VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -oOo - GRADUATION THESIS “STUDY ON psbA-trnH DNA BARCODING CHARACTERISTICS IN SOME SPECIES OF POLYSCIAS GENUS” Student Nguyen Le Tra My Department Biotechnology Supervisor Huynh Thi Thu Hue, PhD Institue of Genome Research, VAST Tran Thi Hong Hanh, MSc Vietnam National University of Agriculture HANOI, 2021 COMMITMENT I hereby declare that the work contained in this thesis has not been previously submitted to meet requirements for an award at this or any other education institution To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made Signature: Date: i ACKNOWLEDGEMENTS This thesis, as any other, would be impossible to complete without kind support from various people First and foremost, I wish to express my greatest thanks to my university, Vietnam National University of Agriculture (VNUA), and particularly Biotechnology department To overcome the those challenging years, I cannot forget the advice and encouragements from all teachers in Microbiology department, especially Ms Tran Thi Hong Hanh Without them, I wouldn't be able to get the opportunity to begin my thesis Besides, I cannot help but mention the Institute of Genome Research (IGR), Vietnam Academy of Science and Technology, where I carry out this research The sincerely thanks and regards goes to my principal supervisor, Ms Huynh Thi Thu Hue, for her relentless mentoring and encouragement Her knowledge, as well as hugely passion in science have enlighted me into the world of science, bring me valuable guidances which enabled me to be the best version of myself It is really my pleasure to finish my work under her supervision Moreover, I would humbly extend my thanks to my friends and colleagues in VNUA, as well as others in IGR laboratory which I cannot mention all of you All the never-ending arguments, enthusiasm cooperation, interesting questions and advice taught me new lessons Last but not the least, I also acknowledge with a deep sense of gratitude for my beloved family, for doubtless assistance both physically and mentally, so that I have remained unwavering in my own path throughout the years Any omission in this brief acknowledgement does not mean lack of gratitude Once again, it is impossible to finish the work without your support, directly or indirectly Hanoi, March 31, 2021 Student Nguyen Le Tra My ii TABLE OF CONTENTS COMMITMENT i ACKNOWLEDGEMENTS ii LIST OF TABLES v LIST OF FIGURES vi LIST OF ABBREVIATIONS vii ABSTRACT ix CHAPTER 1: INTRODUCTION 1.1 DNA barcoding 1.2 Polyscias genus 1.3 Objectives 1.4 Requirements CHAPTER 2: LITERATURE REVIEW 2.1 DNA barcode 2.1.1 Basic features 2.1.2 Nuclear genome sequences 2.1.3 Mitochondrial genome sequences 2.1.4 Chloroplast genome sequences 2.2 Related studies to DNA barcode 2.2.1 Application DNA barcoding in plant 2.2.2 psbA-trnH region – Chloroplast DNA barcode 2.2 Related studies to Polyscias genus 2.3.1 Phytochemical 2.3.2 Biological activity 10 2.3.3 DNA barcode in Polyscias genus 14 CHAPTER 3: MATERIALS AND METHODS 14 3.1 Time and location 15 3.1.1 Time 15 3.1.2 Location 15 iii 3.2 Materials 15 3.3 Methods 15 3.3.1 DNA extraction 15 3.3.2 PCR amplification 16 3.3.3 Purification 18 3.3.4 DNA sequencing 19 CHAPTER 4: RESULTS AND DISCUSSION 19 4.1 DNA extraction 20 4.2 PCR amplification of psbA-trnH fragment 21 4.3 DNA sequencing 23 4.4 Genetic distance and phylogenetic tree 30 4.1.1 Genetic distance 30 4.1.2 Phylogenetic tree 31 CHAPTER 5: CONCLUSION AND SUGGESTION 32 5.1 Conclusion 33 5.2 Suggestion 33 REFERENCES 34 Vietnamese references 34 English references 35 iv LIST OF TABLES Table 2.1: Most relevant reports in pharmacological activities of Polyscias species 13 Table 3.1: Information about studied samples 17 Table 3.2: The psbA-trnH primer 18 Table 3.3: Components of PCR reaction 19 Table 4.1: OD values of five Polyscias samples 23 Table 4.2: Reference sequences list 26 Table 4.3: The polymorphism sites of gene psbA-trnH of 11 Polyscias species 30 Table 4.4: Percentage identities among species 32 Table 4.5: Genetic distances (Pairwise method) 33 v LIST OF FIGURES Figure 2.1: The matrix of genetic information and taxonomic diversity, with DNA barcoding and genomics Figure 2.2: Procedure of DNA barcoding Figure 2.3: General structure organization of the chloroplast psbA-trnH intergenic spacer Figure 2.4: Polyscias balfouriana 12 Figure 2.5 a: Dinh lang la tron: P balfouriana (LT) 15 Figure 2.5 b: Dinh lang la to: P filicifolia (LTO) 15 Figure 2.5 c: Dinh lang la nho: P fruticosa (LN) 15 Figure 2.5 d: Dinh lang la dia: P scutellaria (LD) 15 Figure 2.5 e: Dinh lang la rang: P serrate Balf (LR) 15 Figure 4.1: Total DNA of five leaf samples of Polyscias genus 23 Figure 4.2: Results of purification of five leaf samples of Polyscias genus 25 Figure 4.3: Graphic view alignment of psbA-trnH from Polyscias 26 Figure 4.4: Maximum Likelihood tree of Polyscias based on psbA-trnH sequences 34 vi LIST OF ABBREVIATIONS Abbreviation Meaning BS Bootstrap value BLAST Basic Local Alignment Search Tool Bp Base pair COI Cytochrome C oxidase CpDNA Chloroplast DNA CTAB Cetyl trimethyl ammonium bromide DNA Deoxyribonucleic acid EDTA Ethylenediamine tetracetic acid IC50 The concentration that reduces growth by 50% ITS Internal transcribed spacer ITS1 Internal transcribed spacer ITS2 Internal transcribed spacer Kb Kilo base M Mol concentration MCL Maximum Composite Likelihood matK maturase K minute ml milliliter mtDNA Mitochondrial DNA NCBI National Center for Biotechnology Information nuDNA Nuclear DNA OD Optical Density PCR Polymerase Chain Reaction pH Power of hydrogen/potential of hydrogen psbA-trnH tRNA Phenylanalinephotosystem II protein D1-tRNA Leucine PVP Polyvinylpyrrolidone rbcL ribulose 1,5-bisphosphate carboxylase large subunit rDNA Ribosomal DNA vii RNA Ribonucleic acid rpoB beta subunit of RNA polymerase rpoC1 RNA polymerase subunit C1 rpm Round per minute TAE Tris – Acetate – EDTA Tm Melting temperature of an oligonucleotide trnL-trnF tRNA Leucine - tRNA Phenylanaline µg/ml Microgram/milliliter µl microlitre viii ABSTRACT Polyscias is a flowering genus that belongs to the Araliaceae family, which is commonly used for medical purposes and widely distributed over the world for its economic value due to rich contents of phytochemical DNA barcode is a robust method to identify species and evaluate phylogenetic relationships In this study, the psbA-trnH region was used to investigate the relationship of five species of Polyscias genus The sequencing results showed that the psbA-trnH sequence has about 500 nucleotides as expected length Raw data has been analyzed and these sequences were compared to six reference sequences from NCBI The phylogenetic tree of Polyscias genus based psbA-trnH sequences with Tetraplasandra hawaiensis as out-group species was conducted by bioinformatic tools The combination between the genetic distance method and Maximum-likelihood proposed high confidence results All five studied samples: P balfouriana (LT), P serrata Balf (LR), P fruticosa (LN), P scutellaria (LD), P filicifolia (LTO) were grouped into same clade, which had close relationship with P sp Wen 10765 and P austranliana with reliable bootstrap supported (BS: 72) The psbA-trnH region has been proposed as suitable for DNA barcoding studies Thus, it would be considered widely used in further classification studies ix 250 260 270 280 290 300 | | | | | | | | | | | | Polyscias balfouriana (LT) AATTTAGCTATTTCTTCCCAATCTTTTGGGAAGTTTTTTTTTT-AAAAAAAAGATGAAAA Polyscias filicifolia (LTO) .- Polyscias fruticosa (LN) .- G Polyscias scutellaria (LD) .- G Polyscias serrata Balf (LR) G Polyscias australiana .- G Polyscias sp Wen 10765 G Polyscias macrocarpa - .C . G A.GA Polyscias nodosa - T G TGA Polyscias schultzei C - .C . T A.GA Polyscias spectabilis .AAGA G - G .A 310 320 330 340 350 360 | | | | | | | | | | | | Polyscias balfouriana (LT) TGTTGGACTTTTTACTTAGTTAAGTTAATACTTAATATTTTTATCTCGAAAAAAAAA - Polyscias filicifolia (LTO) G - Polyscias fruticosa (LN) - Polyscias scutellaria (LD) - Polyscias serrata Balf (LR) - Polyscias australiana G - Polyscias sp Wen 10765 G Polyscias macrocarpa . - .G. - Polyscias nodosa . - .AAA Polyscias schultzei . - G G. - Polyscias spectabilis . - .G. - 370 380 390 400 410 420 | | | | | | | | | | | | Polyscias balfouriana (LT) -AAAGATAAGAAAGAAATAATGATGATATGGTAAAAATTTAATCTTTTGAAACGTAAGGA Polyscias filicifolia (LTO) - A Polyscias fruticosa (LN) - Polyscias scutellaria (LD) - Polyscias serrata Balf (LR) - A Polyscias australiana - A Polyscias sp Wen 10765 - Polyscias macrocarpa - . - Polyscias nodosa G .- C T - Polyscias schultzei - . - Polyscias spectabilis - GG 26 430 440 450 460 | | | | | | | | Polyscias balfouriana (LT) AAAAAATAATAAGGGGGCGGATGTAGCCAAGGGGATCAGGGCAG Polyscias filicifolia (LTO) .G .T A Polyscias fruticosa (LN) .G A Polyscias scutellaria (LD) .G T T A Polyscias serrata Balf (LR) C A Polyscias australiana T A Polyscias sp Wen 10765 .G T A Polyscias macrocarpa .G .G T A Polyscias nodosa GAA .T A Polyscias schultzei .G .G T A Polyscias spectabilis .G .G T A Figure 4.3: Graphic view alignment of psbA-trnH from Polyscias The short intergenic region which are non-coding found within genes In Polyscias genus, non-coding psbA-trnH intergenic spacer is located from nucleotides 19 to 429 This spacer is located between the psbA gene and the gene of histidine transfer RNA (trnH), which plays an important role in the regulation of the expression of these genes Degtjareva et al (2012) has been proposed psbA-trnH region as suitable for DNA barcoding studies The polymorphism sites of 11 sequences were observed in figure 4.5 in table 4.3, the results showed that psbA-trnH regions of five studied sequences from our samples were the most similar to P australiana, followed by P sp Wen 10765 Studied sequences had the greatest difference with P nodosa 27 Table 4.3: The polymorphism sites of gene psbA-trnH of 11 Polyscias species Species Polymorphism sites 52 53 54 55 56 136 138 139 140 141 143 144 147 148 T C T T - - A G T T C T A A P filicifolia (LTO) T - T T G - A G T T C T A A P fruticosa (LN) T T T G - - A G T T C T A A P scutellaria (LD) - - T T G - A G T T C T A A P serrata Balf T T T T G - A G T T C T A A P australiana T - T - G - A G T T C T A A P sp Wen 10765 T - T T G - A G T T C T A A P macrocarpa T - - - G C T A C C A G T C P nodosa G - - - A C T A C C A G T C P schultzei T - - - G C T A C C A G T C P spectabilis G - - A A C T A C C A G T C P balfouriana (LT) (LR) 28 Table 4.3: The polymorphism sites of gene psbA-trnH of 11 Polyscias species (continued) Species Polymorphism sites 149 150 156 157 158 169 287 295 296 356 424 432 452 459 P balfouriana (LT) A C T T A G A T G A A A G G P filicifolia (LTO) A C T T A G A T G A A A T A P fruticosa (LN) A C T T A G G T G A G A G A P scutellaria (LD) A C T T A G G T G A G A T A P serrata Balf (LR) A C T T A G G T G A A A T A P australiana A C T T A G G T G A A A T A P sp Wen 10765 A C T T A G G T G A G A T A P macrocarpa T T A A G C G G A G G G T A P nodosa T T A A G C G G A A - G T A P schultzei T T A A G C T G A G G G T A P spectabilis T T A A G C G T A G G G T T We used BLAST tool to calculate percentage identity among species Particularly, one studied sequence was respectively aligned with one reference sequence and then percentage identity was calculated The results indicated that the five studied sequences were the most similar to P australiana and difference with P nodosa The highest similarity was 99.12% between P filicifolia (LTO) and P australiana Pecentage identity was 98.90% found in P cutellaria (LD) and P australiana The lowest similarity was 81.00% between P serrata Balf (LR) and P nodosa Examined samples also had relatively high similarity to P sp Wen 10765 (Table 4.4) 29 Table 4.4: Percentage identity among species Reference species Studied species P balfouriana (LT) P filicifolia (LTO) P fruticosa (LN) P scutellaria (LD) P P sp P P nodosa P schultzei P Australiana Wen 10765 macrocarpa 98.25% 97.82% 88.15% 85.89% 87.50% 88.12% 99.12% 98.04% 88.36% 85.89% 87.72% 88.39% 98.69% 98.69% 88.77% 86.31% 87.90% 88.82% 98.90% 98.47% 88.96% 86.67% 88.10% 89.22% 98.69% 98.25% 88.12% 81.00% 87.26% 87.98% spectabilis P serrata Balf (LR) 4.4 Genetic distance and phylogenetic tree 4.1.1 Genetic distance Genetic distance is a measure of genetic difference between interspecies or intraspecies Genetic distances were calculated automatically by the pairwise distance method in MEGAX software The pairwise distance method is an evaluation of the differences between pairs of sequences and then these differences transform into a distance These distances were used to estimate a tree The results compared the differences between the pairs of sequences according to Kimura model Parameter (Kimura, 1980) indicated that the ranges of genetic difference were from 0.00661 to 0.08506 in which the lowest genetic difference was 0.00661 of P filicifolia (LTO) and P australiana Therefore, two species could have a close relationship 0.00663 was the difference between P australiana and P sp Wen 10765 The genetic difference was 0.00883 found in P fruticosa (LN) and P scutellaria (LD); P fruticosa (LN) and P serrate Balf (LR); P fruticosa (LN) and P sp Wen 10765 (Table 4.5) Thus, P fruticosa (LN) could be grouped into a group with P scutellaria (LD), P serrate Balf (LR) and P sp Wen 10765 The difference between P nodosa and P balfouriana (LT) was 0.08506 showed the highest 30 difference Table 4.5: Genetic distances (pairwise method) 1 Polyscias balfouriana (LT) Polyscias filicifolia (LTO) Polyscias fruticosa (LN) Polyscias scutellaria (LD) Polyscias serrata Balf (LR) Polyscias australiana Polyscias sp Wen 10765 Polyscias macrocarpa Polyscias nodosa 10 Polyscias schultzei 11 Polyscias spectabilis 12 Tetraplasandra hawaiensis 10 11 0.01997 0.01553 0.01773 0.02004 0.01777 0.00883 0.01552 0.01775 0.00883 0.01330 0.01779 0.00661 0.01107 0.01107 0.01107 0.02004 0.01328 0.00883 0.00884 0.01325 0.00663 0.05147 0.04382 0.03631 0.03882 0.04378 0.03635 0.03382 0.08506 0.07656 0.07101 0.07106 0.07672 0.06827 0.06823 0.06306 0.05656 0.04885 0.04382 0.04636 0.05136 0.04387 0.04129 0.01647 0.06300 0.05793 0.05040 0.04544 0.04544 0.05050 0.04299 0.04062 0.03183 0.05243 0.03939 0.05049 0.04301 0.03810 0.03810 0.04309 0.03568 0.03329 0.03465 0.05563 0.04232 0.00692 4.1.2 Phylogenetic tree A phylogenetic tree of 12 sequences was constructed using the MaximumLikelihood (ML) methods based on the Hasegawa-Kishiho-Yano model in MEGAX ML method is one of the most widely used for phylogenetic tree reconstruction Bootstrap Support (BS) values for individual clades were computed by running 1000 bootstrap replicates of the data These values indicate how many times out of 100 the same branch was observed when repeating the phylogenetic reconstruction on a resampled set of data In our study, the tree diagram constructed with psbA-trnH data using the Maximum-Likelihood method indicated that there were two classifications All five examined species: P balfouriana (LT), P serrata Balf (LR), P fruticosa (LN), P scutellaria (LD), P filicifolia (LTO) were grouped in Clade I, which had close relationship with P sp Wen 10765 and P australiana with well supported (BS:76) Clade II included two reference species (P macrocarpa and P schultzei) with strongly supported (BS:96) P nodasa and P spectabilis had a quite distant relationship with studied species In Clade I, P balfouriana (LT) and P serrata Balf (LR) were sister groups- two descends that split from the same node with weak supported (BS:32), P filicifolia (LTO) and P australiana were sister groups (BS=65) (Figure 4.4) 31 Clade I Clade II Figure 4.4: Maximum Likelihood tree of Polyscias based psbA-trnH sequences Note: Branch lengths are to scale Major clades at psbA-trnH tree were indicated Numbers on nodes are bootstrap support values Thus, chloroplast intergenic psbA-trnH spacer has recently become a popular tool for plant identification and phylogenetic analyses This region has been proposed as suitable for DNA barcoding studies Since the psbA-trnH is one of the most rapidly evolving spacers in chloroplast DNA with 75 bp conserved fragments at the ends Besides, it demonstrated good universality and high amplification success (Degtjareva et al., 2012) Therefore, psbA-trnH gene should be widely used for further plant phylogenetic analyses in the world in general, and Vietnam in particular 32 CHAPTER 5: CONCLUSION AND SUGGESTION 5.1 Conclusion - We extracted total DNA from five leaf samples of Polyscias genus: Dinh lang la nho (P fruticosa), Dinh lang la dia (P scutellaria), Dinh lang la to (P balfouriana), Dinh lang la rang (P serrata Balf), Dinh lang la to (P filicifolia) Pu_psbA-trnH primers were specifically designed and successfully amplified the psbA-trnH fragments - The five psbA-trnH fragments were determined in the nucleotide sequence It is about 500 nucleotides in length The raw sequences were edited to remove noise signal at the beginning and the end Finally, we obtained sequences of 457 nucleotides - Bioedit, BLAST, and MEGAX software were used to analysis and construct phylogenetic tree of Polyscias species based on psbA-trnH fragment In the study, five psbA-trnH sequences of the five species were grouped in Clade I and shown the relationship among the five Polyscias with six reference sequences 5.2 Suggestion - Applying other DNA barcoding to exactly evaluate the diversity of these species 33 REFERENCES Vietnamese references Đỗ Văn Mai, Thiều Văn Đường, Vũ Thị Bình, Trần Cơng Luận (2019) Xác Định Tên Khoa Học Của Cây Đinh Lăng Lá Tròn Bằng Phương Pháp GiảiTrình Tự Gen rbcL Tạp chí Nghiên cứu khoa học Phát triển kinh tế Trường 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