MINISTRY OF EDUCATION AND TRAINING NONG LAM UNIVERSITY-HO CHI MINH CITY FACULTY OF BIOLOGICAL SCIENCES INVESTIGATION OF PROLACTIN GENE POLYMORPHISM IN THE TB CROSSBRED LAYER DUCK BY PCR-RFLP TECHNIQUE Major : BIOTECHNOLOGY Student : DO CHI HIEU Student ID : 19126249 Academic year : 2019-2023 Thu Duc City, 08/2023 MINISTRY OF EDUCATION AND TRAINING NONG LAM UNIVERSITY-HO CHI MINH CITY FACULTY OF BIOLOGICAL SCIENCES GRADUATIONTHESIS INVESTIGATION OF PROLACTIN GENE POLYMORPHISM IN THE TB CROSSBRED LAYER DUCK BY PCR-RFLP TECHNIQUE Instructor Student NGUYEN NGOC TAN, Ph. DO CHI HIEU Thu Duc City, 08/2023 ACKNOWLEDGEMENTS First and foremost, I would like to express my special thanks to the teachers of the Faculty of Biological Sciences and Research Institute for Biotechnology and Environment, Nong Lam University — Ho Chi Minh City for their respectful greetings, best wishes, and deep thanks. I received attention, teaching, thoughtful guidance, favorable conditions, and a research environment during the research and implementation of the thesis. I would like to thank my instructor, Nguyen Ngoc Tan, Ph., for providing me with the opportunity to conduct this research.
However, I must admit that I have felt disappointed with his instructions, and I believe that the teaching style may not have been entirely suitable for me. I found it difficult to voice my concerns, as I felt that the teacher did not actively listen or show much interest in the progress of my research. Moreover, I must admit that my instructor did not review this thesis, and I completed it by myself. I want to express my sincerest thanks to my family, who have always been a source of encouragement and concern throughout the journey of preparing this graduation thesis.
Their unwavering support has motivated my continuous efforts to strive and excel on my intellectual path. Furthermore, I would like to acknowledge the support of VIGOVA Poultry Research and Development Center for providing genetic resources and necessary information that enabled the smooth progress of this study. Lastly, I extend my sincere appreciation to my seniors, classmates, and everyone in the research room for sharing and exchanging knowledge and experiences to help me implement this project. AFFIRMATION AND COMMITMENT Name : DO CHI HIEU ID: 19126249 Class: DH19SHC Phone : 0888528341 Email: 19126249@st.vn My major is Biotechnology at Nong Lam University-Ho Chi Minh City.
This is my graduation thesis, which was conducted by myself. All the data and information are completely honest and objective. I take full responsibility in front of the committee for these commitments. Thu Duc City, July 31% 2023 Student’s signature Do Chi Hieu ii ABSTRACT The Prolactin (PRL) gene, encoding the PRL hormone, plays a crucial role in regulating various reproductive processes in animals, including egg production and broodiness in poultry.
Studying the genetic variations and associations of the PRL gene can provide valuable insights into enhancing breeding strategies and optimizing reproductive performance in livestock species. Additionally, the early assessment of reproductive traits, especially in terms of productivity, carries great significance. Therefore, this study aimed to investigate the polymorphism at the PRL/PstI locus of the PRL gene in the second generation of TB crossbred ducks by the PCR-RFLP technique and the association of its polymorphism with reproductive traits. A total of 167 blood samples were collected for DNA extraction and target fragment amplification.
The target gene fragment in the exon 5 region was successfully amplified with a size of 536 bp. The amplified PCR products were subsequently treated for digestion using the Pstl restriction enzyme. The results revealed that polymorphism at the PRL/Pstl locus was detected with two alleles, C and T, and the allele frequencies were 0. Three genotypes were observed; the genotype frequencies were 0.024 for CC, CT, and TT in the total population.
The PIC (Polymorphic Information Content) and He (expected heterozygosity) were 0. In conclusion, polymorphism at PRL/PstI is identified, and the CC genotype is dominant. The distribution of genotypes in the population fits the Hardy- Weinberg equilibrium. Further analysis showed a significant association between the genotypes and egg production.
Specifically, the CC genotypes exhibited higher egg yields up to 38 weeks of age compared to the CT genotype (corresponding to 99.05), following a regression line of y=3.02, with a determination coefficient of 0. Keywords: TB crossbred duck, Prolactin gene, Prolactin Hormone, exon 5, PCR-RFLP, genetic polymorphism, reproductive traits. ill TÓM TẮT Gen Prolactin (PRL), mã hóa hormone PRL, đóng vai trò quan trong trong việc điều chỉnh các quá trình sinh sản khác nhau ở động vật, bao gồm hành vi ấp gia cầm do đó có liên hệ đến năng suất trứng. Nghiên cứu về các biến thể di truyền và mối quan hệ của gen PRL có thé cung cấp cơ sở dữ liệu dé cải thiện các chiến lược nhân giống và tôi ưu hóa hiệu suất sinh sản ở các loài vật nuôi.
Ngoài ra, trong chăn nuôi việc đánh giá sớm các tính trạng sinh sản, đặc biệt là về mặt năng suất, rất có ý nghĩa. Vì vậy, nghiên cứu này nhằm khảo sát tính đa hình tại locus PR1⁄Psí của gen PRL trong thé hé thir hai cua vit lai TB bang ky thuat PCR-RFLP va mối liên hệ của tính đa hình này với các đặc điểm sinh sản. Tổng cộng 167 mẫu máu được thu nhận dé ly trích DNA và khuếch đại đoạn gen mục tiêu. Doan gen mục tiêu trong vùng exon 5 đã được khuếch đại thành công với kích thước là 536 bp.
Sản pham PCR khuếch đại sau đó được tiến hành phân cắt bởi enzym giới han PsíI. Kết quả cho thay tính đa hình tại locus PRL/Pstl được phát hiện với hai allele C và T. Tần suất xuất hiện cua allele này là 0,808 và 0,192. Ba kiểu gen đã được quan sát.
Tần suất kiểu gen tương ứng là 0,641; 0,335 và 0,024 cho CC, CT và TT trong quan thé. Chi số PIC (hàm lượng thông tin đa hình) va He (di hợp tử mong đợi) lần lượt là 0,262 và 0,310. Tổng kết, locus PRL/Pstl đã phát hiện tính đa hình va kiểu gen CC chiếm ưu thế. Phân bố kiểu gen trong quan thé phù hợp với cân bằng Hardy- Weinberg.
Phân tích tiếp theo cho thấy mối quan hệ ý nghĩa giữa các kiểu gen và năng suất trứng. Cụ thé, kiều gen CC cho thấy hiệu suất trứng cao hơn đến 38 tuần tuôi so với kiểu gen CT (tương ứng với 99,25 + 9,82 trứng so với 95,55 + 7,25 trứng; P <0,05), theo đường hồi quy y = 3,39x + §9,02, với hệ số xác định là 0,039. Từ khóa: Vit lai TB, Prolactin gene, Hormone Prolactin, exon 5, PCR-RFLP, đa hình gen, tinh trang sinh san. iv TABLE OF CONTENTS Page ACKNOWLEDGEMENTS caaccesiessbsinnseesssg4102811535013005588.3630/60633340840 i AFFIRMATION AND COMMITMENT.::ceceseeceeeesceeeeseeseeseeseeseeseeseeeeeeeseeeeeeee’ ii ABSTRACT oc.
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Candidate genes are associated with egg-laying performance. The structure of the PRL gene -. THỂ PRI hot ssccen cesses nessa g2 CRDHHGIAXISAS1XSSREBSISSEEIASESESSSESSENSH4ASEEERSBSRSxĐSSE6S 6 22.J, The PRL borin ore: iriTfffiäTHHHđÍS§-=--sez-siesseesesseensssrskkassiLdtkksus. The PRL, hormone 1ñ AVIAN SPECIES sesscsscssscecnsssvessereeseeesesssssmenermanarnasavavencesenes 7 2.
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Classification of molecular Markets ¡á-‹:c--cssccsc5 S161 10141 HẾ Án GI H3 l1 51616008008 12 2. Restriction Fragment Length Polymorphism.-------- 52552552 <+c+sc+sc+sccx2 12 252). RESHEUON ENZYMES ise secesevneeeuern eeermrsecesermenseeneneren verse nnaansneceaneeres 13 2⁄25: REL PO CGSS ss tonnnghöa dư hong G-S48%LG098I0836-H:3053882300GSE3. Polymerase Chain Reaction-Restriction Fragment Length Polymorphism .1, (PC ReREIP process sss sore on ee ats See a ee 15 2.
Advantages and limitations of PCR-RFLP and RFLP techniques. 16 21 RL Seite TS Cat CH usc nance eanmenane ee 16 2. The research status of Vietmamm. The research status of worldwide ssisssizesizs025165561144660661434516480415484GE80 9538148665 G556 18 CHAPTER 3.
MATERIALS AND METHODS .cccceccecceceeeeseeseeseeseeseeseeseeseeseenens 22 1 FeedDieuoeLOOM (Clo 5 (0) ee te ee 22 3,2.1, Sample and data 6öllé€GHOfizseasssesssesesisssiistoottkgEiLSS0103882855135S45954838g6I015380. Equipment and tools used in the research.3: Total DNA 6X ACH Of scccncsssccusecsnnsesr.ciscxeanmeseceenerneeea vane anemenaneseanS 23 3. DNA quality assessment after exfraCfIOT1. Agarose gel electrophoresis of total extracted DNA.
PCR amplification for exon 5 of the PRL gene. RFLP analysis of PCR products sssssspsscss emer ema 26 3 GoD Atal AIL YS ececsaeeieeseribsoesaiezessonosnusipbrasssllEDEMonkEstepisslMemuadkrietodirku8/091-au43581ndluoS44deecasSi 27 CHAPTER 4. RESULTS AND DISCUSSION wesssssssncossssovsnssonsesie se nesncunssesetssceseneseanses 30 4.1 Amplification of the target gene on the exon 5 reg10n. Total DNA €XIE4CLOHisescessssssssssssEss64S6146180414608666539161E558601468558313630Đ55346016G94188.
PCR amplification for exon 5 of the PRL gene. PCR amplification primer 1nfOrIaft1OI. PCR amplification results for exon 5 of the PRL gene. Polymorphic analysis of the PRL/PstI locus by the Ps⁄l restriction enzyme.
Effect of the genotypes at the PRL/PstI locus on some reproductive traits. CONCLUSIONS AND SUGGESTIONS. 40 Bil COnGlUSTONS ssssci-zs2s502580521226n85612 16 EglgiDcsiSdiot8gicuuSdngligdisSnii8daiigatiosidsosciciaidsbizEtdaroiSirdudi0aciimzketsard 40 Su SUD BOS LL ODS cuenndeeanebiuodooottlogiBiSEENGISSSEASSESSSIE-HGGAGSSSEBHMSR3.98S03ã190436100219iJggS0gg88 00380 g9oggssi 40 ;3335i54 0195005 =.Ô 4I ;0 0 x0pINIB)BXSSSS Sa. ca ca cac co 46 vi LIST OF ABBREVIATIONS Abbreviations Description AFLP : Amplified Fragment Length Polymorphism ANOVA : Analysis of variance Bp : Base pair df : Degree of freedom DNA : Deoxyribonucleic acid EDTA : Ethylenediaminetetraacetic acid Ha : The alternative hypothesis He : Expected Heterozygosity Ho : Observed Heterozygosity Ho : The null hypothesis L : Ladder NCBI : National Center for Biotechnology Information OD : Optical Density PCR — RFLP : Polymerase Chain Reaction - Restriction Fragment Length Polymorphism PCR : Polymerase Chain Reaction PIC : Polymorphic Information Content PRL - Prolactin gene PRL : Prolactin hormone QTL : Quantitative Trait Loci RAPD : Random Amplified Length Polymorphism REases : Restriction Enzyme RFLP : Restriction Fragment Length Polymorphism rpm : Round per minute SD : Standard Deviation SNP : Single Nucleotide Polymorphism Vii Ta : Annealing temperature TAE : Tris-Acetate-EDTA Taq : Thermus aquaticus Tm : Melting temperature : Chi-square Vili LIST OF TABLES Table 2.
Summary of Genotypic and Allelic Frequencies at the PRL/PstI Locus. Expected RFLP-digested PCR product sizes and genotype conventions. Information on the L536 primer pair .-------- 5-5525 52 +52 <+£+s£+sc+sccee 31 Table 4. Polymorphic analysis of the PRL/Pstl locus by PstI restriction enzyme.
Effect of genotypes on some reproductive fTa1{S.-- --- ---------+s++++ 36 ix LIST OF FIGURES Page Figure 2. Crossbreeding scheme điagram.-- ---- 2-52 22+ 22+ +22 £+zE++vEezerreezerrreerere 4 Figure 2. Illustrations of portions of the prolactin gene in ducks. IIlustration of RFLP analysis.cccivsssiavssseavesveee oreseervessisessnmmnsensensesaesenses 14 Figure 2.
Illustration of PCR-RFLP anaÌyS1s.------- 55552 +52£+<<+2c+sc+scesrree 15 Figure 4. Representative electrophoresis of the extracted DNA on 1% agarose gel. Representative electrophoresis of the PCR products of PRL in exon 5 on DY APATOSE: GE| picononcrrasemrvsrncevsituerenivnesiiistinses touts witinaitersmtineess viregilinlotristbsbiiltDuairbatt0u-1046 4L 32 Figure 4. Representative electrophoresis PCR-RFLP pattern of the PRL gene digested with Pst] on 2,5% agarose Ø€ÌL.- LG HH TH ng hệt 33 Figure 4.
Genotypic effect of the PRZ/PstI locus on egg yield (EY). Problem statement Along with the wet rice civilization, duck farming has been a long-standing traditional business and played an important role in the agricultural economy in Vietnam. Since 1961, our country has consistently ranked second globally in terms of duck populations. In 2020, Vietnam continued to hold the second position with a population of over 86.5 million ducks, representing 7% of the global total (Jalaludeen et al.
Duck farming contributes remarkably to agricultural productivity by providing a considerable source of duck meat and eggs. Duck meat is highly sought-after for its nutritional value and flavor (Sinanoglou et al. In 2019, Vietnam ranked fourth globally in terms of duck meat production.