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SEX IDENTIFICATION in Canarium Odontophyllum Miq. (DABAI) USING RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) MARKER

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SEX IDENTIFICATION in Canarium Odontophyllum Miq. (DABAI) USING RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) MARKER SEX IDENTIFICATION IN Canarium odontophyllum Miq. (DABAI) USING RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) MARKER TING JEN CHING This thesis is submitted in partial fulfillment of requirements for degree of Bachelor of Science with Honours in Program Resource Biotechnology Faculty of Resource Science and Technology UNIVERSITI MALAYSIA SARAWAK (UNIMAS) 2008 ACKNOWLEGMENT First of all, I would like to express my highest appreciation to my supervisor, Dr. Ho Wei Seng, who gave me the opportunity to work on this final year project. His generosity, patience and sincere guidance had encouraged and motivated me a lot in completing this project. Moreover, my greatest thanks to the master students, Mr. Phui Seng Loi and Mr. Liew Kit Siong for their persistent guidance and assistance throughout my research period, so as their readiness to share their past experiences with me. Nevertheless, I am also grateful to Miss Phang Shek Ling and Miss Tan Sze Leng, research officers of Sarawak Forestry Coporation for their willingness to share their advanced level of knowledge. Special appreciation to my laboratory mate, Miss Iris Tia ak Mekung, Miss Norzaitulrina bt Shamsudin, Miss Suliana Charles and Mr. Henry Lau, for their understanding, kindheartedness and cooperation throughout this research work. Apart from that, my sincere thankfullness to all my lovely, supportive friends especially Miss Un Siew Yien and Miss Harttini Neeni bt Hatta for their enduring supports and boundless helps. Lastly, I would like to dedicate my gratitude with full of my heart to my family for their never ending loves and supports upon completion of this research project. i Sex Identification in Canarium odontophyllum Miq. (Dabai) Using Random Amplified Polymorphic DNA (RAPD) Marker Ting Jen Ching Resource Biotechnology Programme Faculty of Resource Science and Technology Universiti Malaysia Sarawak ABSTRACT Canarium odontophyllum Miq. is a perennial plant native to Malaysia, Indonesia and Philippines. The pistillate plants bear fruits twice a year and are more preferable than staminate plants. Thus, sex determination in Canarium odontophyllum at seedling stage is very important for crop improvement processes because it accelerates the identification of fruitful pistillate plants. In this study, Random Amplified Polymorphic DNA (RAPD) technique was used to amplify DNA segments, with the objective of finding molecular markers associated to sex determination locus in this species. DNA was isolated from one male and three female Dabai trees and subjected to RAPD analysis. Of 52 RAPD primers, one primer (OPL – 03) produced an approximately 1,000 bp amplification band detectable in all pistillate plants, while absent in the staminate plants. The exclusiveness of this female-specific diagnostic band could be used in a breeding program to screen the gender of Dabai trees long before they reach reproductive maturity, resulting in considerable savings of time and economic resources. Key words: Canarium odontophyllum Miq., sex determination, Random Amplified Polymorphic DNA (RAPD), molecular markers, diagnostic band. ABSTRAK Canarium odontophyllum Miq. adalah sejenis tumbuhan yang hanya terdapat di Malaysia, Indonesia dan Filipina. Pokok betina akan menghasilkan buah dua kali setahun dan lebih digemari daripada pokok jantan. Oleh itu, penentuan seks Canarium odontophyllum pada tahap anak benih adalah amat penting untuk proses pemajuan tanaman kerana ia mempercepatkan identifikasi pokok jantan yang berbuah. Dalam kajian ini, teknik Random Amplified Polymorphic DNA (RAPD) telah digunakan untuk mengamplifikasikan segmen DNA dengan objektif untuk mencari marker molekular berkaitan dengan lokus penentuan seks dalam spesies ini. DNA telah dipencilkan daripada satu pokok jantan dan tiga pokok betina Dabai dan digunakan dalam analisis RAPD. Daripada 52 pencetus RAPD, satu pencetus (OPL – 03) telah menghasilkan band amplifikasi bersaiz lebih kurang 1,000 bp yang boleh dikesan dalam semua pokok betina tetapi tidak dalam pokok jantan. Band diagnostik pokok betina yang unik ini boleh digunakan dalam program pembiakan untuk mengesan jantina pokok Dabai sebelum pokok tersebut mencapai tahap matang. Ini akan menjimatkan masa dan sumber ekonomi. Kata kunci: Canarium odontophyllum Miq., penentuan seks, Random Amplified Polymorphic DNA (RAPD), marker molekular, band diagnostik. ii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS i ABSTRACT/ ABSTRAK ii TABLE OF CONTENTS iii LIST OF FIGURES v LIST OF TABLES vii LIST OF ABBREVIATIONS viii CHAPTER I INTRODUCTION 1 CHAPTER II LITERATURE REVIEW 2.1 Canarium odontophyllum Miq. 5 2.1.1 Distribution 6 2.1.2 General Morphology 6 2.1.3 Uses and Economic Importance 8 2.2 Sex Determination in Plant 8 2.3 Polymerase Chain Reaction (PCR) 10 2.4 Random Amplified Polymorphic DNA (RAPD) 11 CHAPTER III MATERIALS AND METHODS 3.1 Plant Material 14 3.2 Total Genomic DNA Isolation of Canarium 14 odontophyllum 3.3 DNA Purification 15 3.4 DNA Quantification 16 3.5 Spectrophotometric Quantitation of DNA 17 3.6 Polymerase Chain Reaction (PCR) Amplification 18 3.7 Selection of Sex-related RAPD Diagnostic Bands in 19 C. odontophyllum iii CHAPTER IV RESULTS 4.1 Total Genomic DNA Isolation 20 4.2 DNA Purification 21 4.3 DNA Quantitation 22 4.4 Polymerase Chain Reaction (PCR) Amplification 24 4.4.1 RAPD Primer Screening 24 4.4.2 Comparison of Male and Female DNA 25 4.4.3 Identification of Female Sex-assiocated RAPD 28 Dignostic Band in C. odontophyllum CHAPTER V DISCUSSION 5.1 Total Genomic DNA Isolation 30 5.2 DNA Purification 31 5.3 DNA Quantitation 32 5.4 RAPD-PCR Amplification 33 5.4.1 Primer Screening 34 5.4.2 RAPD Analysis 35 5.4.3 RAPD Trouble-shooting 37 CHAPTER VI CONCLUSIONS AND RECOMMENDATIONS 38 REFERENCES 40 APPENDICES APPENDIX A The 52 RAPD primer sequences 45 APPENDIX B Primer screening of the 52 RAPD primers through PCR 47 amplification of male DNA sample (D4) of C. odontophyllum iv LIST OF FIGURES Figure No. Page 2.1 20 years old female Dabai trees that achieve up to 20 metres in 6 height 2.2 (A) Mature Dabai Fruits 7 (B) Immature Dabai Fruits 2.3 Edible part of Dabai fruit 7 4.1 0.8% agarose gel showing DNA isolated from three female (D1, 20 D2, D3) and one male leaves (D4) of C. odontophyllum. Lanes 1 to 6 are female DNA samples while lanes 7 and 8 are male DNA sample. 4.2 0.8% agarose gel displaying purified DNA of three female (D1, D2, 21 D3) and male leaves (D4) of C. odontophyllum. L1 and L2 represent the Lambda HindIII DNA marker. Lane 1 to 3 are female DNA samples while lane 4 is male DNA sample. 4.3 (a) 1.5 % agarose gel showing RAPD patterns of combined male 26 and female C.odontophyllum amplified by RAPD primer, OPD – 11. F indicates female tree, M indicates male tree. L1 and L2 as 100 bp DNA ladder. (b) 1.5 % agarose gel showing RAPD patterns of combined male 26 and female C.odontophyllum amplified by RAPD primer, OPE – 04. F indicates female tree, M indicates male tree. L1 and L2 as 100 bp DNA ladder. (c) 1.5 % agarose gel showing RAPD patterns of combined male 26 and female C.odontophyllum amplified by RAPD primer, OPF – 14. F indicates female tree, M indicates male tree. L1 and L2 as 100 bp DNA ladder. (d) 1.5 % agarose gel showing RAPD patterns of combined male 26 and female C.odontophyllum amplified by RAPD primer, OPG – 14. F indicates female tree, M indicates male tree. L1 and L2 as 100 bp DNA ladder. (e) 1.5 % agarose gel showing RAPD patterns of combined male 27 and female C.odontophyllum amplified by RAPD primer, OPI – 12. F indicates female tree, M indicates male tree. L1 and L2 as 100 bp DNA ladder. v (f) 1.5 % agarose gel showing RAPD patterns of combined male 27 and female C.odontophyllum amplified by RAPD primer, OPJ – 19. F indicates female tree, M indicates male tree. L1 and L2 as 100 bp DNA ladder. (g) 1.5 % agarose gel showing RAPD patterns of combined male 27 and female C.odontophyllum amplified by RAPD primer, OPL – 03. F indicates female tree, M indicates male tree. L1 and L2 as 100 bp DNA ladder. 4.4 1.5% agrose gel of RAPD amplication products using primer OPL – 29 03. L1 and L2, 100 bp ladder size marker, M and F, combined DNA samples of 1 male and 3 female C. odontophyllum. The 1000 bp female sex-associated band is indicated with an arrow. vi LIST OF TABLES Table No. Page 3.1 Amount of DNA in each band generated by λ Hind III marker 16 3.2 Thermal cycling profile for RAPD primer screening 18 3.3 Optimized PCR condition for RAPD primer screening 19 4.1 The calculated concentrations of all purified DNA samples of 22 C. odonrophyllum (D1, D2, D3, D4), including reading taken at A260 4.2 Purity calculation of all purified DNA samples of C. odontophyllum 23 (D1, D2, D3, D4), including readings taken at A230, A260, A280 4.3 The calculated concentrations of all purified DNA samples of 23 C. odontophyllum (D1, D2, D3, D4) 4.4 Nucleotide sequences of selected RAPD primers 24 vii LIST OF ABBREVIATIONS CIA Chloroform: Isoamylalcohol CTAB Cetyl Trimethylammonium Bromide DNA Deoxyribonucleic Acid dNTPs Deoxynucleotide-triphosphates OD Optical density PCR Polymerase Chain Reaction PVP Polyvinylpyrrolidone RAPD Random Amplified Polymorphic DNA RNA Ribonucleic Acid SCAR Sequence Characterized Amplified Region TBE Tris-Borate-EDTA UV ultraviolet bp base pair g gram kb kilo base pair ml milliliter mM milimolar ng nanogram nm nanometer rpm revolutions per minute μl microliter β beta viii CHAPTER I INTRODUCTION In Malaysia, Canarium odontophyllum or better known as ‘Sibu Olive’ is a minor tropical fruit which serves as a food source for the local population in Sarawak (Morico et al., 1998). The common names for this wild fruit species are dabai, danau majang, or kedondong.
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