Identification of orange varieties by using its, matk and rbcl primers

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -*** - UNDERGRADUATE THESIS TOPIC: IDENTIFICATION OF ORANGE VARIETIES BY USING ITS, MATK AND RBCL PRIMERS HANOI - 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -*** - UNDERGRADUATE THESIS TOPIC: IDENTIFICATION OF ORANGE VARIETIES BY USING ITS, MATK AND RBCL PRIMERS Student name Student code Class Department Supervisors : VU NGOC HUONG : 620392 : K62CNSHE : Biology : TRAN DANG KHANH, Assoc Prof PhD DONG HUY GIOI, Assoc Prof PhD HANOI - 2022 COMMITMENT I hereby declare that the undergraduate thesis work is mine All research results have been results during the implementation of the topic The results, the data are completely true, have not been appeared in any scientific reports I also guarantee that the references and useful information for the topic are clearly cited and all help is appreciated Hanoi, March 18th , 2022 Student Vu Ngoc Huong i ACKNOWLEDGEMENTS During the process of implementing my undergraduate project, I have received a lot of attention and help from individuals and groups First of all, I would like to express my respect and deep gratitude to Assoc Prof PhD Tran Dang Khanh and Assoc Prof PhD Dong Huy Gioi for giving me the opportunity to carry out this work, and their huge efforts, enthusiasm, and support throughout the duration of the undergraduate thesis Secondly, I would like to express my respect to all of teachers and friends in the Faculty of Biotechnology They have helped and supported me during my training at school Finally, I would like to sincerely thank my family members who always trust, support and encourage me to complete this report Sincerely thank! Hanoi, March 18th , 2022 Student Vu Ngoc Huong ii OUTLINE COMMITMENT i ACKNOWLEDGEMENTS ii OUTLINE iii LIST OF ABBREVIATIONS vi LIST OF TABLES vii LIST OF FIGURES viii ABSTRACT ix CHAPTER I INTRODUCTION 1.1 The urgent of undergraduate thesis 1.2 Objectives and requirements 1.2.1 Objectives 1.2.2 Requirements CHAPTER II LITERATURE REVIEW 2.1 The situation of studying the DNA barcode of Citrus in the world and in Vietnam 2.1.1 The situation of studying the DNA barcode of Citrus in the world 2.1.2 The situation of studying the DNA barcode of Citrus in Vietnam 2.2 Introduction of Citrus (Orange) 2.3 Some methods used in species identification and genetic relationship identification 2.3.1 Classification based on DNA barcode technique 11 2.3.2 Locus used in DNA barcode methods in plants 12 CHAPTER III MATERIALS AND METHODS 17 3.1 Location and time for research 17 3.1.1 Research location 17 3.1.2 Time for research 17 3.1.3 Subjects 17 3.2 Materials 17 3.2.1 Research subjects 17 iii 3.2.2 Chemical reagents 18 3.2.3 Equipment 18 3.2.4 Primers 19 3.3 Methods 20 3.3.1 Total DNA extraction 20 3.3.2 Quantification of DNA by spectrophotometer 20 3.3.3 PCR process (Polymerase Chain Reaction) 21 3.3.4 Purification DNA after gel electrophoresis 24 3.3.5 Sequencing and designing phylogenic tree 25 3.3.6 Analysis the data of SSR gel electrophoresis to identify the genetics of specific Orange varieties in collected samples 26 CHAPTER IV RESULTS AND DISCUSSION 27 4.1 Total DNA extraction of 15 orange leaves 27 4.2 Quantification of DNA by spectrophotometer 27 4.3 PCR amplification using ITS, matK, rbcL primers 29 4.3.1 ITS region sequence results 29 4.3.2 MatK region sequence results 39 4.3.3 rbcL region sequence results 48 4.4 Results of nucleotide sequencing analysis of the ITS, matK, rbcL gene region between 15 studied orange samples and reference samples 54 4.4.1 Using DNA barcode (ITS) to identify studied Orange 54 4.4.2 Using DNA barcode (matK) to identify studied Orange varieties 57 4.4.3 Using DNA barcode (rcbL) to identify studied Orange varieties 60 4.5 Identify specific primer to accurately identify certain varieties of Citrus 62 4.5.1 Identify Cam Tay Giang varieties by the mCrCiR01D06a, CT02 and Ci07B09 primers 62 4.5.2 Duong Orange variety identification by using Ci02F07 primer 64 CHAPTER V CONCLUSIONS AND RECOMMENDATIONS 65 5.1 Conclusions 65 5.1.1 Constructing DNA barcoding 65 iv 5.1.2 Observe genetic diversity of research species 65 5.2 Recommendations 66 REFERENCES 67 APPENDIX 73 v LIST OF ABBREVIATIONS A : Adenine BLAST : Basic Local Alignment Search Tool C : Cytosine CTAB : Cetyltrimethylammonium bromide DNA : Deoxyribonucleic acid dNTPs : Deoxynucleotide triphosphates EDTA : Ethylenediamine Tetra acetic Acid EtBr : Ethidium Bromide Et al : et alii (Latin), and others G : Guanine ITS : Internal transcribed space (Nuclear ribosomal RNA internal transcribed spacer(s) InDel : Insertion Deletion (nucleotide) MEGA : Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment matK : maturase K NCBI : National Center for Biotechnology PCR : Polymerase chain reaction rDNA : Ribosomal deoxyribonucleic acid rbcL : ribulose-1,5-bisphosphate carboxylase oxygenase large subunit SSR : Simple sequence repeats T : Thymine Tris : Tris (hydroxymethyl) aminomethane Tris-HCl : Tris-hydrochloride vi LIST OF TABLES Table 3.1 The list of 15 studied orange samples were collected from provinces in Vietnam 18 Table 3.2 The list of DNA barcodes’ primers was used in the study 19 Table 3.3 The list of SSR primers were used in the study 19 Table 3.4 PCR unit for a reaction with ITS, matK, rbcL primers 21 Table 3.5 PCR program with ITS, matK, rbcL primer 22 Table 3.6 PCR unit for a reaction with SSR primers 23 Table 3.7 PCR program with SSR primers 23 Table 4.1 OD measurement results and concentration of 15 samples’ DNA extraction 28 Table 4.2 Statistical table of different locations in ITS sequences of researched samples and reference samples 31 Table 4.3 Statistical table of different locations in MatKCi1 sequences of researched samples and reference samples 40 Figure 4.5 Alignment of the MatKCi1 sequences of 15 studied samples and reference samples 48 Table 4.5 Evaluation of the identity and coverage between ITS sequences of the studied samples and the corresponding sequence on NCBI 55 Table 4.6 Genetics similarity coefficient between ITS sequences of studied samples and reference samples 56 Table 4.7 Evaluation of the identity and coverage between matK sequences of studied samples and the corresponding sequence on NCBI 58 Table 4.8 Genetics similarity coefficient between matK sequence of studied samples and reference samples 59 Table 4.9 Evaluation of the identity and coverage between rbcL sequences of studied samples and the corresponding sequence on NCBI 60 Table 4.10 Genetics similarity coefficient between rbcL sequence of studied samples and reference samples 61 vii LIST OF FIGURES Figure 4.1 Testing 15 total DNA extraction samples on 1% agarose gel electrophoresis 27 Figure 4.2 1.5% gel electrophoresis of 15 studied samples by using ITS1/ITS4 primers M: Marker generuler 100bp plus DNA 29 Figure 4.3 Alignment of the ITS sequences of 15 studied samples and reference samples 38 Figure 4.4 1.5% gel electrophoresis of 15 studied samples by using MatKCi1 primers M: Marker generuler 100bp plus DNA 39 Figure 4.5 Alignment of the MatKCi1 sequences of 15 studied samples and reference samples 48 Figure 4.6 1.5% gel electrophoresis of 15 studied samples by using RbcLCi2 primers M: Marker generuler 100bp plus DNA 48 Figure 4.7 Alignment of the RbcLCi2 sequences of 15 studied samples and reference samples 54 Figure 4.8 The phylogenic tree generated the ITS gene region of 15 studied orange samples studied with reference samples 57 Figure 4.9 The phylogenic tree generated the matK gene region of 15 studied orange samples studied with reference samples 59 Figure 4.10 The phylogenic tree generated the rbcL gene region of 15 studied orange samples studied with reference samples 62 Figure 4.11 Electrophoresis results of the PCR products with mCrCiR01D06a, CT02 and Ci07B09 primers (M: marker generuler 50bp) 63 Figure 4.12 Electrophoresis results of the PCR products with Ci02F07 primer (M: marker generuler 50bp) 64 viii The second group consists of the remaining 13 samples with a genetic similar coefficient with the reference sample NC_037463.1_Citrus_sinensis ranging from 99.5% (C3) to 99.9% (between C1, C2, C4, C5 and C6 with the reference sample) Table 4.8 Genetics similarity coefficient between matK sequence of studied samples and reference samples Figure 4.9 The phylogenic tree generated the matK gene region of 15 studied orange samples studied with reference samples 59 4.4.3 Using DNA barcode (rcbL) to identify studied Orange varieties The analyzing results of the similar sequencing between the 15 studied samples’ DNA barcode and reference sequence on the NCBI The results in Table 3.7 showed that the comparation of similarity sequences ranged from 98.99% to 99.49% and the coverage was 93% to 98% This result illustrates that the RbcL gene region sequences that amplified 15 studied samples had a high degree of similarity compared to the sequences published in NCBI (Table 4.13) Table 4.9 Evaluation of the identity and coverage between rbcL sequences of studied samples and the corresponding sequence on NCBI The results in Table 4.13 of the analysis of sequences through MEGA v6.06 program obtained showed that there were genetic similar coefficients of 60 the 15 orange varieties ranging from 98.9 to 100% The lowest genetic similarity coefficient was 98.9% between the C15 sample and C3 and C8 Based on the results of DNA sequencing analysis of the rbcL gene region, the 15 varieties of oranges studied were divided into groups (figure 4.10, Table 4.14) The first group consisted of seven samples of C13, C11, C10, C3, C7, C8 and C14 varieties with genetic similar coefficients to the reference sample AB505957.1_Citrus_sinensis of 99.4 (C10) and 99.7% (C7) The second group consisted of three C4, C5 and C15-like samples with a genetic similarity coefficient to the reference sample AB505957.1_Citrus_sinensis of 99.4 (C15) and 99.7% (C4) The third group consists of five varieties: C1, C2, C6, C9 and C12 This group has a genetic similarity coefficient with a very high AB505957.1_Citrus_sinensis reference sample, ranging from 99.7% (between form C9, C12 and reference form) to 100% (between sample C2 and reference form) Table 4.10 Genetics similarity coefficient between rbcL sequence of studied samples and reference samples 61 Figure 4.10 The phylogenic tree generated the rbcL gene region of 15 studied orange samples studied with reference samples The findings showed that matK and rbcL may be utilized to successfully identify Citrus and related taxa However, matK and rbcL only had a valid identification frequency of 55.9% and 37.3%, respectively, at the species level MatK was shown to be more strong than rbcL, meaning that it can be used to identify Citrus species and their related genera (Yu Jie et al., 2011) My data in Citrus spp (Orange) does not agree with this general trend By using matK primers, I can not identify any varieties but by using rbcL primers, samples can be identified 4.5 Identify specific primer to accurately identify certain varieties of Citrus The experience conducted PCR program with SSR primers, but only SSR primers give polymorphism result The remaining primer either not give products or homomorphism at all tapes or give different tape numbers through the runs 4.5.1 Identify Cam Tay Giang varieties by the mCrCiR01D06a, CT02 and Ci07B09 primers Electrophoresis analysis with the mCrCiR01D06a primer showed three types of alleles In particular, the group of 15 orange varieties (symbols from C1 to C15) has samples: C1, C3, C8 and C13 in a heterogeneous state; the 62 remaining samples are in a homogeneous state Tay Giang orange (C1) has the 2nd allele appearance (size about 240bp) different from other varieties in the researched orange group Similarly, in the CT02 primer, Tay Giang orange (C1) also appeared 130bp alleles, lower than other orange varieties With the Ci07B09 primer, Tay Giang orange appears allele at about 150bp, lower than other orange varieties Based on these distinct characteristics, it is possible to identify Tay Giang orange varieties from other varieties in the researched orange group (Figure 4.11) mCrCiR01D06a Figure 4.11 Electrophoresis results of the PCR products with mCrCiR01D06a, CT02 and Ci07B09 primers (M: marker generuler 50bp) 63 4.5.2 Duong Orange variety identification by using Ci02F07 primer Electrophoresis analysis with the Ci02F07 primer (figure 4.12) shows that two types of alleles appear Among the 15 researched samples (symbol C1 C15), only the Duong orange (C11) has a 190bp homogeneous bandage at the first allele The remaining orange samples are heterogeneous or homogeneous at the second allele position So, it is possible to accurately identify the Duong orange variety by the Ci02F07 marker out of the researched group Figure 4.12 Electrophoresis results of the PCR products with Ci02F07 primer (M: marker generuler 50bp) The SSRs in the entire genome sequencing assembly of Clementine mandarin and analyzed their frequency and distribution in different transcript regions has been previously characterized (Sheng-Rui Liu et al., 2013) Most of the SSRs were di-nucleotides and tri-nucleotides, accounting for up to 89% of all of the SSRs identified When examining the repeat number of the SSR motifs, our results showed that the distribution of all di-, tri-, tetra-, penta-, and hexa-nucleotide transcript-SSRs was skewed generally to the smaller number of repeats In my work microsatellite markers were used 4/7 (57.14%) markers that was used in my research agrees apartly with this general trend 64 CHAPTER V CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions 5.1.1 Constructing DNA barcoding The ITS, matK and rbcL gene regions of the studied orange group have been sequenced In which the amplified success rate was 100% for ITS, matK and rbcL primers The sequencing results of gene region named ITS, matK, and rbcL of 15 orange samples showed that ITS had the highest polymorphism in nucleotide sequencing of the three studied gene regions The results of sequencing of the gene regions ITS, MatK, rbcL of 15 orange samples can identify orange samples (C1, C5, C8, C9, C10, C12, C13 and C15) ITS gene region sequence with ITS1/ITS4 marker could accurately distinguished and identified samples of orange as C1 (Cam Tay Giang), C8 (Cam giay), C9 (Cam duong Ha Tinh), C10 (Cam sanh Bo Ha), C12 (Cam sap), C13 (Cam sanh) Based on the chloroplast gene region sequence with the RbcLCi2 marker, it was possible to accurately distinguish and identify samples of orange as C12 (Cam sap), C15 (cam Trung Vuong) By using ITS1/ITS4 marker for 15 researched oranges, the number of orange varieties could be distinguished is the highest, up to 40% distinguished samples 5.1.2 Observe genetic diversity of research species Based on the SSR marker, it was possible to identify the Tay Giang orange variety (C1) by using mCrCiR01D06a, CT02 and Ci07B09 primer Based on the SSR marker, it was possible to identify Duong orange (C11) by Ci02F07 marker 65 5.2 Recommendations Research needs to be expanded with other types of molecular markers and other types of gene regions for better evaluation of the genetic diversity and classification of 15 Citrus spp accessions Research’s data can be analyzed in other software to compare the differences and decide which software is the highest efficiency 66 REFERENCES Vietnamese Bùi Huy Đáp (1960) ệt đới, tập (1), Nxb Nông thôn Nguyễn Thị Thúy Hằng (2012) Xác định tên số loài thuộc chi tre (Bambusa Schreb) biế đổi hình thái Việt Nam kỹ thuật phân tích ADN, Luận văn Thạc sỹ, Trường Đại học Khoa học 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