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.
ii LIST OF TABLES. v LIST OF FIGURES. vi LIST OF ABBREVIATIONS. ix CHAPTER 1: INTRODUCTION.
1 CHAPTER 2: LITERATURE REVIEW. Nuclear genome sequences. Mitochondrial genome sequences. Chloroplast genome sequences.
Related studies to DNA barcode. Application DNA barcoding in plant. psbA-trnH region – Chloroplast DNA barcode. Related studies to Polyscias genus.
DNA barcode in Polyscias genus. 14 CHAPTER 3: MATERIALS AND METHODS. Time and location. 19 CHAPTER 4: RESULTS AND DISCUSSION.
PCR amplification of psbA-trnH fragment. Genetic distance and phylogenetic tree. 31 CHAPTER 5: CONCLUSION AND SUGGESTION. 35 iv LIST OF TABLES Table 2.1: Most relevant reports in pharmacological activities of Polyscias species .1: Information about studied samples .2: The psbA-trnH primer .3: Components of PCR reaction .1: OD values of five Polyscias samples .2: Reference sequences list.3: The polymorphism sites of gene psbA-trnH of 11 Polyscias species .4: Percentage identities among species.
33 v LIST OF FIGURES Figure 2.1: The matrix of genetic information and taxonomic diversity, with DNA barcoding and genomics .2: Procedure of DNA barcoding .3: General structure organization of the chloroplast psbA-trnH intergenic spacer .5 a: Dinh lang la tron: P.5 b: Dinh lang la to: P.5 c: Dinh lang la nho: P.5 d: Dinh lang la dia: P.5 e: Dinh lang la rang: P.1: Total DNA of five leaf samples of Polyscias genus .2: Results of purification of five leaf samples of Polyscias genus .3: Graphic view alignment of psbA-trnH from Polyscias .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 1 ITS2 Internal transcribed spacer 2 Kb Kilo base M Mol concentration MCL Maximum Composite Likelihood matK maturase K min 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. filicifolia (LTO) were grouped into same clade, which had close relationship with 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 CHAPTER 1: INTRODUCTION 1. DNA barcoding DNA barcoding is a modern biological tool for accurate, rapid, and automatable species identification and phylogenetic reconstruction using only standardized piece of DNA sequence. Regions are selected to be barcode is required to universally present in target lineages and have adequate sequence variation to discriminate among species (Hebert et al.
The short DNA sequence from a standard region of the genome is known as a marker which is different for various species. For example, the most commonly used marker for animal is Cytochrome C Oxidase 1 (COI), found in mtDNA. Another suitable marker for fungi is the Internal Transcribed Spacer (ITS) rDNA. Chloroplast DNA barcodes as matK, psbA-trnH, rbcL are used in plants (Kaur, 2015).
Polyscias genus Polyscias is a genus of the Araliaceae family. This genus is widely distributed over the world for economic value and commonly used for medical purposes due to its rich contents of phytochemical. Some Polyscias species are not only functional but also ornamental. Polyscias is named as two Greek words: “poly” means many and “skia” means shade, which indicates the thick foliage characteristic of this genus (Ashmawy et al.
According to the investigation of Vietnamese Ginseng Center in Southern provinces, Polyscias has 6 species: Polyscias fruticosa (L) Harm, Polyscias balfouriana Bailey, Polyscias filicifoli (Merr et Fourn w) Bailey, Polyscias guilfeylei var lacinita Bailey, Polyscias guilfeylei (Cogn et Marche) Bail, Polyscias scutellarie (N.Burn) Fosberg (Nguyen Thuong Dong et al. In this thesis, we carried out the research project: “Study on psbA-trnH DNA barcoding characteristics in some species of Polyscias genus”. Objectives Studying on psbA-trnH DNA barcode to investigate the diversity of five species of Polyscias. Amplification psbA-trnH fragment by PCR 2.
Sequencing psbA-trnH fragment by Sanger Sequencing 3. Analysis and evaluating the diversity based on psbA-trnH sequence 1 CHAPTER 2: LITERATURE REVIEW 2. Basic features DNA barcode is a fast and reliable method to classify species based on nucleotide diversity of short DNA segments. Universality, specificity on variation, and easiness on employment are remarkable features of a DNA barcode.
This means that the region considered as a barcode should be used for a wide range taxa, contains a sufficient number of variations between different species but also ensures conservative characteristics within species, thus variability in interspecies will be insignificant. Therefore, an ideal marker should be routinely retrievable with a single pair of primers and can be sequenced in two-dimensional which has fewer requirements for the manual editing of sequence traces. Additionally, the gene sequences used for barcoding must be short enough to facilitate PCR amplification. Generally, DNA barcoding is based on the use of a short, standardized region that allows for cost- effective species identification (Vijayan and Tsou, 2010).
There are four main factors in selecting a plant DNA barcode namely: universal PCR amplification, range of taxonomic diversity, power of species differentiation, bioinformatics analysis and application (Kress et al. DNA barcoding is considered a modern and accurate method for identifying organisms, using DNA regions in the nucleus, mitochondria, and chloroplasts (Nguyen Van Viet et al.1: The matrix of genetic information and taxonomic diversity, with DNA barcoding and genomics (Kress et al.2: Procedure of DNA barcoding (Abdul Bari Chowdhury, 2020) 2. Nuclear genome sequences Barcoding based on the nuclear DNA segment is expected to provide more information on species identity because of its biparentally inherited characteristic. Until now, Internal Transcribed Spacer (ITS) regions of the ribosomal DNA (rDNA) is the only nuDNA that has been tested for suitability as a barcode in plants (Vijayan and Tsou, 2010).
This region consists of one conserved 5.8S sequence and two variable partitions namely ITS1, ITS2. Due to the convenience of PCR amplification, the ITS regions are widely used for performing taxonomy analysis of the fungi, monocot, and dicot. However, a quite complex evolution pattern is found in the ITS that relates to the nuclear genome and causes difficulties for analysis (Huynh et al. Mitochondrial genome sequences Analysis in mitochondrial DNA (mtDNA) has had a greater impact on phylogenetic studies in animals which have more studies than plant mtDNAs (Palmer et al.
The mitochondrial genome in plants is likely to be unstable for barcoding due to its frequent intermolecular and intramolecular recombinations, which constantly change gene orders (Palmer, 1992; Palmer et al. However, nucleotide substitution rates of plant mtDNA are lower than chloroplast DNA, plant nuDNA, and animal mtDNA about 3-4 times, 12 times, and 40–100 times, respectively (Cho et al. There are a few mitochondrial markers showing promise for phylogenetic purposes. The mitochondrial DNA cytochrome C oxidase subunit I (COI) gene is commonly used for species identification of birds (Yoo et al., 2013), insects (Hajibabaei et al., 2006), and fishes (Ward et al.
Chloroplast genome sequences Barcode regions found in Chloroplast DNA have been used very frequently in plant systematics and phylogenetic studies. The chloroplast genome has a highly conserved structure, for example, it is relatively free of large deletions, insertions, single nucleotide polymorphism (SNPs), transpositions, and inversions. This characteristic makes it useful for studies about phylogenetic. Chloroplast DNA is relatively abundant compared to nuclear DNA.
In general, 50 chloroplast per cell multiplied with 50 cpDNA copy per chloroplast, while nuclear DNA is 2n. Therefore, DNA extraction and analysis from cpDNA are more facilitate. Many noncoding cpDNA regions are also useful targets of study such as the intergenic spacer of atpB-rbcL, matK, ndhF, ycf6-psbM, psbM-trnD, rps16, rpL16 intron, trnL-F and psbA-trnH spacer by using universal primers. For phylogenetic researches, cpDNA has been more utilized, than the nuclear genome and mitochondrial genomes of animals for barcoding (Ali et al.
Related studies to DNA barcode 2. Application DNA barcoding in plant DNA barcode is convenient and cost-effective for the plant taxonomist. The most significant benefit of using DNA barcoding is the wide range of applicable plant 4 samples. This approach can be applied for DNA samples obtained from different parts of the plant as leaf, root, and flower in various kinds of preservation conditions.