Analysis of nucleotide seqences in the d loop region the mitochondrial gene of bang troi chicken

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY  UNDERGRADUATE THESIS TITLE: “ANALYSIS OF NUCLEOTIDE SEQUENCES IN THE D-LOOP REGION OF THE MITOCHONDRIAL GENE OF BANG TROI CHICKEN” Student name : NGUYEN THI THUY DUONG Class : K62CNSHE Student code : 620572 Faculty : BIOTECHNOLOGY Supervisor : Nguyen Thi Dieu Thuy, PhD Nguyen Huu Duc, PhD Hanoi – 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY  UNDERGRADUATE THESIS TITLE: “ANALYSIS OF NUCLEOTIDE SEQUENCES IN THE D-LOOP REGION THE MITOCHONDRIAL GENE OF BANG TROI CHICKEN” Student name : NGUYEN THI THUY DUONG Class : K62CNSHE Student code : 620572 Faculty : BIOTECHNOLOGY Supervisors : Nguyen Thi Dieu Thuy, PhD Nguyen Huu Duc, PhD Hanoi-2022 DECLARATION First of all, I hereby declare that the graduate thesis work is mine All research results have been results during the implementation of the topic The results, the data are completely true, never appeared in any scientific report Finally, I also guarantee that the references and useful information for the topic are clearly cited and all help is appreciated Hanoi , 2022 Student Nguyen Thi Thuy Duong i ACKNOWLEDGEMENTS During the process of implementing my graduation 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 Dr Nguyen Thi Dieu Thuy and Dr Nguyen Huu Duc 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 thanks the teachers in the Faculty of Biotechnology have helped and taught me during my training at school Especially the teachers of the Biology department who gave me advice during carrying out Finally, I would like to sincerely thanks my family members and friends who always trust, support and encourage me to complete this report Sincerely thank! Hanoi, March, 14 th , 2022 Student Nguyen Thi Thuy Duong ii CONTENTS DECLARATION i ACKNOWLEDGEMENTS ii LIST OF ABBREVIATION v LIST OF TABLES vi LIST OF FIGURES vii PART I INTRODUCTION 1.1.Preface 1.2 Objective and requirements 1.2.1 Objective 1.2.2 Requirements PART II LITERATURE REVIEW 2.1 Overview of the origin and classification of domestic chickens 2.2 Some characteristics of Bang Troi chicken breed 2.3 Mitochondrial genome characterization and the role of B-loop in chicken identification .6 2.3.1 Mitochondrial structural features, genetic mechanism of chicken mitochondrial genome 2.3.1.1 Mitochondrial structural features 2.3.1.2 Chicken mitochondrial genome structure 2.3.1.3 Genetic mechanism of mtDNA 10 2.3.2 Structure and role of the D-loop region in genetic diversity assessment .11 PART III MATERIALS AND METHODS 14 3.1 Location and time for research 14 3.1.1 Research location 14 3.1.2 Time for research 14 3.1.3 Subjects 14 3.2 Materials 14 iii 3.2.1 Materials .14 3.2.2 Chemicals 14 3.2.3 Equipments 15 3.3 Methods .17 3.3.1 DNA extraction from blood samples .17 3.3.2 Agarose gel electrophoresis 17 3.3.3 Quantitative, qualitative DNA by spectrophotometer 18 3.3.4 Specific amplification of the D-loop region of the mitochondrial gene using specific primers 19 3.3.5 Analyze gene sequences and build genetic family trees .23 3.3.5.1 Sequencing the D-loop region 23 3.3.5.2 Data analysis with specialized software 24 PART IV RESULTS AND DISCUSSION 25 4.1 Extraction of total DNA from chicken blood samples, evaluation of DNA .25 quantity and quality 25 4.2 Amplification of the mitochondrial D-loop region from chicken samples by PCR using specific primer pairs 27 4.3 Analysis gene sequences, build genetic family tree between samples .30 PART V CONCLUSION AND SUGGESTION 35 5.1 Conclusion 35 5.2 Proposal for further work 35 REFERENCES 36 iv LIST OF ABBREVIATION µl Microlitre kb Kilobase bp Base pair DNA Deoxyribonucleic acid PCR Polymerase Chain Reaction F Forward primer R Reverse primer UV EDTA Ultra Violet Ethylendiamin tetracetic acid TBE Tris-Borate-EDTA dNTP Deoxynucleoside triphosphate Tris mtDNA COI D-loop Aminomethane Mitochondrial DNA Cytochrome oxidase I Displacement loop A Adenin C Cytosine G Guanine T Thymine TAE Tris – acetate – EDTA NCBI National Center for Biotechnology Information ATP Adenosine triphosphate RNase Ribonuclease v LIST OF TABLES Table Components of PCR reaction .20 Table 2: PCR reaction heat cycle 20 Table 1: The genetic similarity of the mitochondrial D-loop region among chicken breeds .33 vi LIST OF FIGURES Figure 1.1: Structural diagram of mitochondria (https://www.sciencefacts.net/wpcontent/uploads/2020/04/Mitochondria.jpg ) Figure 1.2 : The chicken mitochondrial genome (https://www.researchgate.net/figure/The-Genomic-Organisat-ion-of-the-ChickenMtDNA-Compared-with-that-of-other-Vertebrates_fig1_15472316 ) 10 Figure : Gene electrophoresis machine - England 16 Figure : Centrifuge machine Hettich – Germany 16 Figure 3: PCR PTC-100 (MJ Research, Inc.,USA) 16 Figure 1: Electrophoresis of total DNA products extracted from Bang Troi chicken blood 26 Figure 2: Electrophoresis of PCR products amplifying the D-loop region of Bang Troi chicken mitochondrial gene 29 Figure 3: The results of comparing the gene sequences of the D-loop region of the chicken mitochondrial gene Bang Troi Figure 4: Genotypic tree of chicken breeds based on mitochondrial D-loop sequence built with MEGA 5.1 software, using UPGMA with bootstrap value 1,000x 33 vii PART I INTRODUCTION 1.1 Preface The domestic chicken (Gallus gallus domesticus) is the most popular domesticated chicken in the world According to the World Food and Agriculture Organization (FAO), the global number of chickens in 2007 was estimated at 17 billion, more than half of which were in Asia It is one of the essential food sources for humans, especially in developing countries, providing almost all of the meat and egg needs in remote rural areas and about 20% of the food needs urban areas In addition to the purpose of food, domestic chickens are also raised as pets, cockfighting or medicinal Not only that, chicken is also widely used in biomedical research In the agricultural sector of our country, chicken raising accounts for 72-73% of the total poultry flock annually In 2006, the Ministry of Agriculture and Rural Development developed a strategy to develop Vietnam's poultry production for the period 2006 2015 Accordingly, the livestock industry must strive to increase the proportion of poultry meat by 2015 to 32 % of total meat production of all kinds, in which chicken production accounts for 88% of total poultry flocks, reaching 350 million heads, meat weight 1,992,000 tons, egg production 9,236 billion eggs To achieve the above goal, it is necessary to improve the source of breeding stock, and at the same time to conserve and develop precious local poultry breeds In our country, there are 27 chicken breeds, including 16 domestic chicken breeds Domestic chicken breeds such as Ri chicken, Dong Tao chicken, H'Mong chicken, Tre chicken are varieties with delicious egg meat quality, tolerance to hardship, high disease resistance, which are a source of good quality precious genes and need to be invested in selection and breeding to improve productivity and use crossbreeding with other varieties to improve productivity, creating high-yield hybrids to provide breeding stock for production However, due to the tradition of small-scale farming by households, these breeds are often grazed freely with other domestic chicken breeds in the locality, along with the introduction of new genetic material due to the introduction of new genetic material Massively imported varieties are imported, so they are at risk of being hybridized and lost gradually Therefore, the issue of preserving genetic resources of precious poultry breeds is an urgent requirement of reality Research on µm containing POP-4™ of ABI, Korea The D-loop control region was sequenced on an ABI 3730XL DNA Analyzer, Applied Biosystems (Macrogen, Korea) 3.3.5.2 Data analysis with specialized software - The mitochondrial D-loop region-specific PCR product was sequenced directly on the ABI 3730XL DNA Analyzer, Applied Biosystems (Macrogen, Korea) - Results of reading gene sequences were analyzed using BioEdit software (Hall, 1999), BLAST tool, and then compared with reference gene sequences obtained from GenBank - Building a genetic pedigree tree by Neighbor-Joining method using MEGA 5.1 software, with a bootstrap value of 1,000 sampling times (Tamura et al., 2007) 24 PART IV RESULTS AND DISCUSSION 4.1 Extraction of total DNA from chicken blood samples, evaluation of DNA quantity and quality With poultry: Blood can be obtained immediately before or after the animal's death For live poultry, regularly take two blood samples a few days apart to determine whether the antibody levels rise or fall for diseases tested by serology Blood is usually drawn from the jugular or wing veins or directly into the heart This makes it safe for the birds to take a second blood draw In this experiment, we used wing venous blood collection: usually the best and simplest method for chickens, turkeys and most other poultry raised in the wild The vein is exposed by plucking some of the hair on the surface of the wing vein, the vein is clearly visible below the indentation of the wing between the wing joint and the brachial muscle If you use 70% alcohol or a colorless antiseptic to wet the veins, the veins will be more visible The person taking blood uses one hand to hold the two wings together, grasp the two wings in the upper area and insert the needle into the vein on the right arm, the other hand in the middle of the syringe insert the needle into the vein, the needle is directly opposite the vein flow to the heart There are also a number of other methods such as blood collection from the heart: Poultry are blood drawn by the person holding the back lying down on the table with the abdomen facing up with the left hand Before taking human blood, draw the feathers of the kite area and the entrance to the chest, disinfect it to dry The needle is inserted from the thoracic line anteriorly parallel to the spine, slightly to the left until the needle penetrates the heart Blood will flow into the syringe, use your right hand to pull the syringe to draw blood out slowly During the blood collection process, pay attention that the needle must be sterilized, sharp, and disposable needles are the best When the needle hits a vein, blood will flow out, collecting blood in a sterilized test tube Collecting approximately ml of blood is sufficient for most testing requirements Serum testing requires the blood sample to clot so that all red and white blood cells and fibrin clots, and the rest is serum To so, place the blood test tube slightly tilted for a large surface area, at ambient temperature avoid too hot places and direct sunlight for about 1-2 hours, then place it in the lower compartment of the refrigerator at 4°C, after a few hours or overnight, use a pipette or syringe to remove the serum Or you can use a centrifuge at 25 1000 rpm for 10-15 minutes, extract the serum The removed serum is stored in a fresh clean tube It is possible to send a blood sample that has not been segregated for testing, but there is a risk of hemolysis due to hemoglobin leakage from the red blood cells into the serum If the sample is sent day late, the blood must be serumcoagulated and kept in the refrigerator until the sample is sent If sample submission is delayed by weeks or more, serum samples should be stored in a negative refrigerator until needed for testing Some tests require a sample of uncoagulated whole blood In this case, the sample vial must contain anticoagulant, provided by the laboratory Several anticoagulants are available, and which one to use depends on the test being performed In the experiment we used antifreeze tubes containing EDTA After extraction and purification, the DNA samples were checked for concentration and purity by measuring ultraviolet absorption spectra at two wavelengths 260 nm, 280 nm on a Nanodrop 1000 The extracted DNA samples had only A260/280 number ranged from 1.81 to 2.02 with concentrations ranging from 37.0 ng/μl to 295.0 ng/μl The DNA samples all have smooth, unbroken, angled, and angular absorption curves, and the absorption peak corresponds to the wavelength of 260 nm Figure 4.1: Electrophoresis of total DNA products extracted from Bang Troi chicken blood 1→6: Total DNA of samples 26 Total DNA of chicken samples, after being extracted from fin samples, was checked by electrophoresis on 1% agarose gel The image on the gel electrophoresis shows that the total DNA bands are clearly dyed, indicating that the high DNA concentration is sufficient to perform the PCR reaction in the next steps 4.2 Amplification of the mitochondrial D-loop region from chicken samples by PCR using specific primers The D-loop is the only non-coding region on vertebrate mitochondrial DNA This region contains the transcriptional initiation of the heavy chain and the transcriptional promoters of mitochondrial DNA With the characteristic that the accumulation of mutations is - 10 times higher than that of other genes in mitochondria and compared to nuclear DNA, the D-loop becomes an important region in the study of biodiversity and phylogeny species of organisms Therefore, in order to evaluate the genetic diversity of chickens belonging to Bang Troi chicken, we chose the nucleotide sequence of the D-loop region of the mitochondrial genome as the research thesis Gene cloning by PCR is successful when the reaction is highly specific Therefore, to give the best product, the reaction conditions must be optimized To multiply the D-loop region requires template DNA, primers, dNTPs, Taq DNA polymerase, MgCl2, buffer, and a suitable thermal cycle Components of PCR reaction - Buffer: PCR reaction is carried out in buffer medium with pH 7.2 to stabilize the activity of Taq polymerase In this experiment, we used a buffer containing NH4+ but not containing Mg2+ from Fermentas, which is suitable for Taq polymerase activity and a wide range of MgCl2 concentrations Moreover, this type of mattress gives better product quality than the non-NH4+ mattress - Mg2+ concentration: in the experiments, we used a buffer that did not contain Mg2+ to be able to investigate its influence on the reaction The concentration of Mg2+ ions (provided as MgCl2) can affect the denaturation temperature (Tm) of the template DNA, Taq polymerase activity and the accuracy of the results Too high a concentration of Mg2+ will increase the stability of the double-stranded DNA and prevent complete denaturation to release the single-strand in each PCR cycle, making the PCR product poorer, it also increases the phenomenon of pseudo-pairing at nonhomologous positions, leading to the appearance of non-specific products Conversely, if the concentration of Mg2+ is too low, it will adversely affect DNA synthesis 27 because Mg2+ acts as a co-factor of Taq polymerase Therefore, it is necessary to determine the optimal concentration of Mg2+ to ensure the amplification efficiency and specificity of the PCR product After investigating a number of different Mg2+ concentrations, we found that the 10 mM Mg2+ concentration was most suitable for amplifying the D-loop region by PCR technique -dNTPs: concentrations of suitable dNTPs will give stable, accurate and specific results The four types of dNTP should be used in equal concentrations to minimize any misincorporation of the genetic code High concentrations of dNTP are not good for the reaction as it will sequester Mg2+ ions The concentration of each type of dNTP also depends on the size of the DNA fragment to be replicated In this experiment, we used a concentration of 10 mM dNTPs (0.25 mM each) Pair of primers used to clone the D-loop region: HGal-F: 5’-AGGACTACGGCTTGAAAAGC-3’ LGal-R: 5’-CATCTTGGCATCTTCAGTGCC-3’ Here, H (Heavy chain) and L (Light chain) are the symbols for the heavy chain and light chain, and the digit is the number indicating the 3' end nucleotide position of the primer in the complete sequence of the chicken mtDNA The adjustment of the annealing temperature and primer concentration determines the amount of product obtained The two primers H and L must have equal concentrations and must be adjusted appropriately so as not to be too high to avoid primer-dimer phenomenon and the phenomenon of primers misplaced on the template low to avoid reducing the amount of product formed In this experiment, we used primers with a concentration of 10 pmol/ul for each of H and L - Concentration of template DNA: PCR consists of two stages: screening phase and amplification phase If the reactants (including primers) are in high concentration while the template DNA is in low concentration, the screening phase of PCR becomes more difficult as the frequency with which primers and template DNA meet is reduced markedly, while the primer-to-primer contact increased, causing primerdimer phenomenon in the amplification phase Typically, the concentration of template DNA used ranges from 10 to 100 ng/25 µl of reaction solution The template DNA used in the study is 71 total DNA samples of chicken breeds: Ri, Dong Tao and Tre, these samples have high purity and little breakage 28 - Taq polymerase: too high an enzyme concentration can cause non-specific PCR products to appear, leading to erroneous results Conversely, if the enzyme concentration is too low, it will not be enough to catalyze the production of the desired amount of product In this experiment, we used Taq polymerase with a concentration of units/ul 1270 bp Figure 4.2: Electrophoresis of PCR products amplifying the D-loop region of Bang Troi chicken mitochondrial gene M: standard DNA scale 1kb; 1-6: Amplification product of Bang Troi chicken mitochondrial gene D-loop region; 7: DC: Amplification product of the chicken mitochondrial gene D-loop region of Lien Minh chicken After optimizing the conditions for PCR reaction, we carried out PCR amplification of this gene fragment in all obtained samples and conducted electrophoresis to check the product on 1% agarose gel The obtained results show that the PCR product band is clear, no by-products appear, the amplified gene fragment size is in the range of 1270 bp consistent with the previously designed PCR product size Thus, the length of the amplified gene fragment is suitable and the expected size The PCR1 product was then purified and PCR2 reaction (PCR sequencing) performed 29 with the forward primer PCR2 products were purified and sequenced according to the steps mentioned in the research methods section Therefore, it can be roughly concluded that this is the D-loop region of the chicken mitochondrial genome 4.3 Analysis gene sequences, build genetic family tree between samples The D-loop segment was sequenced according to Sanger 's method The sequence of 940 nucleotides in the D-loop region from position 21 to position 960 on mtDNA of samples of Bang Troi chicken breeds was determined directly by dideoxy method, BigDye Terminator v3.1 Cycle Sequencing Kit on Automated DNA sequence analyzer ABI 3730XL DNA Analyzer, Applied Biosystems (Macrogen, Korea) Nucleotide sequence data, after being processed by DNA Sequencing Analysis v5.3.1, SeqScape® v2.6 and BioEdit v7.0.9 software were compared with that of the Lien Minh chicken sequence The 940 nucleotide sequences in the D-loop region of individual samples of the studied chicken breed were processed and compared as follows: 30 31 Figure 4.3: The results of comparing the gene sequences of the D-loop region of the chicken mitochondrial gene Bang Troi We analyzed the genetic relationship of chicken samples with Lien Minh chicken In the chicken samples studied, GBT 01 chicken has the closest genetic relationship to AB268541 Gallus E03 chicken, the nucleotide sequence similarity between GBT 01 chicken samples compared to AB268541 Gallus E03 chicken is 100%, GBT02 and GBT 03 chickens are genetically distant from AB268542 Gallus E04 and GBT01 chickens are nearly equivalent, the similarity of nucleotide sequences of these two chicken breeds is 100% and 99.8%, respectively The genetic relationship between the chicken samples studied with each other and with Lien Minh chickens is shown in Table: 32 Seq-> AB268541 Gallus E03 AB268539 Gallus E01 AB268542 Gallus E04 GBT 01 GBT 02 GBT 03 GBT 04 GBT 05 GBT 06 ID 0.996 0.996 0.996 0.998 0.985 0.985 0.992 ID 0.997 0.996 0.997 0.995 0.984 0.982 0.995 ID 0.996 0.997 0.986 0.984 0.993 ID 0.996 0.998 0.985 0.985 0.992 ID 0.997 0.986 0.984 0.993 ID 0.986 ID 0.986 0.992 ID 0.991 0.987 0.986 ID Table 1: The genetic similarity of the mitochondrial D-loop region among chicken breeds Figure 4.1: Genotypic tree of chicken breeds based on mitochondrial D-loop sequence built with MEGA 5.1 software, using UPGMA with bootstrap value 1,000x On the phylogenetic tree, comparative samples were divided into separate large groups, group I consisted of samples of Bang Troi chickens (species GBT04, GBT05) Group II includes the remaining samples of the Bang Troi chicken breed This group split into subgroups, one consisting of AB268541 Gallus E03 and GBT 01, the other consisting of samples AB268542 Gallus E04 and GBT 03, D9C 05 and, G9C 42F This result is also consistent with the genetic relationship analysis Thus, in 33 chicken breeds, GBT 01, GBT02, GBT 03, GBT 06 have a closer relationship with each other than between them with GBT 04, GBT05, both of which belong to the same branch Building a genetic pedigree tree by Neighbor-Joining method using MEGA 5.1 software, with a bootstrap value of 1,000 sampling times (Tamura et al., 2007) A genetic classification tree between chicken breeds based on the D-loop gene sequence has been built, which is classified into the common branches of Asian domestic chickens (branches E and B) where GBT01 is closely related to AB268541 Gallus E03, GBT02 is closely related to AB268542 Gallus E04, GBT03 is closely related to GBT 01, GBT04 is closely related to GBT05 Among the local chicken breeds we studied, there were some individuals that were particularly different from those of the same breed (for example, G9C 05, G9C 42F) It can be said that in the process of evolution and development, there was another maternal ancestor mixed in or crossed 34 PART V CONCLUSION AND SUGGESTION 5.1 Conclusion Extracted total DNA from 06 chicken blood samples, evaluated the quantity and quality of DNA Amplified the mitochondrial D-loop region from chicken samples by PCR using specific primer pairs The nucleotide sequence of the D-loop region of the Bang Troi chicken mitochondrial gene with a molecular size of 1,270 bp has been determined Analyzed gene sequences, built genetic family trees between samples A 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