MINISTRY OF EDUCATION AND TRAINING NONG LAM UNIVERSITY-HO CHI MINH CITY FACULTY OF BIOLOGICAL SCIENCES EVALUATION OF GENETIC DIVERSITY OF TEA TREE (Melaleuca alternifolia) COLLECTED FROM DONG THAP MUOI CENTER BASED ON ISSR (INTER SIMPLE SEQUENCE REPEATS) MARKER Majors : BIOTECHNOLOGY Student : VO LAM HAI NAM Student code : 19126251 Academic year : 2019 - 2023 Thu Duc City, 03/2024 MINISTRY OF EDUCATION AND TRAINING NONG LAM UNIVERSITY-HO CHI MINH CITY FACULTY OF BIOLOGICAL SCIENCES GRADUATION THESIS EVALUATION OF GENETIC DIVERSITY OF TEA TREE (Melaleuca alternifolia) COLLECTED FROM DONG THAP MUOI CENTER BASED ON ISSR (INTER SIMPLE SEQUENCE REPEATS) MARKER Advisor Student Pham Duc Toan, Ph.D Vo Lam Hai Nam Thu Duc City, 03/2024 ACKNOWLEDGEMENT Fixed: The thesis was completed at Nong Lam University. Throughout the thesis completion process, I received invaluable assistance. First and foremost, I want to express my sincere gratitude to Mr. Pham Duc Toan for his guidance, knowledge, and experience during the thesis's creation.
I also extend my appreciation to the teachers of the Faculty of Science and Biotechnology at Nong Lam University, who shared valuable knowledge with me during my last academic term. Lastly, I want to thank my family and friends who consistently encouraged and supported me during the writing of this thesis. Additionally, I'd like to express my gratitude to the respondents who enthusiastically participated in answering the survey questions, contributing significantly to the completion of this graduate thesis. Once again, I sincerely thank you all! CONEIRMATION COMMITMENT My name is Vo Lam Hai Nam, Student ID: 19126251, Class: DH19SHC, Faculty of Biological Sciences, Nong Lam University.
I hereby affirm that this is my personally conducted Graduation Thesis. All data and information presented in the research are completely truthful and objective. I fully take responsibility before the Council for these commitments. Thu Duc City, February 2024 Student’s signature 1 ABSTRACT The tea tree (Melaleuca alternifolia), which has strong antibacterial therapeutic qualities is a valuable essential oil tree in Australia.
Subsequently, the tea tree was brought into Vietnam with the intention of using its medical qualities, particularly at the Dong Thap Muoi medicinal reserve located in Moc Hoa district, Long An province. In order to support acclimatization and conservation efforts, the study's objective was to assess the genetic diversity of 50 melaleuca samples that were gathered within the reserve using ISSR molecular biological markers. DNA was taken from leaf samples for this investigation, and 20 ISSR primers were used in PCR investigate any sample. Used all 50 tea tree samples, a PCR-ISSR reaction was performed using the 12 primers (UBC811, UBC814, UBC825, UBC841, UBC845, UBC848, UBC850, UBC854, UBC856, UBC873, UBC880, and UBC890) that were chosen for their high polymorphism rate following the reaction.
In order to create a genetic classification tree, the outcomes will be based on the DICE similarity coefficient and UPGMA clustering type using NTSYSpc 2. Out of the 104 bands that were generated, 93 (88. The tea tree samples have genetic distance coefficients ranging from 0.77 and genetic similarity coefficients from 0. A genetic distance coefficient of 0.44 was used to split the 50 tea tree samples into 5 groups based on the research results.
The results showed that the genetic diversity was not too high among the 50 tea tree samples used in the study. Keywords: Dong Thap Muoi medicinal revese, genetic diversity, ISSR primer, Melaleuca alternifolia, The tea tree 11 TÓM TẮT Cây tram trà (Melaleuca alternifolia), một loài cây cho tinh dau có giá trị kinh tế cao của nước Úc bởi tính chất được liệu kháng khuẩn tốt mà nó mang lại. Sau đó, cây được du nhập về Việt Nam nhằm khai thác tính chất được liệu này, đặc biệt là tại khu bảo tồn dược liệu Đồng Tháp Mười ở huyện Mộc Hóa, tỉnh Long An. Do đó, nghiên cứu được tiễn hành nhằm mục tiêu đánh giá sự đa dạng di truyền của 50 mẫu tràm trà được thu thập tại khu bảo tồn dựa trên chỉ thị sinh học phân tử ISSR để phục vụ cho công tác di thực và bảo tồn.
Trong nghiên cứu này, tiễn hành ly trích DNA từ mẫu lá và thực hiện phản ứng PCR khảo sát một mẫu bất kì với 20 primer ISSR. Sau phản ứng, chọn lọc được 12 primer (UBC811, UBC814, UBC825, UBC841, UBC845, UBC848, UBC850, UBC854, UBC856, UBC873, UBC880, UBC890) cho tỉ lệ đa hình cao và thực hiện tiếp phản ứng PCR — ISSR với tat cả 50 mau tram trà. Kết qua thu được dựa vào hệ số tương đồng DICE, kiểu phân nhóm UPGMA trên phần mềm NTSYSpe 2.1 dé dựng cây phân nhóm di truyền. Tổng cộng có 104 băng được tạo ra trong đó 93 băng đa hình chiếm tỷ lệ 88,6%.
Hệ số khoảng cách di truyền của các mẫu tram trà nằm trong khoảng từ 0,19 — 0,77 (hệ số tương đồng di truyền từ 0,23 — 0,81). Kết quả nghiên cứu đã chia 50 mẫu tràm trà thành 5 nhóm ở hệ số khoảng cách di truyền trung bình 0,44. Kết quả cho thấy sự đa đạng di truyền không quá cao giữa 50 mẫu tràm trà được sử dụng trong nghiên cứu. Từ khóa: Cây tràm trà, đa dạng di truyền, ISSR primer, khu bảo tồn dược liệu Đồng Tháp Mười, Melaleuca alternifolia.
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Results of total DNA extraction from tea tree leaves.3, Results of PCR-ISSR T634GLDDssssssssesssssitiaisoiiGi13045E633684G8488553854301551484BESLGESSES3SG8333584868 23 4. Measured primer temperatures for ISSR-PCR reaction. PCR product with ISSR: HIẢIGBOeeeeeeieenigi41150210011110145021011045010215T1L6T215EEEEESE SE 25 4. Genetic diversity analysis of 50 tea tree safnpÏes.
2s re 27 VyAb on. CONCLUSION AND SUGGESTIONS. 2c 2S Series 35 So, loyal! Vics (0): |e eee ee ee ee ees 35 5,2, SUSPESHONS seconsencseeennnemnnmenenenem mene eee aOR 35 REFERENCES csscsssessussseswecusaneenenen rene EA03831015005E28EL-ISGISLRSHOINSSHESEESS14SE34SESEESEEHSSAESSTSSSESESEEESBESSSENGE 36 44ND eee ee ee eT ee ee ee 39 VI LIST OF ABBREVIATIONS Bp Base pair DNA Deoxyribonucleic acid dNTP Deoxyribonucleotide triphosphate RAPD Random Amplified Polymorphic DNA RFLP Restriction Fragment Length Polymorphisms AFLP Amplified Fragment Length Polymorphism ISSR Inter Simple Sequence Repeat SSR Simple Sequence Repeat SDS Sodium dodecyl sulfat EDTA Ethylene Diamine Tetra acetic Acid PCI Phenol/Chloroform/Isoamyl alcohol Cl Chloroform/Isoamyl alcohol TAE Tris — Acetate -EDTA TE Tris - EDTA NTSYSpc Numerical Taxonomy System personal computer UPGMA Unweighted Pair -group Method with Arithmetic Mean SAHN Sequential agglomerative hierarchical non-overlapping Vii LIST OF TABLES Page Table 3. List of 50 tea tree samples collected at Dong Thap Muoi Medicinal Materials Conservation Center, Long An province used for research.(Continue) List of 50 tea tree samples collected at Dong Thap Muoi Medicinal Materials Conservation Center, Long An province used for research.
List of 20 primers for PCR reaction - ISSR. cece cece ceseeecesceeeeeeeeeeeeeeeseteeeeeeees 18 Table 3. Thermal cycling for POR TeaCtl OD wx. cxesvessessasenverenssaxsnexnsesansnace exeastenessiraoaviannesecerenses 18 Table 4.1 Records the leaf dimensions of 50 Melaleuca samples.(countine) Records the leaf dimensions of 50 Melaleuca samples.1(countine) Records the leaf dimensions of 50 Melaleuca samples.
12 primer ISSR were selected to preformed ISSR-PC. Summary of band numbers of 12 primers surVeyed. -- --- + s+sc+xs+scsesexss 25 Table 4. Genetic distance coefficient of 50 tea tree saimpÏes.(countine) Genetic distance coefficient of 50 tea tree samples.(countine) Genetic distance coefficient of 50 tea tree samples.(countine) Genetic distance coefficient of 50 tea tree samples.
------‹ 31 Vili LIST OF FIGURES Page Figure 21. THC t a từ ÊnseseesesesessetoniiEnsgtiSiESGSSE3.Đ073HI2ESSREG/2871800/G002080808 3 Figure 2. Diagram illustrating ISSR primer fVp€s. Representative tea tree leaf sarmpÏÌe.
Total DNA electrophoresis results of 50 tea tree samples. Electrophoresis results of PCR products of 1 DNA sample with 20 ISSR BEIGES: At S54 Gace ascecuresasssesemneresnsayse exseeraness quarsenarraseunemensasmaneseerserasnsmercneneeens 24 Figure 4. Electrophoresis results of PCR products of 50 tea tree samples with primer UB C811. cssssecsusscssseusesseseameanenscsnaats ste mason eitanea man naam emer mae 80005 00550 26 Figure 4.
Electrophoresis results of PCR products of 50 tea tree samples with primer IBC 8205 ongeiisxii6 0E G52000705.5:LS582158 TV8860067300XLXSR02Đ5HSSSS0WGGEAE2/2EL498320128E09Đ8I70007E080516:38980 27 Figure 4. Genetic clustering dendrogram drawn using UPGMA method.Introduction Tea Tree (Melaleuca alternifolia) is a species of tree in the Myrtaceae family, mainly grown to produce essential oil. Melaleuca alternifolia, an Australian native, prefers wet soil and lots of sunshine. At the moment, this plant is widely grown in numerous locations including Vietnam.
Melaleuca is a small woody plant 2 - 3 m tall and up to 15 m tall often grown on coastal sandy soil or alkaline soil in high terrain. Tea tree has many nutritional values and today is used in many different fields such as cosmetics and pharmaceuticals. Tea tree has two main active ingredients that play an important role in disinfecting: Eucalyptol accounting for 42-52% and a- Terpineol accounting for 5- 12%. While Eucalyptol has a pleasant fragrance and mild antiseptic properties that are often used by many cosmetic companies, a- Terpineol is used to produce antiseptic and antifungal products.
Because this tree species has highly valuable essences, it is exploited and imported to Vietnam research and discovery are still restricted due to the paucity of information on the genetic resources of Melaleuca trees in our nation. It's essential to comprehend how trees behave. However, it is sometimes exceedingly challenging to identify the links between Melaleuca varieties when just using phenotypic criteria to evaluate Melaleuca variations. In addition, Moc Hoa Tram 1s an expert in manufacturing products using essential oils that completely use the Dong Thap Muoi medicinal reserve region in Moc Hoa district, Long An province, where the alum soil terrain is well-suited for the benefits of tree cultivation.
There are numerous techniques available now to investigate plants genetic diversity. Here are resources for crop genetics, including Simple Sequence Repeat (SSR), Random Amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), and Inter Simple Sequence Repeat (ISSR). These techniques are interconnected, and their analysis contributes to understanding genetic diversity. Additionally, morphological markers are used to assess genetic diversity, although they can be influenced by environmental factors, resulting in phenotypic variations.Objective Genetic analysis and phylogenetics of Melaleuca alternifolia individual based on ISSR molecular markers in order to migrate and conserve the Melaleuca alternifolia for medicinal purposes in Dong Thap Muoi Medicinal Conservation, Moc Hoa district, Long An province.Research contents Content 1: Evalution of growth and development of survey 50 Melaleuca alternifolia collected from Dong Thap Muoi Center for Reseacher Conservation and Development of Medicinal Plants.
Content 2: Using the ISSR marker to evaluate the genetic diversity of 50 Melaleuca alternifolia samples. Introduction to Melaleuca alternifolia 2.1 Origin and Distribution of Tea Tree Gender: Plantae Branch: Angiospermae Class: Eudicots Ordo: Myrtales Familia: Myrtaceae Genus: Melaleuca Figure 2.