we compared the ability of species delimitation between thecomplete mitochondrial genome sequences and the partial (COX1 gene, 12S rRNA, 16S rRNA)using the meatIDNGS. The results showed that the software could be used for thequantitative analysis of mixed sample without prior assumption. It is capable to be appliedto the species identification of Chinese patent medicine ingredients, which needs to beexperimental verification.

IC18-A13

DNA Technologies for Quality Control of Botanicals: An Industry Perspective

Zhengfei Lu

Herbalife Nutrition, Suite 650, 990 West 190th Street, Torrance, CA 90502.

Presenting Author Email: yanjunzh@herbalife.comCorresponding Author E-mail: yanjunzh@herbalife.com

Abstract

Current good manufacturing practices (cGMPs) require dietary supplement manufacturersto conduct at least one appropriate test to verify the identity of any component to be usedin their products. Identification and authentication of botanical ingredients presentchallenges due to profile complexity and damage to DNA during processing. Because of thespecificity of DNA sequences, DNA-based methods are valuable contributions in the toolboxof botanical authentication, which previously contained only morphological and chemicalmethods. Depending on the circumstances, DNA technologies may be the most viable toolto precisely define the target botanical in supply chain management, as well as tracingmaterial identity throughout the manufacturing processes and to detect potentialadulterants in finished products. Herbalife leads the dietary supplement industry in DNA-based botanical authentication. Here, we present an example on how different DNAtechnologies are applied to trace and qualify botanical ingredients from starting botanicalparts before manufacturing to finished products delivered to the consumer.

IC18-A16

Identification and Spatiotemporal Regulation of the Patchoulol Synthase Gene Responsible for Sesquiterpene Biosynthesis in Different PogostemonCablin (Blanco) Benth. Cultivars

Xindan Liu1, Ying Zhang1,*, Menghua Wu2, Hui Cao2

1. College of Pharmacy, Jinan University, Guangzhou, China.2. Research Center for Traditional Chinese Medicine of Lingnan, Jinan University,

Guangzhou, China.

Presenting Author Email: 627983246@qq.comCorresponding Author E-mail: zhangying@jnu.edu.cn

Abstract

Pogostemon cablin (patchouli) is an aromatic medicinal plant which accumulates largeamounts of essential oils. The tertiary sesquiterpene alcohol patchoulol is one of the majorbioactive constituents. The profile of phytochemical volatiles in various patchouli cultivarsand their importance as aroma compounds have been studied in detail, but much is stilllacking in our understanding of the genetic regulation of their production. Here, we reporton the identification and spatiotemporal regulation of PTS, the patchoulol synthase-encoding gene, involved in sesquiterpene aroma synthesis. The recombinant PTS wasidentified as patchoulol synthase by liquid chromatography-mass spectrometry (LC-MS) andwestern blotting. Real-time PCR analyses indicated that the optimum harvest season(reflecting the appropriate developmental stage of the plant) of the HN-1, HN-2, SP-1, YN-1,YN-2, and GY cultivars was in November, whereas the best time to harvest the SP-2 cultivarwas in August. In addition, phytochemical assay revealed that the patchoulol concentrationsin stem were positively associated with those in leaf. This work will not only be beneficial tothe patchouli resources conservation but also provide a basis for the full development andutilization of patchouli resources.

IC18-A31

Zhen-Tracking: A Block-Chain Based Tracking System Based on Genetic Fingerprints

Chang Liu1, Weiwei Fang2, Feng Xue2

1. Institute of medicinal Plant Development, Chinese Academy of Medical Science, 151 Malinawa North Road 151, Beijing, China.

2. School of Computer and Information Technology, Beijing Jiaotong University, 3 Shang Yuan Cun, Haidian District, Beijing, China.

Presenting Author Email: cliu6688@yahoo.comCorresponding Author E-mail:

Abstract

The Chinese Food and Drug Administration has issued the guidance for constructing trackingsystems to ensure the quality and safety of drugs. However, the classical tracking system hasdifficulty for wide application due to the lack of trust among the participants. Recently, theblock chain technology has been used in many areas due to its decentralized, distributedand immutable characteristics. Here, we constructed a tracking system based on geneticfingerprints of medicinal plants and the block-chain technology, called “Zhen-Tracking”,using the Python programming language. The system includes a mobile client and a block-chain based data storage system. The client app can be used by everyone, for example,those involved in the production, sale and consumption of the products by scanning the QRcode containing the genetic information of the products to be tracked. For the producers,they provide the genetic fingerprints of the plant materials to construct a QR code, whoseinformation will be stored on the chain. For the sale persons, they scan the code to store therelevant circulation information on the block chain. For the consumers, they can scan the QRcode to obtain the information about the sources and circulation paths of the products. TheZhen-Tracking system have several advantages such as large capacity, high degree of security,free of centralized management and etc. In a decentralized global environment, the systemwill be invaluable for ensuring of the safety and efficacies of products of health food andtraditional medicines.

IC18-A40

Molecular Authentication of the Crude Snake Drugs of Zaocys and Its Adulterants

Nana Zhou, Qing He, Menghua Wu, Ying Zhang, Hui Cao

College of Pharmacy, Jinan University, Guangzhou, China.

Presenting Author Email: 1109905561@qq.comCorresponding Author E-mail: kovhuicao@aliyun.com

Abstract

Zaocys, a kind of snake from colubridae family, had a long history for clinical use in China. Itsdry body and the processed products were usually used for dispelling wind, unblocking thechannels, and stopping spasms. Nowadays, distinguishing the crude snake drugs was mainlydepended on well experienced in the outer appearances of kinds of snakes, and which madeit a confused challenge to identify Zaocys and its adulterants of Ptyas korros (Schlegel), Ptyasmucosus (Linnaeus), Naja naja (Linnaeus), Dinodon rufozoatum (Cantor), Enhydris chinensis(Gray) et al. We designed a pair of specific primers for distinguishing the crude snake drug ofZaocys from adulterants based on the sequence data of cyt b gene of snake samples. TotalDNAs were extracted from 11 species of crude snake drugs, and PCRs were performed usingthe primers with these total DNAs as a template. Positive amplifications were obtaineduniquely from the DNA template of Zaocys, and the specific band can be distinguished byelectrophoresis pattern of the amplified products. To explore a distinguish method toidentify Zaocys and its adulterants in the medical market quickly and accurately. The resultsindicated that the primers designed in this study can be used to identify Zaocys and itsadulterants specifically and efficiently. And the methods can provide a valuable reference toidentify other animal crude drugs.

IC18-A43

Assessing Species-admixtures in Herbal Products Using DNA Barcoding

Krishna Chaitanya M J1, Santhosh Kumar J U2, Ravikanth G3, Uma Shaanker R2*, Prathiba R D1

1. St. Joseph’s College (Autonomous), Bangalore-560027, India.2. School of Ecology and Conservation, Department of Crop Physiology, University of

Agricultural Sciences, GKVK, Bangalore-560065, India.3. Ashoka Trust for Research in Ecology and the Environment, Royal Enclave Srirampura,

Jakkur Post, Bangalore-560064, India.

Presenting Author Email: mjkchaitanya@gmail.comCorresponding Author E-mail: umashaanker@gmail.com

Abstract

The effectiveness of the bioactive compounds from medicinal plants has gained aconsiderable attention in the global market towards their use as therapeutic agents andpopular food-supplements. Consequently, an upsurge of 15% in the world economy ofinternational trade in herbal products is observed each year. The wild populations of Southand Southeast-Asian countries are the major source of the raw materials, for most herbalproducts. India, the second largest active-trader, exports nearly 10% of cultivated medicinalplants. Growing demands for such natural-products is a stimulus for adulteration andsubstitution, threatening consumer health and herbal industries of economic-fraud. Toprotect from associated risks and consequences, this study demonstrates the application ofDNA barcoding technique, to detect admixtures in the processed herbal product. BiologicalReference Material (BRM) developed from taxonomically authenticated leaves ofChyawanprash product listed (n=52 species) plant species, was used to amplify andsequence DNA barcodes for two regions: nuclear region ITS and chloroplast region psbA-trnH. Chyawanprash is highly sold products in India, was used to detect the presence oflisted plants species. This study followed two methods, BLAST similarity-based identificationand developed Phylogenetic tree based identification approach. In our results shows thatTerminalia chebula, Sida cordiflolia, Tinospora cordifolia, and Aegle marmelos species weredetected from Chyawanprash products. To ensure higher quality in herbal trade network, astringent evaluating structure is an urgent need. This provides an incentive to develop arobust regulatory framework for processed products and also advocating herbal industriesto have an authentication mechanism, to increase consumer confidence.

IC18-A46

Molecular Authentication of Commercial Pheretimaaspergillum (Guang Dilong) and Its Adulterants

Jie Sun, Menghua Wu, Ying Zhang, Hui Cao, Zhiguo Ma*

Research Center for TCM of Lingnan (Southern China), College of Pharmacy, Jinan University, China.

Presenting Author Email: 1491295760@qq.comCorresponding Author E-mail: mzg79@hotmail.com

Abstract

Guang Dilong is the dried body of Pheretima aspergillum (E.Perrier), one species ofearthworm. Through market research and literature retrieval, it was found that the source ofcommercial Guang Dilong was chaotic and there were many counterfeits. Some earthwormswas similar to the original animal form of Guang Dilong, but it was not Pheretimaaspergillum and lacked effective identification methods. DNA barcode was the main mostimportant method for identifying of Guang Dilong. To identifies39 batches of commercially available Pheretima medicinal materials. First, we extractedgenome DNA from the samples. The extracted genomic DNA was used as a template foramplifying the sample 16S rRNA gene through the polymerase chain reaction (PCR). Theresult of the identification was verified by DNA sequencing. The sequencing results weremanually corrected and spliced, comparing with the 16S rRNA public sequence ofearthworm in the GenBank database. The species can be confirmed when the similarity ofthe gene sequence was above 98%. The DNA barcode technology identified a total of 16batches of Pheretima aspergillum, the rest are spurious breed, of which 9 batches wereMetaphire magna. The method can be used for the authenticity identification and qualityevaluation of Guang Dilong,.

IC18-A47

Combined DNA Barcoding and HPTLC for Differentiation of the Poisonous Urobotrya Samensis from the Edible Melientha Suavis and Sauropus Androgynus for Food Safety

Kannika Thongkhao1,2, Thatree Padungchareon3, Suchada Sukrong1,2*

1. Research Unit of DNA barcoding of Thai Medicinal Plants, Chulalongkorn University Drug and Health Product Innovation Promotion Center, Chulalongkorn University, Bangkok, Thailand.

2. Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, 3Rangsit University, Pathumthani, Thailand.

Presenting Author Email: kannika.thongkhao@gmail.comCorresponding Author E-mail: suchada.su@chula.ac.th

Abstract

Urobotrya siamensis Hiepko (Phak Wan Mao) was a cause of death in Southeast Asiacountries due to the confusing with Melientha suavis Pierre (Phak Wan Pa). Both plantsbelong to Opiliaceae family and their leaves look very similar. M. suavis is now popularserved as vegetable for dishes and production of this species tends to increase according tothe demand of the market. However, Sauropus androgynous (Phak Wan Ban), edible plant inthe Phyllanthaceae, also shares the same Thai vernacular name with U. siamensis and M.suavis. Leaves of S. androgynus have been reported to have nutritive value. Hence, the aimof this study is to differentiate the poisonous U. siamensis from the edible species, S.androgynus and M. suavis, using the DNA barcoding couple with HPTLC techniques. CoreDNA barcode regions, rbcLa, matK, ITS and psbA-trnH, were amplified and sequenced.Polymorphic sites were detected from rbcLa gene. Indels were observed in psbA-trnH regionof the U. siamensis and led to size different of the PCR products. The chemical profiles of thetoxic and edible species showed unique patterns. This study is the first report that combinedDNA barcoding and chemical profile for differentiation of poisonous plant from the edibleones. The successful of this study will support us the knowledge for developing a routinedetection tool to detect the poisonous U. siamensis contamination for food safety.

IC18-A59

Chloroplast and Mitochondrial Genome Flux Makes Confusion for Plant DNA Barcoding

Hyun-Seung Park, Sae Hyun Lee, Jae-Hyeon Jeon, Jee Young Park and Tae-Jin Yang*

Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea.

Presenting Author Email: fgenesis@snu.ac.krCorresponding Author E-mail: tjyang@snu.ac.kr

Abstract

Food adulteration, also known as economically motivated adulteration (EMA), of herbalproducts is a severe threat both to human health and to herb-based industries. AlthoughDNA barcoding is a promising approach for preventing EMA, major unaddressedshortcomings of barcoding techniques have caused substantial interruption of the herbalindustry, including recent cases in Korea and the USA. Here, our de novo assembly of theplastid and mitochondrial genomes of the Cynanchum wilfordii and C. auriculatum in Korearevealed that their organellar genomes contain a substantial proportion of mitochondrialsequences of plastid origin (MTPTs). A broad survey of MTPTs in more than 80 plantsincluding model and non-model species found dynamic and lineage-specific plastid–mitochondrial genome flux that has diversified the mitochondrial genome structure. Further,we demonstrate a DNA marker paradox derived from co-amplification of MTPTs can causemis-authentication of herbal products. We propose a guideline of using multiple targetmarkers for EMA regulation to protect both human health and the integrity of the herbalsupplement and functional food industry.

Acknowledgement: This work was carried out with the support of the Cooperative ResearchProgram for Agriculture Science & Technology Development (grant number, PJ013238) RuralDevelopment Administration, Republic of Korea.

IC18-A61

Phylogenetic Signal of Steroidal and Glyco-alkaloids in Genus Solanum Based on DNA Barcodes and HPTLC Fingerprints

U. Senthilkumar1,2*, N. Kasthuri Bai2, R. Pandian2, K. Ilango2, M. Parani1

1. Centre for DNA Barcoding, Department of Genetic Engineering, School of Bioengineering, SRM Institute of Science and Technology, Kattankulathur, Chennai-603 203, India.

2. Interdisciplinary Institute of Indian System of Medicine (IIISM), SRM Institute of Science and Technology, Kattankulathur, Chennai- 603 203, India.

Presenting Author Email: sensonsam85@gmail.comCorresponding Author E-mail: sensonsam85@gmail.com

Abstract

Plants have evolved with high diversity of alkaloids either for their defenses (allelochemicals)or survival, which leads to evolution of specialized metabolites and its natural derivatives.The steroidal alkaloids and glycol-alkaloids are such ones. Phylogenetic approach offers apredictive approach on selection of plants based on the evolutionary relatedness that leadsto discovery of novel drugs. However, the analysis of combined data from both phylogenyand phytochemical investigations are poorly established. In this study, we have created thephylogenetic framework for Solanum (Solanaceae), which is an important genus bothmedicinally and commercially. Maximum likelihood and Bayesian analyses were carried outusing both nuclear (ITS2) and plastid (ndhF, trnL-trnF) DNA sequences for 25 species fromIndia. We have investigated the steroidal-alkaloides and glyco-alkaloids i.e. α-Solanine,Solasodine, Solanidine from the leaves and berries based on High Performance Thin LayerChromatography (HPTLC) and found significant phylogenetic signal at interspecific level. Inthis study, we have also have mapped the metabolites to derive the phylogeneticrelationships between crop and non-crop species based on the presence and absence dataas well as the percentage of the metabolites (quantified) present. In conclusion, the studyreveals that the phylogenetic signals suggest in selection of non-poisonous wild Solanumspecies and to avoid the excessive intake of crops (edible Solanum) based on the content ofsteroidal alkaloids that are known to be poisonous at higher dosage.

IC18-A62

DNA Barcoding of Hong Kong Ilex Species

Lim-Ho Kong1,3, Tai-Wai David Lau3,4, Zhixiu Lin2, Pang-Chui Shaw1,2,3,4

1. Li Dak Sum Yip Yio Chin R & D Centre for Chinese Medicine, The Chinese University of Hong Kong Hong Kong, China.

2. School of Chinese Medicine, The Chinese University of Hong Kong Hong Kong, China.3. Institute of Chinese Medicine, The Chinese University of Hong Kong Hong Kong, China.4. School of life Sciences and Shiu-Ying Hu Herbarium, The Chinese University of Hong Kong

Hong Kong, China.

Presenting Author Email: konglimho@yahoo.com.hkCorresponding Author E-mail: pcshaw@cuhk.edu.hk

Abstract

According to recent studies, Ilex asprella shows good antiviral activity and the Ilex pubescenspossess excellent anti-inflammatory response. Since Ilex is a genus of herbs which arecommonly used in Traditional Chinese Medicine, and there are 20 Ilex species commonlyfound in Hong Kong and nearby regions, we set forth to clarify the relationship of theseherbs by DNA barcoding. In our study, all of the 20 species were collected and their DNA wasextracted. Gene segments rbcL, psbA-trnH, matK and ITS2 were amplified by polymerasechain reaction and sequenced. We found that matK was not amplified in most of the Ilexspecies. rbcL was quite conserved and only showed single nucleotide polymorphism in a fewbase pairs. ITS2 gave the greatest variation and a region was identified for authenticationpurpose. By using the ITS2 sequences and the Maximum Likelihood model, phylogenetictree was constructed. Their relationship among themselves and with other published Ilexspecies was compared.

IC18-A67

Jiang San, A Traditional Multi-herb Formulation, Exerts Anti-Influenza Effects in vitro and in vivo Via Neuraminidase Inhibition and Immune Regulation

Tianbo Zhang1, Mengjie Xiao2,3, Chun-Kwok Wong4,5, Ka-Pun Chris Mok6, Xin Zhao2,7, HuihuiTi2,6*, Pang-Chui Shaw1,5,7,*

1. School of Life Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR 999077, China.

2. State Key Laboratory of Respiratory Disease, Guangdong Provincial Key Laboratory of Molecular Target & Clinical Pharmacology, School of Pharmaceutical Sciences and The Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou 510632, China.

3. Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Science, Guangzhou 510632, China.

4. Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, N.T., Hong Kong SAR 999077, China.

5. Institute of Chinese Medicine and State Key Laboratory of Phytochemistry and Plant Resources in West China, the Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR 999077, China.

6. HKU-Pasteur Research Pole, School of Public Health, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR 999077, China.

7. Li Dak Sum Yip Yio Chin R & D Centre for Chinese Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR 999077, China.

Presenting Author Email: konglimho@yahoo.com.hkCorresponding Author E-mail: pcshaw@cuhk.edu.hk

Abstract

Background:Sheng Jiang San (SJS), a multi-herb formulation, is used in treating high fever, thirsty andanxiety in ancient China and it is sometimes used to treat seasonal influenza nowadays.However, there is no evidence-based investigation and mechanism research to support theanti-influenza efficacy of SJS. This study aims at evaluating the anti-influenza effect of SJSand investigating its possible mechanism.

IC18-A68

Methods:The inhibitory effect of SJS against different influenza virus strains on MDCK cells wasexamined. Influenza virus infected BALB/c mice were employed to evaluate the efficacy as invivo model. Mice challenged with A/PR/8/34 (H1N1) were orally administrated 1 g/kg/dayof SJS for seven days and monitored for 14 days. The survival rate, body weight changes,lung index, lung viral load, histopathologic changes and immune regulation of the mice weremeasured. The underlying anti-influenza virus mechanism of SJS was studied by a series ofbiological assays to determine if hemagglutinin, ribonucleoprotein complex orneuraminidase were targets of SJS.

Results:Results showed SJS exerted a broad-spectrum of inhibitory effects on multiple influenzastrains in a dose-dependent manner. IC50 of SJS against A/WSN/33 (H1N1) was lower than35 μg/ml. SJS also protected 50% of mice from A/PR/8/34 (H1N1) infection. The lung indexand the lung viral load of SJS treated mice were significantly decreased compared withuntreated mice. Meanwhile, SJS targeted on neuraminidase of influenza virus as SJS at 2mg/ml inhibited 80% of neuraminidase enzymatic activity. SJS also significantly down-regulated TNF-α and up-regulated IL-2 of influenza virus induced mice.

Conclusions:Thus, SJS is a useful formulation for treating influenza virus infection.

Keywords:Sheng Jiang San; anti-influenza activity; neuraminidase inhibition; immune regulation

IC18-A68

Development of KASP Markers Derived From Single-Locus-unique Nuclear Genome and Chloroplast Genome of Allotetraploid Panax Ginseng

Yeeun Jang, Woojong Jang, Won-Kyung Lee, and Tae-Jin Yang*

Department of Plant Science and Plant Genomics and Breeding Institute, College of Agriculture and Life Sciences, Seoul National University, Seoul, 08826, Korea.

Presenting Author Email: whatsupyen@gmail.comCorresponding Author E-mail: tjyang@snu.ac.kr

Abstract

Korean ginseng (Panax ginseng C. A. Meyer) is a valuable medicinal herb, containing variousginsenosides which have benefits to human health. A lot of pharmacological studies for theplant have been performed for a long time, however, genetic studies are fewer because ofcomplex genome characteristics and unusual slow growth. To construct molecular breedingbasis on ginseng, genetic researches using various genetic resources are demanded. Weanalyzed various 119 ginseng breeding lines with genotyping by sequencing (GBS) techniqueto research intraspecific variations. 2,574 SNPs were detected as high quality of SNPs(MAF≥0.25, read depth≥5). Considering the character of allotetraploid of ginseng, additionalsteps were carried out for filtering out SNPs that are likely amplify a unique locus byavoiding paralogous sequences. As a result, 10 KASP markers were developed on nucleargenome using the selected SNPs and we identified that they worked properly. Also, wecompleted chloroplast genomes of 10 wild ginsengs and detected 6 SNPs and 1 sing indel.Using the variations, 5 KASP markers were developed on chloroplast genome. Consequently,we developed KASP markers derived from each single locus on nuclear genome and alsofrom polymorphic chloroplast genome sequence. We performed phylogenetic analysis usingthe KASP markers. The information will support genetic studies on ginseng and thesemarkers could be applied to other ginseng genetic resources for breeding and classification.This work was carried out with the support of "Cooperative Research Program forAgriculture Science & Technology Development (PJ01311901)" Rural DevelopmentAdministration, Republic of Korea.

IC18-A69

Identification of Gene Inducing Variegated Chimeric Leaves Based on Complete Sequencing of Two Types Chloroplast Genomes Derived from Single Plant in Reynoutria Japonica

Jae-Hyeon Jeon1, Hyun Jo Koo1, Jee Young Park1, Hyun-Seung Park1, Jung Hwa Kang2, TaekJoo Lee2 and Tae-Jin Yang*

1. Department of Plant Science and Plant Genomics and Breeding Institute, College of Agriculture and Life Sciences, Seoul National University, Seoul, 08826, Korea.

2. Hantaek Botanical Garden, Yongin, Gyeonggi-do, 17183, Republic of Korea.

Presenting Author Email: wwjun436@gmail.comCorresponding Author E-mail: tjyang@snu.ac.kr

Abstract

In nature, many plants forming albino leaves exist. It is known that plants with albino leavesare caused by the mutation in the chloroplast, resulting in abnormal formation ofchlorophyll. Reynoutria japonica is frequently observed with albino and normal leaves on asingle plant, referred to as “Patterned R. japonica”. In this study, the chloroplast genomesequences were completed using NGS from the leaves of patterned R. japonica and analyzedto elucidate the reason of this phenomenon. Only a single SNP was identified between thetwo chloroplast genomes, located in rpoC2 gene, a subunit gene of plastid-encoded RNApolymerase (PEP). qRT-PCR was performed using the specific primers of each genetranscribed by PEP and nuclear-encoded RNA polymerase (NEP) to examine differences inRNA expression. The genes transcribed by PEP were transcribed much less in the whiteleaves than green leaves. On the other hand, genes transcribed by NEP showed littledifference in transcription level between green and white leaves. In the protein structuremodel, the SNP caused the substitution of histidine to proline in albino leaf. Thus, it appearsthat the hydrogen bond between the changed amino acid and the adjacent amino acid isremoved and α-helix structure is destroyed, leading to misfolding of the protein structure.The result of this research will extend understanding about the effects of chloroplastgenome mutation. This work was supported by the Bio & Medical Technology DevelopmentProgram of the NRF, MSIP(Grant No. NRF-2015M3A9A5030733), Republic of Korea

IC18-A70

Inter- and Intra- Chloroplast Genome Diversity and Classification of Germplasm in Cynanchum Species

Sae Hyun Lee, Hong-Seob Yu, Ho Jun Joh, Hyun-Seung Park, Jee Young Park, Tae-Jin Yang *

Department of Plant Science and Plant Genomics and Breeding Institute, College of Agriculture and Life Sciences, Seoul National University, Seoul, 08826, Korea.

Presenting Author Email: whyskyisgray@naver.comCorresponding Author E-mail: tjyang@snu.ac.kr

Abstract

Cynanchum wilfordii has long been regarded as a medicinal plant in Korea. Because of itsvarious pharmacological effects, especially for menopause, the annual production hasincreased continuously. However, morphological similarity between C. auriculatum, and C.wilfordii may cause Economically Motivated Adulteration (EMA) in the market. Therefore, itis necessary to develop authentication methods to distinguish two species. Most of themolecular markers are designed from chloroplast genome sequences for the identificationof plant species. For Cynanchum species, several markers have been developed for speciesidentification, but they often fail to do so which might be caused by intra-species variationsfrom natural diversity. Therefore, to develop more stable authentication markers to protectEMA issue, we assembled complete chloroplast genome and 45s rDNA of four C. wilfordiiusing NGS data with de novo assembly of low coverage WGS sequence. By comparativeanalysis, we identified six SNPs and five InDels in the chloroplast genome of four C. wilfordiiaccessions. Four SNPs were identified in the intergenic region and two SNP was found in theexon region. We also identified five InDels, two in the intergenic region and three in theexon region. Markers that can verify inter- and intra- chloroplast genome diversity in twoCynanchum species were developed and applied to breeding line germplasms collected fromall over Korea. As a result, the germplasms were grouped into thirteen types. This researchwas supported by “Cooperative Research Program for Agriculture Science & TechnologyDevelopment (Project No. PJ013238)”, Rural Development Administration, Republic of Korea.

IC18-A71

Reconfiguration of Complex Inheritance of Chloroplast and Mitochondrial Genomes Using Whole Genome Sequences of Reciprocal Cross Hybrids in Cucumber

Won-Kyung Lee1, Jee Young Park1, Sang-Choon Lee1, Ho Jun Joh1, Jaehyeon Jeon1, KihwanSong2*, Tae-Jin Yang1*

1. Department of Plant Science and Plant Genomics and Breeding Institute, College of Agriculture and Life Sciences, Seoul National University, Seoul, 08826, Korea.

2. Department of Bioresources Engineering, Sejong University, Seoul 05006, Republic of Korea.

Presenting Author Email: lwk4279@naver.comCorresponding Author E-mail: khsong@sejong.ac.kr, tjyang@snu.ac.kr

Abstract

Cucumber (Cucumis sativus var. sativus) organelle genomes have been known to showexceptional inheritance patterns. Because of its economic importance at vegetable marketand scientific value as a model plant for sex determination and vascular biology study,reconfiguration of cucumber’s organelle genome inheritance pattern is necessary. To verifyinheritance pattern of organelle in cucumber, two cucumber inbred lines of Korean solidgreen and Chinese long green types and their reciprocal F1 hybrids were re-sequenced andused to analyze their organelle genome sequences.

First, the complete chloroplast (cp) genomes were de novo assembled with next generationsequencing (NGS) data of whole genome. There was one SNP among cp genome sequencesof four cucumber samples and it was inherited maternally to F1 hybrids.

Second, because of its large size and complexity,mitochondrial (mt) genomes of fourcucumber samples were resequenced using reference cucumber mt sequence. 292polymorphic sites were found between two parental lines. With whole mt genomeresequencing and marker test, all of the polymorphic sites were verified to be inheritedpaternally to F1 hybrids.

Consequently, this study confirmed that cucumber cp genome was maternally inherited likemost plant species, whereas mt genome was paternally inherited. This study would providevaluable genetic information for understanding organelle-derived traits and molecularbreeding in cucumber.

This work was supported by Korea Institute of Planning and Evaluation for Technology inFood, Agriculture, Forestry and Fisheries (IPET) through Agri-Bio industry TechnologyDevelopment Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA)(grant number 116076-03-3-HD0b0).

IC18-A72

Random Primed Polymerase Chain Reaction (RP-PCR) To Differentiates Codonopsis Pilosula From Different Localities and Polymerase Chain Reaction-restriction Fragment Length Polymorphism (PCR-RFLP) To Identify Adulterants

Yan-Bo Zhang1, Par-Sim Ho1, Fai-Ngor Ngan2, Tzi-Bun Ng3, Paul Pui-Hay But4, Ning Li2, Jun Wang2, Pang-Chui Shaw PC2*

1. School of Chinese Medicine, the University of Hong Kong, 10 Sassoon road, Pokfulam, Hong Kong;

2. School of Life Science, The Chinese University of Hong Kong, Hong Kong, China.3. School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China. 4. Department of Biology, The Chinese University of Hong Kong, Hong Kong, China.

Presenting Author Email: ybzhang@hku.hkCorresponding Author E-mail: pcshaw@cuhk.edu.hk

Abstract

DNA fingerprints distinctive among the samples from different localities in China weresuccessfully reproduced for the Chinese herb Dangshen, the roots of CODONOPSIS PILOSULA,(Campanulaceae). Similarity index (S.I.) analysis revealed that C. PILOSULA samples from thesame province generated similar DNA fingerprints, while samples of different provincesdisplayed different DNA fingerprints. This method may be a general and valuable tool forlocality authentication of other Chinese herbal medicinal materials.

DNA sequence analysis of rDNA internal transcribed spacer (ITS) and polymerase chainreaction-restriction fragment length polymorphism (PCR-RFLP) were exploited for theirapplications in differentiating medicinal species Codonopsis pilosula, C. tangshen, C. modesta,and C. nervosa var. macrantha, from two related adulterants Campanumoea javania andPlatycodon grandiflorus. The data demonstrated that the rDNA ITSI and ITSII sequences ofthe four Codonopsis are highly homologous but not identical, and are significantly differentfrom those of the two adulterants. The sequence difference allows effective and reliabledifferentiation of Codonopsis from the adulterants by PCR-RFLP.

Key words:Chinese medicine, Codonopsis pilosula, differentiation, RP-PCR, PCR-RFLP.

IC18-A75

Medicinal Materials DNA Barcode Database (MMDBD) v1.5 – One-stop Platform for Storage, BLAST, Alignment & Primer Design

HY Wu1,2, TH Wong1,3, Grace WC But2, Stacey SK Tsang3, David TW Lau3 and, PC Shaw1,2

1. Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong, China.2. LDS YYC R & D Centre for Chinese Medicine, The Chinese University of Hong Kong, Hong

Kong, China.3. SY Hu Herbarium, School of Life Sciences, The Chinese University of Hong Kong, Hong

Kong, China.

Presenting Author Email: karenwhy@cuhk.edu.hkCorresponding Author E-mail: pcshaw@cuhk.edu.hk

Abstract

Authentication of medicinal materials by DNA technology is gaining popularity in herbalindustry. In 2010, our team has created Medicinal Materials DNA Barcode Database(MMDBD) version 1.0, an interactive database of DNA barcode sequences of medicinalmaterials. MMDBD now contains DNA barcode sequences of medicinal materials listed inthe Chinese Pharmacopoeia, Dietary Supplements Compendium and Herbal MedicineCompendium of the US Pharmacopoeia and selected adulterants. The data archive isregularly updated and currently it stores 65 520 DNA sequences of 2225 medicinal materials.A major improvement has been carried out on the interfaces and essential bioinformaticstools were incorporated to facilitate the authentication work.

MMDBD version 1.5 contains detailed information of each medicinal material, including thematerial name, medical part, pharmacopeia information, biological classification in rank offamily and its status on the Convention on International Trade in Endangered Species of WildFauna and Flora and the International Union for Conservation of Nature’s Red List ofThreatened Species, if any. User can retrieve DNA sequences by search in Latin scientificname, Chinese name, family name, material name, medical part and simplified Chinesecharacter stroke. A `BLAST’-based engine is included in MMDBD v1.5 to allow searching DNAsequence based on similarity. In addition, we have integrated the `Clustal Omega alignmenttool’ and `Primer3’ in the form of web interface for easy primer design. These new toolsfacilitate multiple sequence comparison and the design of primers for amplification of atarget DNA barcode region, allowing DNA barcoding authentication.

IC18-A78

A Rapid Sample-to-answer Analytical Detection of Genetically Modified Papaya Using Loop-mediated Isothermal Amplification Assay on Lab-on-a-discfor Field Use

Jacky FC Loo1,2 *, Grace WC But1 *, HC Kwok3, PM Lau1, SK Kong1, HP Ho2, PC Shaw1,4

1. School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China.2. Department of Biomedical Engineering, The Chinese University of Hong Kong, Hong

Kong, China.3. Department of Electronic Engineering, The Chinese University of Hong Kong, Hong Kong,

China.4. Li Dak Sum Yip Yio Chin R & D Centre for Chinese Medicine and Institute of Chinese

Medicine, The Chinese University of Hong Kong, Hong Kong, China.

Presenting Author Email: gracebut@link.cuhk.edu.hkCorresponding Author E-mail: pcshaw@cuhk.edu.hk

Abstract

With genetically modified (GM) food circulating on the market, a rapid transgenic foodscreening method is needed to protect consumer rights. The on-site screening efficiency ofGM food testing is low. We report rapid sample-to-answer detection of GM papayas withloop-mediated isothermal amplification (LAMP) and a compact, portable, integratedmicrofluidic platform using microfluidic lab-on-a-disc (LOAD). GM samples weredifferentiated 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. Thedetection limits for DNA and juice from papaya were 10 pg/μL and 0.02 μL, respectively. OurLOAD platform is a simple and robust solution for GM screening, which is anticipated to be afoundation for on-site testing of transgenic food.

IC18-A79

Multidisciplinary Protocol for Authentication of Placenta Hominis

Yat-Tung Lo, Mavis Hong-Yu Yik and Pang-Chui Shaw*

Li Dak Sum Yip Yio Chin R & D Centre for Chinese Medicine, State Key Laboratory of Phytochemistry and Plant Resources in West China (CUHK) and School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China.

Presenting Author Email: ytlo@cuhk.edu.hkCorresponding Author E-mail: pcshaw@cuhk.edu.hk

Abstract

Placenta Hominis, a medicinal product made from human placenta, is used for treatingvarious diseases among Asian countries. With its therapeutic benefits and limited supply,intentional or inadvertent adulteration is frequently found in the market. In order tosafeguard people’s health and protect customer rights, we established a hierarchicalprotocol involving morphological, chemical, biochemical and molecular diagnosis toauthenticate ten placenta samples collected from herbal shops in China, Hong Kong andTaiwan. Species-specific diagnostic primers for human, cow, deer and sheep were designedfor PCR amplification and subsequent DNA sequencing for species identification.Commercially available pregnancy test strip was used to detect human chorionicgonadotropin (hCG), and progesterone competitive ELISA kit was used to detect thepresence of progesterone in samples. Small amount of iodine solution was also added ontothe samples to detect the presence of starch. Among the ten samples studied, resultsshowed that no cow, deer and sheep DNA was presented in all samples. Five samples weregenuine with the presence of human DNA, hCG and progesterone accompanied with theabsence of starch fillers. On the other hand, four samples were adulterants which may bemade from starch products. In addition, a sample was found as a mixture of PlacentaHominis and starch fillers, and it did not conform to the product requirement of PlacentaHominis. The multidisciplinary protocol developed involving morphological, chemical,biochemical and molecular diagnosis provides an accurate method to regulatory bodies andtesting laboratories for the quality control of Placenta Hominis.

IC18-A80

DNA Authentication of Herbal Products with Hedyotis diffusa

Hong-Yu Yik2, Yat-Tung Lo2 and Pang-Chui Shaw1,2 *

1. School of Life Sciences, Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong, China.

2. Li Dak Sum Yip Yio Chin R & D Centre for Chinese Medicine, The Chinese University of Hong Kong, Hong Kong, China.

Presenting Author Email: mavisyik@cuhk.edu.hkCorresponding Author E-mail: pcshaw@cuhk.edu.hk

Abstract

Hedyotis diffusa Willdenow (Baihuasheshecao in common name) is a well-known traditionalChinese medicine for treating different cancers including colorectal cancer, leukemia,prostate cancer and multiple myeloma etc. It has the pharmacological effects of anti-oxidation, anti-tumor and anti-inflammation. There are a number of herbal products in formof drinks, granules and ampules on the market. The high medicinal values of H. diffusa,however lead to the emergence of intentional or inadvertent adulteration in the products.For instance, a closely related species in Rubiaceae family named Hedyotis corymbosa(Linnaeus) Lamarck (Shuixiancao in common name) is frequently found as adulterant. Thesetwo species look alike morphologically and only different form several features such as theshape of stems, inflorescence and texture, etc. These unapparent features makemorphological identification difficult for layman and may result in misidentification andmisuse. DNA barcoding is a cutting-edge technique for identification and authentication ofbiological specimens by using short DNA sequences. Based on the genome variation, speciesidentity can be revealed accurately without morphological and physiological restriction. Inthis project, the species identity of H. diffusa or H. corymbosa in the herbal products wererevealed by DNA barcoding, so as to enhance the quality control and assurance of herbalproducts in the market.

IC18-A97

Molecular Quantification, Strategy for Quality Control of Chinese Patent Medicine Containing Animal-derived Crude Drugs

Haoting Ye, Xiaolei Li, Chan Li, Enwei Tian, Zhi Chao

School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China.

Presenting Author Email: chaozhi@smu.edu.cnCorresponding Author E-mail: chaozhi@smu.edu.cn

Abstract

Quality control of Chinese patent medicines is the premise to ensure their efficacy. However,for most animal medicines, the fundamental research is weak, the chemical composition isnot clear, and the quality standards are relatively backward, which depend mainly onmacroscopic inspection and impurity examination. Usually, the specificity of inspection isnot ideal, and quantitative evaluation is lacking. Quality control of animal-derived crudedrugs and their preparations is confronted with great difficulties.

Jinlong capsule is a patent medicine comprised of Peking gecko, many-banded krait, andsharp-nosed viper. Four items, macroscopic character, identification, test and assay, werelisted in its earliest quality standard; and protein content and activity of arginine esterasewere taken as indices for quality assay. Further improvement of the standard mainlyembodied in the establishment of assays for amino acids, free monosaccharides andpolysaccharides, small molecular substances such as uracil and hypoxanthine in eachmaterial crude drug of Jinlong capsules and their mixtures. Similar to other Chinese patentmedicines containing animal crude drugs, the quality control of Jinlong capsule also defectsin high dependence on experience in identification, poor specificity of qualitative inspectionand quantitative assays, etc.

We here propose a new strategy for quality control of Jinlong capsule by molecularquantification. In Jinlong capsule, there are still remnant DNAs of each biologicalcomponents. We designed species-specific primers and probes for Peking gecko, many-banded krait, and sharp-nosed viper respectively, and determined the copy number ofspecific DNA fragments of each component by fluorescence quantitative PCR technology.The copy numbers can be used to reflect the quantity and proportion of each material crudedrug fed, and their qualitative and quantitative analysis could be completed in one step, thusthe stability of drug quality could be ensured. The study is expected to provide a newmethod for quality control of Jinlong capsule.

Keywords: Molecular quantification, Chinese patent medicine, animal drugs

IC18-A125

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