Application of molecular marker start codon target (scot) in individual identification of cat hoa loc mango (mangifera indica l)

NGUYEN TAT THANH UNIVERSITY /TVgaSV* NGUYEN TAT THANH True learning - True practice - True success - True future FACULTY OF BIOTECHNOLOGY GRADUATION THESIS APPLICATION OF MOLECULAR MARKER: START CODON TARGET (SCOT) IN INDIVIDUAL IDENTIFICATION OF CAT HOA LOC MANGO (Mangifera indica L) Student’s name : Pham Thi Hoa Mai Student ID :1611540685 Supervisor : MSc Nguyen Thi Nha Ho Chi Minh City, 2020 TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS ii ABSTRACT iv LIST OF FIGURES V LIST OF TABLES vi LIST OF ACRONYMS vii INTRODUCTION viii CHAPTER LITERATURE REVIEW 1.1 Introduction of Cat Hoa Loc Mango tree 1.1.1 Ecological characteristics 1.1.2 Distribution place .2 1.1.3 The values of Cat Hoa Loc mango 1.2 Techniques used in research 1.2.1 Molecular marker technique 1.2.2 Molecular marker SCoT 1.3 Research situation of SCoT molecular indicator and application of molecular indicator in individual identification 1.3.1 Studies ofSCoT directive application 1.3.2 Application of molecular markers in individual identification CHAPTER CONTENTS AND METHODS 2.1 Place of administration 2.2 Contents 2.3 Methods ii 2.3.1 DNA extraction 2.3.2 Screening polymorphic SCoT markers 12 2.3.3 PCR data analysis 14 CHAPTER RESULTS AND DISCUSSION 17 3.1 Sample collection 17 3.2 DNA extraction 20 3.3 Evaluate the polymorphism of 27 primers on 15 individuals of Hoa Loc mango 21 3.4 Identify specific DNA bands to demonstrate certain individuals 27 CONCLUSIONS AND RECOMMENDATION 29 REFERENCES 30 APPENDICES 32 iii ABSTRACT The study “Application of molecular marker: start Codon Target (SCoT) in individual identification of Cat Hoa Loc mango (Mangiferaindica £)" was carried out from October 2019 to July 2020 in Molecular Biology lab, Faculty of Biotechnology, Nguyen Tat Thanh Univerisity with an aim to based on Start Codon Target (SCoT) marker system A total of 15 Cat Hoa Loc mango cultivars included in the study were performed DNA extraction, polymorphic selection, and amplification of SCoT markers, and genetic diversity analysis using NTSYSpc software The polymorphism of 27 primers on 15 individuals of Hoa Loc mango was evaluated The study resulted in the successful identification of specific DNA bands for 15 individuals IV LIST OF FIGURES Figure 1.1 Picture of Cat Hoa Loc mango Figure 2.1 Preparation and data entry in Microsoft Excel 15 Figure 2.2 Example of similarity matrice of sample 15 Figure 2.3 Example of dendrograms drawing (phylogenetic trees) 16 Figure 3.1 Total DNA on electrophoresis gel 20 Figure 3.2 PCR products of SC0TO6, SCoT14, SC0TI8, and SC0T44 across 15 Cat Hoa Loc mango cultivars 23 Figure 3.3 Similarity matrix of 27 polymorphic SCoT marker across 15 Hoa Loe cultivars using SM coefficient 24 Figure 3.4 Phylogenetic tree of 27 polymorphic SCoT marker across 15 Cat Hoa Loc mango cultivars 24 Figure 3.5 Similarity matrix of 27 polymorphic SCoT marker across 15 Hoa Loc cultivars from SOFR1 using SM coefficient 26 Figure 3.6 Phylogenetic tree of 27 polymorphic SCoT marker across 15 Cat Hoa Loc mango cultivars from SOFRI 26 Figure 3.7 PCR products of SCoT19 across 15 Cat Hoa Loc mango cultivars from SOFRI 27 V LIST OF TABLES Table 2.1 Sequences of SCoT markers using in this study 12 Table 2.2 Component content of PCR reaction 13 Table 2.3 PCR cycling conditions 13 Table 3.1 List of mango cultivars using in this study 17 Table 3.2 OD concentration of 15 DNA samples 20 Table 3.3 List of polymorphic SCoT markers selected 22 Table 3.4 List of SCoT identified cultivars 27 vi LIST OF ACRONYMS bp Base pair DNA Deoxyribonucleic acid SCoT Start Codon Target UPGMA Unweighted Pair Group Method with Arithmetic mean Rnase Ribonuclease PLS Plant Lysis buffer PBB Plant Binding buffer PWB Plant Wash buffer EB Elution Buffer PCR Polymerase Chain Reaction uv Ultraviolet TBE Tris-Borate - EDTA OD Optical Density Vil INTRODUCTION Rationale for this thesis Identifying varieties is very important in choosing which varieties of plants to take the best advantages of the natural conditions and other resources of the region The best way to utilise that advantages is choosing the plants with the most favorable conditions for them to nuture, develop, and produce high yields Besides, identification of the varieties is important to the management and exploitation of its use-value Identifying varieties with agronomic properties such as appearance observing, color, taste, still faces with many difficulties and inaccuracy when identifying seed samples with similar characteristics in shapes and sizes Molecular technology has appeared and become an effective application for the selection of plant varieties, genomic, genetic diversity, and phylogenetic research in many types of plants Several crops such as tomato, rice, maize, wheat, soybean that are resistant to pests, salinity, drought with high productivity have been successfully selected with the help of molecular markers Currently, compared to other methods to evaluate genetic diversity, molecular techniques help US to the task easier and faster More and more researches apply molecular markers in the study of plant molecular genetics In particular, the SCoT molecular marker technique is a simple technique based on the PCR reaction of amplifying DNA fragments that contain the ATG codon SCoT advantages includes highly polymorphic amplification which is related to functional genes, ability to perform reactions without genetic information, and application in genetic diversity analysis, QTL mapping as well as bulked segregant analysis The study of using SCoT on mangoes in Vietnam has been conducted, besides the genetic grouping for 30 cultivar samples, this study has discovered some specific DNA bands that can be used to identify individuals of mango Among the current mango varieties grown in Vietnam, Cat Hoa Loc mango (Mangiferci indica L) is a fruit food favored by both its taste and the nutritional value This mango is high in nutrients such as calcium, potassium, carbohydrates, phosphorus, magnesium, vitamins such as vitamins A, and c Besides, Hoa Loc viii mango has a high economic value in exporting big markets such as the United States, Singapore, China, Europe, Korea, Japan, Australia, New Zealand, etc The identification of Cat Hoa Loc mangoes is very crucial in the management and exploitation of the use-value of Hoa Loc mangoes Objectives The aim of this study is to identify specific DNA bands that identify certain individuals and evaluate the polymorphism of 27 primers on 15 individuals of Hoa Loe mango IX Chapter Literature review CHAPTER LITERATURE REVIEW 1.1 Introduction of Cat Hoa Loc Mango tree Scientific name and location of Mango in the classification system: Kingdom Plantae Phylum Mangoliophyta Class Mangoliopsida Order Sapindales Family Anacardiaceae Genus Mangifera Species Itĩdica L Figure Picture of Cat Hoa Loc mango Hoa Loc mango was first grown in Hoa Loc village, Giao Duc district, Dinh Tuong province in 1930 (now Hoa hamlet, Hoa Hung village, Cai Be district, Tien Giang province) Mango characteristics involve moderate growing plants, oblique branches, umbrella-shaped canopy, oblong leaves, wavy leaf covers, pointed tail, 400 - 600 grams in weight of fruit, oblong fruit, near-inflated stems, bright yellow skin when ripping, 28 - 32 mm in thick and fleshy fruit, 78 - 80 % edible fleshy rate, low fiber, firm and smooth flesh, a sweet bar, Brix degree from 20 - 22 %, 105 - 120 days in flower-tofruit process from, good drought tolerance L 1.1.1 Ecological characteristics Cat Hoa Loc Mango has average growth and development rate Suitable soil is alluvial soil in the riverside, rich in nutrients Besides, Cat Hoa Loc mango can also be grown on acidic or saline soils The appropriate temperature for growing Hoa Loc mangoes is between 18 - 35 °C but still tolerant at temperatures of °C and above 40 °C for several weeks Terrain altitude 1300 m, where precipitation is about 1000 mm or more, however, 2-4 month drought period will help in fruiting better Chapter Results and Discussion X8 1.796 135.7 X9 1.985 199.5 X10 2.023 154.8 Xll 1.821 160.35 X12 1.753 107.34 X13 1.869 113.46 X14 1.708 117.15 X15 2.091 116.05 Al 1.852 172.6 A2 1.965 148.25 A3 1.971 203.55 A4 1.874 143.7 A5 1.965 148.25 A6 1.819 130.95 A7 1.791 158.65 A8 1.896 144.8 A9 2.003 150.15 A10 1.807 160.35 All 1.957 162.85 A12 2.015 101.5 A13 1.866 123.15 A14 1.814 155.7 A15 1.826 191.3 DNA concentration is a range of 101.5 - 203.55 (ng/pl), OD value is indicated with 260/280 ratio of 1.753 - 2.091 3.3 Evaluate the polymorphism of 27 primers on 15 individuals of Hoa Loc mango Table 3.3 List of polymorphic SCoT markers selected 21 Chapter Results and Discussion Total Polymorphic bands bands CAACAATGGCTACCACCC SCoT03 CAACAATGGCTACCACCG SC0TO4 CAACAATGGCTACCACCT SCoT05 CAACAATGGCTACCACGA SC0TO6 CAACAATGGCTACCACGC SCoT07 CAACAATGGCTACCACGG SC0TO8 CAACAATGGCTACCACGT SCoT09 CAACAATGGCTACCAGCA SCoTIO CAACAATGGCTACCAGCC SC0TI2 ACGACATGGCGACCAACG SCoT14 ACGACATGGCGACCACGC SCoT15 ACGACATGGCGACCGCGA SC0TI6 ACCATGGCTACCACCGAC SCoT17 ACCATGGCTACCACCGAG SC0TI8 ACCATGGCTACCACCGCC SCoT19 ACCATGGCTACCACCGGC SCoT20 ACCATGGCTACCACCGCG SC0T22 AACCATGGCTACCACCAC SC0T23 CACCATGGCTACCACCAG SC0T25 ACCATGGCTACCACCGGG SCoT26 ACCATGGCTACCACCGTC SC0T33 CCATGGCTACCACCGCAG SCoT36 GCAACAATGGCTACCACC 11 SC0T44 CAATGGCTACCATTAGCC SC0T6I CAACAATGGCTACCACCG SC0T66 ACCATGGCTACCAGCGAG SCoT77 CCATGGCTACCACTACCC Sum — 161 93 Primer ID Sequence (5’ to 3’) SCoT02 22 Chapter Results and Discussion Max 11 Min Average 5.962963 3.444444 The analysis of 27 SCoT markers on 15 Hoa Loe mango cultivars yielded a total of 161 obvious and bright bands, which their sizes ranged between 150 bp and 3000 bp Ninety-three bands from the total were demonstrated as polymorphism The number of the polymorphic bands for each SCoT markers varied from to bands SCoT77 created the most polymorphic bands at while SCoT19 created the least polymorphic bands at The average bands of SCoT were scored at 5.96 bands per marker and 3.44 polymorphic bands per marker markers SC0TO6, SCoT14, SC0TI8, and SC0T44 provided the clearest polymorphic band patterns Figure 3.2 PCR products of SC0TO6, SCoT14, SC0TI8, and SC0T44 across 15 Cat Hoa Loc mango cultivars 23 Chapter Results and Discussion Figure 3.3 Similarity matrix of 27 polymorphic SCoT marker across 15 Hoa Loc cultivars using SM coefficient Similar matrix analysis showed that the genetic similarity of 15 Cat Hoa Loc mango cultivars collected from Tien Giang, Ben Tre, Long An, and Khanh Hoa was quite high, ranging from 0.95 to 0.98 The highest similarity was found between X13 and X14 at 0.98 while the lowest similarity was scored between X2 and XI5 at 0.93 X9 Tien Giang IIA - X10 Long An XII Ben Tre XI2 Ben Tre IIB |XB Khanh Hoa 'xu Khanh Hoa X15 Khanh Hoa 0.96 —r— 0.9? COtttÓM —r • - - - I 0.97 0.98 Figure 3.4 Phylogenetic tree of 27 polymorphic SCoT marker across 15 Cat Hoa Loc mango cultivars 24 Chapter Results and Discussion Fifteen Hoa Loc mango cultivars were divided into two main groups: group I from Tien Giang from XI to X9 and group II from other areas from XI0 to XI5 Group I continued to divide into two subgroups at 0.96 in similarity consisted of IA and IB, which IA included only XI Tien Giang and IB included X2 to X9 (eight cultivars) In the group IB, eight cultivars were grouped into two branches at 0.965 in similar index which the IB(a) consisted of X2 to X5 and the IB(b) consisted X6 to X9 Among that, two X7 and X8 cultivars were demonstrated to have the highest similarity at 97.8% Group II comprised X10 to X15, which continued divided into two subgroups at 0.962 in similarity Group IIA included only XI0 Long An and group IIB included XI1 to X15, in which the highest similarity was belonged to X13 and X14 at 98% There are differences between the two groups of Cat Hoa Loc Mango in Tien Giang and other areas Because of its geographic location, climate, and other natural factors, it may have a tendency to separate species 25 Chapter Results and Discussion Al A2 A3 A4 A5 A6 A7 AS A9 A10 All A12 A13 AU A15 Al A2 A3 A4 A5 A6 A7 1.0000000 0.9985185 1.0000000 0.9985185 1.0000000 1.0000000 0.9985185 1.0000000 1.0000000 1.0000000 0.9970370 0.9985185 0.9985185 0.9985185 1.0000000 0.9970370 0.9985185 0.9985185 0.9985185 1.0000000 1.0000000 0.9970370 0.9985185 0.9985185 0.9985185 1.0000000 1.0000000 1.0000000 0.99'0370 0.9985185 0.9985185 0.9985185 1.0000000 1.0000000 1.0000000 0.9970370 0.9985185 0.9985185 0.9985185 1.0000000 1.0000000 1.0000000 0.9970370 0.9985185 0.9985185 0.9985185 1.0000000 1.0000000 1.0000000 0.9955556 0.99'0370 0.99'0370 0.9970370 0.9985185 0.9985185 0.9985185 0.9940'41 0.9955556 0.9955556 0.9955556 0.9970370 0.9970370 0.9970370 0.9940'41 0.9955556 0.9955556 0.9955556 0.9970370 0.9970370 0.9970370 0.9940'41 0.9955556 9955556 9955556 0.9970370 0.9970370 0.9970370 09940741 0.9955556 0.9955556 0.9955556 0.9970370 0.9970370 0.9970370 AS A9 A10 1.0000000 1.0000000 1.0000000 1.0000000 1.0000000 1.0000000 0.9985185 0.9985185 0.9985185 0.9970370 0.9970370 0.99703'0 0.99'0370 0.9970370 0.9970370 0.9970370 0.9970370 0.9970370 0.9970370 0.9970370 0.99703'0 All A12 A13 AU A15 1.0000000 0.9985185 1.0000000 0.9985185 1.0000000 1.0000000 0.9985185 1.0000000 1.0000000 1.0000000 0.9985185 0.9970370 0.9970370 0.99'0370 1.0000000 Figure 3.5 Similarity matrix of 27 polymorphic SCoT marker across 15 Hoa Loc cultivars from SOFRI using SM coefficient Figure 3.6 Phylogenetic tree of 27 polymorphic SCoT marker across 15 Cat Hoa Loc mango cultivars from SOFRI A similar matrix analysis showed that the genetic similarity of 15 Cat Hoa Loc mango cultivars from SOFRI was very high, ranging from 0.99 to 1.00 These results illustrated the homogeneity of 15 SOFRI cultivars, which is considered to originate from one original ortet 26 Chapter Results and Discussion Phylogenetic tree of 15 cultivars from SOFRI illustrated that A2, A3, and A4 had homogeneity in data of DNA fragments amplified by SCoT markers Similar to that, group of A5, A6, A7, A8, A9, and A10 and group of A12, A13, and A14 also share the homogeneity when analyzing DNA fragment around ATG codon by SCoT markers Figure 3.7 PCR products of SCoT19 across 15 Cat Hoa Loc mango cultivars from SOFRI 3.4 Identify specific DNA bands to demonstrate certain individuals All 15 Cat Hoa Loc samples are identified by two markers SCoT02 and SC0T33 and Cat Hoa Loc cultivars were separated into small clusters as same as Nga’s results 20 SCoT is a highly polymorphic marker at an individual level, which is evident by amplifying different bands for individuals in the same breed Table 3.4 List of SCoT identified cultivars Primers Identified cultivars Locus (bp) SC0TO2 X15 2000 X10 2100 XI 400 SCoT03 27 Chapter Results and Discussion SCOT05 X9 2000 SCOT08 X8, X12 1750 SCOT09 X9, X10 600 SCoTIO X10 1100 SCoT14 X3 2000 SCoT15 X13 2000 SCoT17 X4 1250 X2 1500 X5 200 SC0T25 X2, X3, X4 2000 SCoT26 X4 1500 SCoT36 XI,X6 1750 SC0T6I XI1, X12 600 SCoT77 X4, X10 1250 SC0TI8 Summarize: Based on Table 3.4, there are specific primers used to identify individual - SCoT02 and SCoT15 identified three mango cultivars collected from Khanh Hoa at 2000 bp; - SC0T6I identified two mango cultivars collected from Ben Tre at 600 bp; - SCoTIO identified one mango cultivar collected from Long An at 1100 bp; - SC0TI8, SC0T25, SCoT26, and SCoT36 identified nine mango cultivars collected from Tien Giang at 200, 2000, 1500 and 1750 bp respectively 28 CONCLUSIONS AND RECOMMENDATION Conclusions Selection of 27 polymorphic markers from 46 SCoT markers Fifteen Cat Hoa Loc mango cultivars were divided into main groups: group I involved XI to X9 from Tien Giang and group II from other areas consisted of X10toX15 specific primers used to identify individual: - SCoT02 and SCoT15 identified three mango cultivars collected from Khanh Hoa at 2000 bp; - SC0T6I identified two mango cultivars collected from Ben Tre at 600 bp; - SCoTIO identified one mango cultivar collected from Long An at 1100 bp; - SC0TI8, SC0T25, SCoT26, and SCoT36 identified nine mango cultivars collected from Tien Giang at 200, 2000, 1500 and 1750 bp respectively Recommendations Continuing research on screening markers to identify individuals for Cat Hoa Loc mango cultivars group from Southern Horticultural Research Institute (SOFRI) 29 REFERENCES Nguyễn Minh Châu Giới Thiệu Các Giống Cây An Quả Phô Biến Ở Miền Nam NXB Nông Nghiệp 2009 Truơng Hồng Võ Tuấn Kiệt, Dương Ngọc Thành Phân tích chuồi giá trị xồi cát Hịa Lộc (Mangifera indica L) tỉnh Đơng Tháp Tạp chí Khoa học Trường Đại học Cần Thơ 2014:35 Nguyễn Đức Thành DNA marker techniques in study and selection of plant Tạp Sinh học 2015;36(3) Luo Cong, He Xin-Hua, Chen Hu, Ou Shi-Jin, Gao Mei-Ping Analysis of diversity and relationships among mango cultivars using Start Codon Targeted (SCoT) markers Biochemical Systematics and Ecology 2010;38:1176-1184 Chai Xutian, Dong Rui, Wenxian Liu, Wang Yanrong, Liu Zhipeng optimizing Sample Size to Assess the Genetic Diversity in Common Vetch (Vicia sativa L.) Populations Using Start Codon Targeted (SCoT) Markers Molecules 2017;22 Luo Cong, He Xin-Hua, Chen Hu, et al Genetic diversity of mango cultivars estimated using SCoT and ISSR markers Biochemical Systematics and Ecology 2011;39:676-684 Youxiong Que, Yongbao Pan, Yunhai Lu, et al Genetic analysis of diversity within a Chinese local sugarcane germplasm based on start codon targeted polymorphism BioMed research international 2014;2014:468375 Seyedimoradi Hiva, Talebi Reza, Fayaz Farzad Geographical diversity pattern in Iranian landrace durum wheat (Triticum turgidum) accessions using start codon targeted polymorphism and conserved DNA-derived polymorphism markers Environmental and Experimental Biology 2016; 14(2):63-68 Yan Guo, Lina Zhai, Hao Long, et al Genetic diversity of Bletilla striata assessed by SCoT and IRAP markers Hereditas 2018; 155:35 10 Collard Bertrand c Y., Mackill David J Start Codon Targeted (SCoT) Polymorphism: A Simple, Novel DNA Marker Technique for Generating GeneTargeted Markers in Plants Plant Molecular Biology Reporter 2008;27( ):86 11 H p Gajera, R p Bambharolia, Rinkal K Domadiya, s V Patel, B A Golakiya Molecular characterization and genetic variability studies associated with fruit quality of indigenous mango (Mangifera indica L.) cultivars Plant Systematics and Evolution 2014;300(5):1011-1020 12 Fahad Al-Qurainy, Salim Khan, Mohammad Nadeem, Mohamed Tarroum SCoT marker for the assessment of genetic diversity in Saudi Arabian date palm cultivars 2015;47(2):637-643 13 Lê Ngọc Triệu, Nguyền Hoàng Phong, Mai Tiến Đạt Khảo sát đa dạng di truyền xác lập thị phân tử cho việc nhận dạng so dòng bơ (Persea americana 30 Miller) qua sơ tuyển chọn Lâm Đồng Tạp chí Khoa học Đại học Đà Lạt 2016;6(4) 14 Nguyễn Văn Trứ, Nguyễn Đức Thành, Hồ Hữu Nhi, Lê Thị Bích Thủy Ket đánh giá đa dạng di truyền số giống mía tập đồn Việt Nam chọn lọc thị SSR nhận biết dòng giống có hàm lượng đường cao Tạp Khoa học Công nghệ 2012;50(2) 15 Jingjing Yang, Jian Zhang, Ruixi Han, et al Target SSR-Seq: A Novel SSR Genotyping Technology Associate With Perfect SSRs in Genetic Analysis of Cucumber Varieties Frontiers in plant science 2019; 10:531 16 Risliawati Andari, Riyanti Eny L, Lestari Puji, Utami Dwinita w., Silitonga Tiur s Development of SSR Marker Set to Identify Fourty Two Indonesian Soybean Varieties Jurnal AgroBiogen 2016;l 1(2):49 17 Nam-Hoon Kim, Hong-Il Choi, In-Ok Ahn, Tae-Jin Yang EST-SSR Marker Sets for Practical Authentication of All Nine Registered Ginseng Cultivars in Korea Journal ofginseng research Jul 2012;36(3):298-307 18 Jamshidi s NTSYSpc 2.02e Implementation in Molecular Analysis(Clustering, Screening, and Individual Selection) 2011 19 Voytas Daniel Agarose Gel Electrophoresis Current Protocols in Molecular Biology 2000;51(l):2.5A 1-2.5A.9 20 Le Thi Thanh Nga Genetic diversity of mango cultivars in Central and Southern Vietnam using Start Codon Target markers 2019 31 Biodata APPENDICES wf xĩ » ỉĩ ữ X? 1’4 L J X8 xé' X*' J r • xỉ’ xíó' xíí' xú xíĩ XÌ4 xis Xi X4 x& N6 JW MỀO Mil MS X12 M13 M14 XIS I-ja 1 j L L Jl Figure PCR production of SC0TO2 Figure PCR production of SC0TO3 Figure PCR production of SC0TO4 Figure PCR production of SC0TO5 Figure PCR production of SC0TO6 M *1X2 &F XI X2 X3 X4 X5 X£ W MS x» ** —- •• Ì? ***** MS Xll X13 X12 XI4 Figure PCR production of SC0TO7 XIS X2 XI Xi X4, x% < IB y® HU XẬỈ Xỉi Xi4 XJ5 Ị — - TT *4 ỆỆ it Figure PCR production of SC0TO8 Figure PCR production of SCoT09 32 M Figure PCR production of SCoTIO Figure 10 PCR production of SCoT12 Figure 11 PCR production of SCoT14 Figure 12 PCR production of SCoT15 XI Xi XỈ X4 xs xs X7 xs M X9 xlo Xll XÍ2 X13 XI4 XIS ■ |'| M XI XỈ X3 XI XS XT X/ Xi X3 xli XÍỈ XJ3 x>4 XIS Figure 13 PCR production of SCoT 16 Figure 14 PCR production of SCoT 17 Figure 15 PCR production of SC0TI8 Figure 16 PCR production of SCoT19 33 M XI xa XÀ Xà ỈỌL X4 í15 I xg u M XI X2 X3 X4 Xb XB Xì X8 X9 XÍO Xll XV XI3 X14 Figure 17 PCR production of SCoT20 Figure 18 PCR production of SC0T21 Figure 19 PCR production of SC0T22 Figure 20 PCR production of SC0T23 M XI XI X3 x» M M «» -M » no «11 xn xn X1« XIS Ml xa Xi & XJ «1 a* ** XL a* —* ~ ••••••• Figure 21 PCR production of SC0T25 M X1S XL XL XX M XL Xd XM Xil XiZ Figure 22 PCR production of SCoT26 M3 X44 Figure 23 PCR production of SCoT36 XI X2 X4 X5 X/ X9 X1O Xll XU Xlỉ X14 XIS Figure 24 PCR production of SC0T44 34 Figure 25 PCR production of SC0T61 M Xi M X5- Xfe life- X8 x» X1O.X11 xu X13 X14 Figure 26 PCR production of SC0T66 XIS Figure 27 PCR production of SC0T77 35 ... molecular indicator and application of molecular indicator in individual identification 1.3.1 Studies ofSCoT directive application 1.3.2 Application of molecular markers in individual. .. study ? ?Application of molecular marker: start Codon Target (SCoT) in individual identification of Cat Hoa Loc mango (Mangiferaindica £)" was carried out from October 2019 to July 2020 in Molecular. .. medicinal plants9, and others 1.3 Research situation of SCoT molecular indicator and application of molecular indicator in individual identification 1.3.1 Studies of SCoT directive application In