Identification of tangerine varieties in vietnam using its, matk, rbcl primers

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY UNDERGRADUATE THESIS TOPIC: IDENTIFICATION OF TANGERINE VARIETIES IN VIETNAM USING ITS, MATK, RBCL PRIMERS Student : TO HOANG ANH MINH School year : 2017 – 2022 Major : Biotechnology Lecture : Assoc Prof Dr TRAN DANG KHANH Agricultural Genetics Institute Assoc Prof Dr DONG HUY GIOI Vietnam National University of Agriculture HANOI – 2022 COMMITMENT I hereby declare that all results in this thesis are my own work The data and results published in the thesis are completely honest, accurate and have not been published in any other works Hanoi, March 2022 Student To Hoang Anh Minh i ACKNOWLEDGMENTS First of all, I would like to express my deep respect and gratitude to Assoc Prof Dr Tran Dang Khanh - Head of Genetic Engineering Department - Agricultural Genetics institute and Assoc Prof.Dr Dong Huy Gioi - Head of Department of Biology - Vietnam national university of Agriculture who has directly guided, enthusiastically instructed and created all the best conditions for me during my study and scientific research I would like to thank the staff of the Department of Genetic Engineering Agricultural Genetics institute for always encouraging me as well as providing valuable professional contributions for me to complete this thesis I would also like to express my deep gratitude to the teachers of faculty of Biotechnology - Vietnam National University of Agriculture for helping me have the right and correct orientation to carry out my thesis Finally, I would like to express my deep gratitude to my father, mother, family members as well as friends who have always supported and encouraged me to be stable throughout the study and research process I sincerely thank! Hanoi, March 2022 Student To Hoang Anh Minh ii CONTENTS COMMITMENT i ACKNOWLEDGMENTS ii LIST OF TABLES vi LIST OF FIGURES vii SUMMARY viii SECTION I: INTRODUCTION 1.1 The urgency of the subject 1.2 The goal of the subject 1.3 Research range SECTION II: OVERVIEW 2.1 Overview of tangerine 2.2 Methods used in determining genetic relationships in citrus fruits 2.2.1 Genetic diversity, classification of citrus fruit groups based on morphological markers 2.2.2 Genetic diversity, classification of citrus fruit groups based on isozyme markers 2.2.3 Genetic diversity, classify citrus fruit groups based on DNA molecular markers 2.3 DNA barcode 11 2.3.1 The loci used in the method DNA barcoding in plants 14 2.3.2 Nuclear gene sequence 14 2.3.3 Ribosome coding region 15 2.3.4 Chloroplast gene sequence 16 2.3 Research on genetic diversity and classification in Vietnam citrus fruit trees 17 SECTION III: RESEARCH MATERIALS AND METHODS 19 3.1 Research meterials 19 iii 3.2 Chemicals 19 3.3 Research methods 20 3.3.1 Total DNA extraction 20 3.3.2 PCR reaction 22 3.3.3 PCR cycle 22 3.3.4 Electrophoresis to check PCR products 22 3.3.5 Electrophoresis method on gel agarose 23 3.3.6 Electrophoresis method on gel polyacrylamide 24 3.3.7 Purification gel of kit Qiagen 24 3.3.8 Sequencing 25 3.3.9 Data analysis: 25 SECTION IV: RESULTS AND DISCUSSION 26 4.1 Results of nucleotide sequence analysis of ITS gene region between 14 studied tangerine varieties 26 4.2 The results of nucleotide sequence analysis of the matK gene region between 14 researched tangerine varieties 39 4.3 The results of nucleotide sequence analysis of the rbcL gene region between the studied tangerine samples 51 SECTION V: CONCLUSIONS AND PETITIONS 61 REFERENCES 62 iv ABBREVIATION Acronyms Full name AFLP Amplified Fragment Length Polymorphism CTAB Cetrimonium bromide DNA Deoxyribonucleic acid EDTA Ethylenediaminetetraacetic acid F Forward FAO Food and Agriculture Organization ISSR Inter-Simple Sequence Repeats ITS Internal transcribed spacer PCR Polymerase Chain Reaction PCR Polymerase Chain Reaction PVP Polyvinylpyrrolidone R Reverse RAPD Randomly Amplified Polymorphic DNA RFLP Restriction Fragment Length Polymorphism SCAR Sequence Characterized Amplification Regions SDS Sodium dodecyl sulfate SNP Single Nucleotide Polymorphism SSR Simple Sequence Repeats SSR Simple sequence repeat TAE Tris-acetate-EDTA v LIST OF TABLES Table 3.1 List of tangerine samples leaves in the study 19 Table 3.3 List of components in PCR reaction 22 Table 3.4 List of phase in PCR cycle 22 Table 3.5 List of components in Electrophoresis 23 Table 4.1 Evaluation of the similarity and coverage of the rbcL region DNA sequences of the research samples with the corresponding sequences on NCBI 27 Table 4.2 Some position differences in nucleotide sequences of tangerine samples research and reference samples 29 Table 4.3 Genetic similarity coefficient between 14 samples of tangerine studied and reference gene 38 Table 4.4 Coefficient of nucleotide sequence similarity in the matK gene region between 14 studied tangerine varieties and reference samples 40 Table 4.5 Some position differences in nucleotide sequences of tangerinelike samples research and reference samples 42 Table 4.6 Genetic similarity coefficient between 14 samples of tangerine studied and reference gene 51 Table 4.7 Evaluation of the similarity and coverage of the rbcL region DNA sequences of the research samples 53 Table 4.8 Some position differences in nucleotide sequences of the research and reference samples 55 Table 4.9 The nucleotide sequence similarity coefficient of the RbcL gene region between the studied tangerine samples and the reference sample 60 vi LIST OF FIGURES Figure 2.1 Lists some of the studies using DNA barcoding as a plant identification tool( Peter M Hollingsworth, 2011) 14 Table 3.2 List of primer pairs used in the study 20 Figure 4.1 PCR results of 14 samples with primers ITS1/ITS4 26 Figure 4.2 Images compare the nucleotide sequences of the ITS region of the studied tangerine samples using the primer pairs ITS1/ITS4 37 Figure 4.3 Phylogenetic tree of the ITS gene region of 14 studied tangerine varieties and reference genes 39 Figure 4.4 PCR results of 14 samples studied with matKCi1 primer pair 39 Figure 4.5 Some images comparing matK region nucleotide sequences of studied tangerine samples with reference samples using primer pairs 50 Figure 4.6 Phylogenetic tree of the matK gene region of 14 studied tangerine varieties and reference genes 51 Figure 4.7 PCR results of 44 research samples with primer pair rbcLCi2 52 Figure 4.8 Some images comparing the nucleotide sequences of the Rbc region of the studied tangerine samples 59 Figure 4.9 Phylogenetic tree of the rbcL gene region of the studied and reference samples 60 vii SUMMARY Using the ITS genomic region is one of the most useful tools for phylogenetic assessment in both plants and animals because it is common in nature to have genotypes associated with highly conserved chloroplast genomes and Because of the specificity of each species, the use of chloroplast genome analysis results in phylogenetic studies and plant taxonomy is of great interest to scientists as a method widely applied in many different plant species In particular, with the situation that Vietnam's agricultural products are being interested in promoting export, so as to improve the ability to conserve, classify, exploit and ensure the origin of mandarin varieties Therefore, the topic " Identification of tangerine varieties in Vietnam using ITS, matK, rbcL primers " was carried out to identify local mandarin varieties, aiming to build a database of genetic resources of these species native tangerine varieties Through the process of research, comparison and reference to published studies, it shows the ability to identify some citrus varieties in the sample group based on ITS, matK, rbcL gene regions On that basis, continuing to research, perfect and expand the orientation of building DNA barcodes for tangerine sources, thereby helping to classify and identify genetic resources viii SECTION I: INTRODUCTION 1.1 The urgency of the subject Tangerine belongs to the group of citrus fruit trees, is the most widely produced fruit tree in the world There are two clearly differentiated markets: the fresh fruit market and the processed juice market The increase in the production of citrus in the world was relatively stable in the last decades of the twentieth century, and brought about great economic resources they are produced in a variety of countries around the world, with a total production The citrus fruit quantity in 2019 is about 158.9 million tons (FAOSTAT, 2020) In Vietnam, there are many famous tangerine growing regions with an annual output of up to tens of thousands of tons However, due to the rapid increase in the area of citrus trees, despite the recommendations of the authorities and localities, farmers have begun to have consequences such as price reductions and price differences between provinces citrus growing area According to statistics of the Ministry of Industry and Trade, in 2018, the area of fruit trees increased, concentrated in the citrus group (oranges, tangerines, pomelos) As of September 2018, the area of citrus trees in the whole country reached 192,700 hectares, an increase of 3% over the same period in 2017 Meanwhile, most citrus trees are only consumed domestically and exported insignificantly With fruit products that meet the requirements of importers on food safety and hygiene criteria, high quality, branded Vietnam, the quantity of goods is not enough to satisfy the importer The biggest limitation of fruit production, including tangerines, is the small scale, making it difficult for investment, quality control and product consumption In order to promote the goal of meeting domestic demand, towards the export of Vietnam's agricultural products, including tangerines It is necessary to develop identification and study of each variety and genetic resources to determine the origin to help preserve, identify and develop mandarin varieties with economic value in each region Since then, I have done showed monomorphic bands with the size of about 800 bp Figure 4.7 PCR results of 44 research samples with primer pair rbcLCi2 M: Marker generuler 100bp plus DNA The results of homologous sequence analysis of the DNA barcodes of the studied tangerine samples and the sequence on NCBI showed that the similarity of the samples ranged from 95.11% to 99.62% and the coverage was 94% to 98% of the reference sample The similarity of Q1 variety with reference sample AB505952.1 Citrus reticulata is 95.11% The similarity of the sample Q2 (with the reference sample AB505952.1_Citrus_reticulata is 99.62%(table 4.3.1) 52 Table 4.7 Evaluation of the similarity and coverage of the rbcL region DNA sequences of the research samples No Name Code Reference sequence Similarity Coverage (%) (%) Tangerine duong Q1 AB505952.1_Citrus_reticulata 95,11 97 Q2 AB505952.1_Citrus_reticulata 99,62 96 Q3 AB505952.1_Citrus_reticulata 99,37 94 Q4 AB505952.1_Citrus_reticulata 99,37 96 Q5 AB505952.1_Citrus_reticulata 99,49 96 Tangerine chieng co Tangerine hong(Tieu son) Tangerine Tich Giang Tangerine huong can Tangerine Trang Dinh Q6 AB505952.1_Citrus_reticulata 99,61 98 Tangerine lua Q7 AB505952.1_Citrus_reticulata 99,49 98 Q8 AB505952.1_Citrus_reticulata 99,49 96 Tangerine Dong Khe Tangerine ngoc hoi Q9 AB505952.1_Citrus_reticulata 99,37 96 10 Tangerine hoi Q11 AB505952.1_Citrus_reticulata 98,74 76 Tangerine ngot 11 Ha Giang Q12 AB505952.1_Citrus_reticulata 99,36 95 12 Tangerine bop Q13 AB505952.1_Citrus_reticulata 98,98 94 The results obtained show that because there is a deletion or an addition at 53 some positions on the gene fragment, the total number of nucleotides of each sequence segment is different among the studied samples Statistical results of the sequences in the RbcL region have 14 different positions in the nucleotide sequence in the studied samples In which, cultivars, Q1 and Q8, had different nucleotide changes compared to the other cultivars and reference samples AB505955.1 Citrus maxima, AB505952.1 Citrus reticulata and AB505957.1 Citrus sinensis Based on the sequence of the chloroplast gene region with marker rbcLCi2, it is possible to distinguish and accurately identify two samples of the studied tangerine, Q1 and Q8 The Q1-like sample has nucleotide substitution G to T at positions 420, 492 and C to A at position 704 All bouncing nucleotide transformation sites are unique and different from the samples remainder and the reference sample Besides, at position 708, there is a substitution of nucleotide G to C in samples like C9 and Q1 However, there were differences in the Q1 seed samples at positions 420 and 492, so it was possible to recognize the C9 variety more than the rest of the cultivars and the reference samples Sample like Q8 (Dong Khe tangerine) has nucleotide substitutions at positions 304, 362, 654 and 766 However, at position 304, substitution A to T is unique and different from other samples back and reference form(table 4.3.2) 54 Table 4.8 Some position differences in nucleotide sequences of the research and reference samples Location difference in nucleotide sequence Name 60 30 36 42 49 65 70 70 76 76 76 76 76 4 G A A G G G A C G G T A T - T G - - Q1 A T T T G A C A Q3 T G A T - Q4 T G A T - Q5 T G T Q7 T G T Q8 T T G A Q9 T G A T A - Q6 T G T Q2 T G T Q11 A G T A T A Q12 A T G T A T A Q13 A T G T A T A AB505955.1 Citrus maxima AB505952.1 Citrus reticulata AB505957.1 Citrus sinensis 55 56 57 58 Figure 4.8 Some images comparing the nucleotide sequences of the Rbc region of the studied tangerine samples The analysis results based on the DNA sequence of the Rbc region showed that the genetic tree branching had a separation into groups.The first group consisted of the only tangerine variety sample Q1 whose genetic similarity coefficient with reference sample AB505952.1_Citrus_reticulata was 99.1% The second branch includes varieties, namely Q7, Q5, Q2, Q6, Q8, Q4, Q3 and Q9 This group has similarity coefficients with reference sample 59 AB505952.1_Citrus_reticulata ranging from 99.6% to 99.9% The third branch consists of varieties, namely Q13, Q12 and Q11, with similarity coefficients with reference sample AB505952.1_Citrus_reticulata ranging from 99.2% to 99.4% Table 4.9 The nucleotide sequence similarity coefficient of the RbcL gene region between the studied tangerine samples and the reference sample Figure 4.9 Phylogenetic tree of the rbcL gene region of the studied and reference samples 60 SECTION V: CONCLUSIONS AND PETITIONS 5.1 Conclusion ITS gene sequence with marker ITS1/ITS4 can accurately distinguish and identify samples of tangerine, studied as Q5 (Huong Can tangerine), Q9 (tangerine Ngoc Hoi), Q11 (tangerine hoi), Q12 (tangerine ngot Ha Giang), Q13 (tangerine ngot) Based on the sequence of the chloroplast gene region with marker MatkCi1, it is possible to identify the Q14 variety (tangerine Bac Kan) Based on the sequence of the chloroplast gene region with marker RbcLCi2, it is possible to distinguish and accurately identify samples of mandarin and pomelo varieties studied, namely Q1 (tangerine duong), Q8 (tangerine Dong Khe) 5.2 Petitions It is necessary to continue to expand the assessment of genetic diversity and develop DNA barcodes for many indigenous specialty genetic resources of Vietnam in order to serve early identification, diversity research, conservation and selection of new plant varieties with economic value 61 REFERENCES VIETNAMESE Lê Thi Thu Trang, Đàm Thị Thu Hà, Lã Tuấn Nghĩa, Nguyễn Mạnh Điệp, Vũ Thị Thảo Mi, Hoàng Trọng Cảnh (2020), “Đánh giá đa dạng di truyền số giống quýt địa phương Việt Nam thị SSR”, Tạp chí Khoa học Nông nghiệp Việt Nam số 9(118)/2020 Nguyễn Đức Thành (2014), “Các kỹ thuật thị dna nghiên cứu chọn lọc thực vật”, Tạp chí 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