MINISTRY OF EDUCATION AND TRAINING NONG LAM UNIVERSITY-HO CHI MINH CITY FACULTY OF BIOLOGICAL SCIENCES GRADUATION THESIS GENETIC DIVERSITY OF FIFTEEN DENDROBIUM SPECIES USING SRAP MARKERS Major : BIOTECHNOLOGY Student implementation : NGUYEN TRAN TUYET NHI Student code : 18126117 Academic year : 2019-2023 Thu Duc City, 03/2024 MINISTRY OF EDUCATION AND TRAINING NONG LAM UNIVERSITY-HO CHI MINH CITY FACULTY OF BIOLOGICAL SCTENCES GRADUATION THESIS GENETIC DIVERSITY OF FIFTEEN DENDROBIUM SPECIES USING SRAP MARKERS Supervisor Student Assoc. DrNGUYEN BAO QUOC NGUYEN TRAN TUYET NHI Thu Duc City, 03/2024 ACKNOWLEDGEMENTS First and foremost, my deepest heartfelt gratitude goes to my Supervisor, Assoc. Nguyen Bao Quoc, for his patience and wisdom, his gentle guidance, and for helping me evaluate my thesis in a detailed and analytical way. I would want to thank my wonderful senior, Nguyen Mai Nghiep.
For him deep, fascinating discussions with me about this thesis throughout my last college semester. I consider myself really fortunate to have had such amazing labmates with whom I exchanged views and ideas. I would also like to thank my pals and wish them the best for all of the laughs and great times. Furthermore, I have spent four years of my life studying here.
Through it all, I have been fortunately to learn and be a better version of myself. I also deeply thank everyone at Faculty of Biological Sciences whom I met and learned knowledge and skills from them. I must say I am proud to be a student of Nong Lam University. In addition to the help in terms of knowledge, I also thank Proffesor Bui Cach Tuyen for the support facilitate and support the process of collecting dendrobium samples.
Thank you to the Faculty of Biological Sciences, Nong Lam University lecturers for their enthusiastic help and guidance in achieving the best results for this thesis. Last, but never least, my family have been enormously supportive of me. So, to my parents, thank you for always being by my side and for being completely gorgeous, funny and loving. AFFIRMATION AND COMMITMENT I am Nguyen Tran Tuyet Nhi, student code: 18126117, Class: DH19SHB, Faculty of Biological Sciences, Nong Lam University, Ho Chi Minh City.
[hereby declare: This 1s my own graduation thesis, the data and information in the research are completely honest and objective. I take full responsibility to the board for these commitments. Thu Duc City, April 2024 Performer Nguyen Tran Tuyet Nhi ll ABSTRACT Dendrobium species have been widely utilized for various purposes, not just for their aesthetic appeal but also for their nutritional and medicinal properties. The identification and evaluation of genetic diversity within denrobium species play a crucial role in supporting conservation efforts aimed at preserving the genetic resources of these unique species.
In this study, SRAP — Sequence Related Amplified Polymorphism molecular markers will be used to study the genetic diversity present in currently conserved orchid in Vietnam. Sixteen highly polymorphic primes out of thirty SRAP markers employed amplifying in 30 individuals from 15 dendrobium species generate 142 polymorphic fragments. The analysis revealed that a total of 198 bands were amplified, of which 142 polymorphic bands. SRAP markers showed the values of polymorphism information content (PIC) ranged from 0.5 with the mean value of 0.
The average value of Shannon's Information Index (I) and Nei (H) were 0. Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustering and principal coordinate analysis was performed based on the similarity matrix used to analyze specifically the genetic relationship between denrobium species. The results show that the species in this study are divided into five groups with the lowest similarity coefficient of 0.724 and the highest of 0. Keywords: dendrobium, SRAP, genetic diversity.
1H TÓM TẮT Dendrobium được sử dụng rộng rãi không những vì vẻ đẹp thâm mỹ mà còn vì tính chất dinh dưỡng và được học. Nhận biết và đánh giá đa dạng di truyền trong các loài déndrobium đóng vai tri quan trọng trong việc hỗ trợ bảo về nguồn gen của các loài động vật quý hiếm. Trong nghiên cứu này chỉ thị phân tử SRAP sẽ được sử dụng dé nghiên cứu sự đa dạng di truyền của 1 số giống dendrobium ở Việt Nam. Trong số 30 primer SRAP được sử dụng, 16 môi có biểu hiện các đoạn sản pham khuéch dai.
Khuéch dai thanh cong 198 band với 142 band đa hình. Chi thi phân tử SRAP biểu hiện giá tri thong tin da hình (PIC) từ 0,08 đến 0,5 với giá trị trung bình 0,4. Tat cả 15 giống dendrobium trong nghiên cứu bang chi thị phân tử SRAP có mức độ da dang thấp, chi số đa dạng di truyền theo Shannon (I) và chỉ số đa dạng di truyền theo Nei (H) trung bình lần lượt là 0,402 và 0,190. Dựa vào phương pháp UPGMA và PcoA được thực hiện dựa trên ma trận tương đồng được sử dung dé phân tích mối quan hệ di truyền giữc các nhóm.
Nghiên cứu này được chia làm năm nhóm với hệ số tương đồng thấp nhất là 0,724 và cao nhất là 0,827. Từ khóa: Lan dẹndrobium., SRAP, da dang di truyền. IV TABLE OF CONTENTS Page ACKNOWLEDGEMENTS.cscsisnssesscnnssssstnseasscononsnasinassnnvheensnasineassneutnnentietausnasnlsnucsvebaansinns 1 AFFIRMATION AND COMMITMENT .:c:ccecssceceseeseseeeeseeceseeceeceseeeeseseeeeeeeseeeeeeeees il CHAPTER 00010949)9)0/9019)01525. 1 pI Melba hi coXeh Nic bic) i eee ee eee ee ee 1 1D, OUCCV Cement eee een ee 2 IE6NeU.
2 CHAPTER 2 CV ERNE W vsesass: conan nner ere EET 4 2 eADCNGPODIOT OV LVIEW toanggiidtstiistlllitiiBdiusSiablptitb84DBndNSibSgpsfiistioislsskGlskbäseisistrisosskodna 4 2. Classification of dendrobium in Viet Nam. S- SĂcSc Series 5 2:1»3; MOTPh0]G6VOE(G6HNHODHÍTT tudnng tù dh tt gi GIENHAS43.6036401381G013/G013638014008339000 0008 6 2a, ROOUS scenceuserescssenssnamnsnscasma (GG8019095008386.ES0010020Đ818 6 la 905: Dị TT casxseeeevisssvbihtigiaESkEASAG1SESGCETTESSGIS5/006GSEVESRESIMSREENESLSESSIIDRGIGISS.HBSISSUĐRMIGEAHESEESEESES 6 2c 1231 LOBV CS secsscsseeeer T015 00SEÄBHĐ4USEESEESS3R0ST3S3SSESECSGEEESABSSSESSSSESAEHSABSEBRESHGS8236:S.S83880/8S0/20805 6 Pd 350i LÔ WIDÍÑỸ tung nhang gà ti 5 :Ô: FEUHS 8S sennansenninbiiendnndibnliS115513304455855E5ESSESSESESESSSTSSEESG)SSSG5SSES5189EESEVESEEESSE vị 222:: CIGIGHIGTGIUETSTE St E003 GR HH NGAHIIGIGSGGINSHEXGDASRIBBEESEISDEASSEAHGEEEIEIHIIGIIGNHBNSSiNBSN. LJEHTHTHOÍsss6sáxss65208 0116101111 1215358304361551615189181495345313338482535455E4BELASSHEEG43461043353604483586858004658 8 2.
Lhe signticance of genetic diversity vcscosescesssvessessnesnsounnsssacommnsenscsmnscesmreemmpemsuenerars 8 2.3: Markers forassessment Of GIVGrSuty ssc. ccrssesacessessecuanecee ceevteeaerecssacaman mexacmaaanomaaass 9 2. Some molocular markers used to study the genetic dIVeTSIfy. SRAP - Related Amplified Polymorphism.
RAPD — Random Amplified Polymorphic DNA. RFLP — Restriction Fragment Length Polymorphism. AFLP - Amplified Fragment Lenght Polymorph1Isims. SCAR - Sequence Characterized Amplified Region.
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RESULTS AND DISCUSSIONS.Ặ LH, 22 A LAINGSULIS! esp aces career tee ae Ee 22 4. DNA extraction of 15 dendrobium species. Quanility DNA: Che Khosessissasiaisiaesoealisriosgbeuskvesuonlndindnouili-iuueadaLgkudutiokesoodigircbazrôstiourdsd2gi 22 4. Genetic diversity analysis among dendrobium SpeC1€S.
Genetic distance and cluster analysis of 15 dendrobium specl1es. 28 UP co on. The effectiveness of using the SRAP marker in genetic dIVerSIty. Genetic diversity among 15 dendrobium species.
Genetic relationships of 15 dendrobium species. CONCLUSION AND SUGGESTION .cccscssscssseceseesesecsseeeseeseeneoes 31 REFERENCES saceeaaaaadeeiiuiiiobibodietoosbobiEESL101358058DA421615SELISS42383383898383355SG:XBESE4E.SE08 32 VI LIST OF ABBREVIATIONS AFLP: Amplified Fragment Length Polymorphism bp: Base pair CTAB: Cetyltrimethylammonium bromua DNA: Deoxyribo nucleic acid EDTA: Ethylenediamine Tetraacetic Acid et al: and others Kb: Kilobase pairs MI: Marker Index OD: Optical Density GCP: Generation challenge programme PCR: Polymerase chain reaction RAPD: Random amplification of polymorphic DNA SSR: Simple Sequence Reapead SRAP: Sequencce — Ralated Amplified Polymorphism TBE: Tris — Borate — EDEA TE: Tris/EDTA UPGMA: Unweighted pair group method of arithmetic averages UV: Untralviolet VI LIST OE TABLES Page Table 3. List of dendrobium species used in the study. Sequence information for COX Universal primers (Dickinson, 2015).
DNA absorbance ratio and concentration measurermenIfS. Primer information, scored bands and discriminating power statistics. Genetic diversity parameters of dendrobium species analyzed. 26 Vill LIST OF FIGURES Page Figure 2.
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Introduction Dendrobium is the second largest genus in the Orchidacease (flowing by Bulbophyllum), which is one of the largest genera of tropical orchids and 1s widely grown commercially, with a significant economic impact on several ASEAN countries. Some denrobium species is used as medicinal herbs, including D. Many Dendrobium plants have been used as ingredients for nutraceutical beverages and food products, that make a market and medicinal demand. In line with the trend of human consumption, dendrobium products have grown a diverse range of uses (Bao et al.
In general, the Orchidaceae family comprises perennial herbaceous plants (occasionally partially woody at the base). They either inhabit soil, crevices, rocky surfaces, or adopt epiphytic 66 lifestyles, often clinging and hanging suspended on the bark of other woody plants. Orchids are widely distributed across the Earth, making them a global family. They are found from 68th parallel north latitude to 56th parallel south, spanning from near the North Pole to the southernmost islands.
However, the central distribution of this family is 1n tropical regions, especially in the America and Southeast Asia. Asia 1s particularly rich in species, including the genera dendrobium (1400 species), Coe/ogyne (200 species), and Vanda (60 specie coconut genetic resources). When there is a clear understanding, the work of storing and utilizing crop biodiversity will be optimized. The natural habitats of native dendrobium species in Vietnam are increasingly diminishing due to climate change and excessive human exploitation.
This poses a risk of losing the genetic resources of not only orchids in general but also specifically dendrobium species. Additionally, the introduction and hybridization of numerous new dendrobium varieties contribute to the gradual neglect of many indigenous species. Therefore, the timely identification and assessment of genetic diversity in dendrobium species in Vietnam are crucial for the preservation of this valuable orchid group. In light of this reality, numerous research initiatives have been undertaken to preserve the diversity of orchid species, notably within the Orchid Collection of the Biotechnology Center in Ho Chi Minh City (Duong Hoa Xo ef a/.
As of the end of 1 2019, this collection has amassed nearly 400 orchid varieties, with 190 of them belonging to the dendrobium genus. These specimens have undergone identification and classification based on morphological characteristics. There are many studies in application Several marker systems, including morphological, biochemical, and molecular (DNA) markers, can be utilized to evaluate genetic diversity. In light of recent developments in genomics research, the value of DNA markers has greatly increased.
Utilizing multiple marker systems, the genetic diversity of crop plants can be accessible at the species level, the population level, among germplasm accessions, and at the individual genotype level, such as among pure lines or clones. The advantages of the SRAP technology include suitability for work on anonymous genomes; applicability to problems where only limited quantities of DNA are available; efficiency; and low expense. SRAP markers were used to conduct research on the diversity and genetics of some coconut varieties mentioned in the topic. The goal in the study is to improve the quality of the seeds and get a high yield while preserving genetic diversity coconut varieties in Vietnam.
Several studies have been conducted to apply molecular markers to support diversity assessment, genetic mapping, and construction of genetic linkage maps.