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Isolation and Characterization of Microsatellite Loci in an Endangered Palm, Johannesteijsmannia Lanceolata (Arecaceae)

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Isolation and Characterization of Microsatellite Loci in an Endangered Palm, Johannesteijsmannia Lanceolata (Arecaceae) ISOLATION AND CHARACTERIZATION OF MICROSATELLITE LOCI IN AN ENDANGERED PALM, JOHANNESTEIJSMANNIA LANCEOLATA (ARECACEAE) ANG CHENG CHOON FACULTY OF SCIENCE UNIVERSITY OF MALAYA KUALA LUMPUR 2012 ISOLATION AND CHARACTERIZATION OF MICROSATELLITE LOCI IN AN ENDANGERED PALM, JOHANNESTEIJSMANNIA LANCEOLATA (ARECACEAE) ANG CHENG CHOON DISSERTATION SUBMITTED IN FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE FACULTY OF SCIENCE UNIVERSITY OF MALAYA KUALA LUMPUR 2012 ABSTRACT Johannesteijsmannia lanceolata J. Dransf. (Arecaceae) is a rare and endangered palm species which is endemic to Peninsular Malaysia. It was listed as an endangered species in the 1997 IUCN red list of threatened plants owing to its substantially reduced population size. Hence, understanding of the population structure and genetic diversity by using microsatellite markers is important to postulate scientific conservation and restoration strategies for this species. In this study, a set of 33 novel polymorphic microsatellite loci had been successfully isolated and characterized for J. lanceolata from a genomic library enriched for AG/CT repeats via the selective hybridization of biotinylated (CT)15 probes and streptavidin-coated magnetic beads. The microsatellite primers were screened on 24 samples collected fresh from the Angsi Reserve Forest. In general, the microsatellite loci exhibited high level of polymorphism with an average number of 10 alleles per locus and mean polymorphic information content (PIC) of 0.771. Two loci (Jla130b and Jla332a) showed significant departures from the Hardy-Weinberg equilibrium with (P < 1.515 x 10-3) after Bonferroni correction. Micro-checker analysis suggested the presence of null alleles in both the Jla332a and Jla118 loci. No significant linkage disequilibrium was detected between any pair of loci. All 33 microsatellite loci were confirmed to be conforming to the Mendelian mode of inheritance in a test with an open-pollinated half-sib family where all the progenies inherited at least one maternal allele. Transferability of the microsatellite loci were checked across other Johannestsijsmannia species. All loci showed positive amplifications in four samples from each of the three Johannesteijsmannia species, except for loci Jla124 (J. magnifica J. Dransf.) and Jla168b (J. magnifica and J. perakensis J. Dransf.). i Of the 33 newly isolated microsatellite loci, twenty-eight loci are in Hardy- Weinberg equilibrium, highly polymorphic, absence of linkage disequilibrium and null alleles. Therefore, they could be used as markers for the investigation of the population genetic structure, gene flow, parentage, and mating system of J. lanceolata across its distribution range in the near future. ii ABSTRAK Johannesteijsmannia lanceolata J. Dransf. (Arecaceae) merupakan spesis palma yang jarang ditemui dan endemik di Semenanjung Malaysia. Ia telah didaftarkan sebagai tumbuhan terancam oleh ‘1997 IUCN red list of threatened plants’ berdasarkan saiz populasinya yang semakin berkurangan. Oleh itu, pemahaman tentang struktur populasi dan diversiti genetik dengan menggunakan penanda mikrosatelit amat diperlukan untuk proses konservasi dan sebarang strategi bagi pemulihan spesies tersebut. Dalam penyelidikan ini, sebanyak 33 lokus microsatelit baru yang polimorfik telah berjaya diasingkan dan dicirikan untuk J. lanceolata daripada sumber genomik yang diperkaya dengan ulangan AG/CT melalui hibridisasi penunjuk biotinylated (CT)15 dengan manik-manik magnet yang dilapisi streptavidin. Primer mikrosatelit telah disaring daripada 24 sampel dari Hutan Simpan Angsi, Negeri Sembilan. Secara umumnya, lokus mikrosatelit tersebut menunjukkan tahap polimorfik yang tinggi dengan bilangan purata 10 alel bagi setiap lokus dan purata 0.771 kandungan informasi polimorfik (PIC). Dua lokus (Jla130b dan Jla332a) telah menunjukkan keberangkatan yang signifikan dari Keseimbangan Hardy-Weinberg di mana P < 1.515 x 10-3 setelah pembetulan Bonferroni. Analisis perisian Micro-checker mencadangkan kehadiran alel null dalam kedua-dua lokus Jla118 dan Jla332a pada aras kepercayaan 99 %. Tiada sebarang hubungan yang tidak seimbang yang signifikan didapati antara setiap pasangan lokus tersebut. Kesemua 33 lokus mikrosatelit ini mematuhi mod warisan Mendelian melalui ujian dengan keluarga separa- beradik-pendebungaan terbuka di mana semua progeni mewarisi sekurang-kurangnya satu alel ibu. Lokus amplifikasi bersilang mikrosatelit diperiksa bagi semua spesis dalam genus Johannestsijsmannia. Semua lokus telah menunjukkan amplifikasi positif dalam ketiga-tiga iii spesies Johannesteijsmannia yang lain kecuali lokus Jla124 (J. magnifica J. Dransf.) dan lokus Jla168b (J. magnifica dan J. perakensis J. Dransf.). Daripada 33 penanda mikrosatelit baru ini, 28 lokus berada dalam keseimbangan Hardy-Weinberg, sangat polimorfik, ketiadaan ketakseimbangan hubungan dan alel null. Oleh itu, mereka boleh digunakan sebagai penanda untuk mengkaji taburan populasi spesis ini dari segi struktur genetik populasi, aliran gen, keturunan, dan sistem kacukan J. lanceolata pada masa akan datang. iv ACKNOWLEDGEMENTS I would like to thank my supervisors, Dr. Ng Ching Ching and Associate Professor Dr. Rozainah Mohamad Zakaria for their advice, tremendous support, constant encouragement, and valuable criticism during the preparation of this thesis. I wish to express my gratitude and appreciation to Genetics Laboratory of Forest Research Institute Malaysia (FRIM) for providing the place and facilities to conduct this project. Many thanks go to Dr Lee Soon Leong for his valuable advice, constructive suggestions and critical comments on the preparation of the manuscript during paper submission. I would like to extend my appreciation to Dr Lee Chai Ting and Dr Tnah Lee Hong for their guidance, patient and assistance during the construction of enriched-library. Not forgetting to thank Dr. Kevin Ng Kit Siong for sharing his knowledge on fragment analysis, Dr. Ng Chin Hong for introducing the MISA software, Ms Chan Yoke Mui for describing the ecology and morphological features of Johannesteijsmannia lanceolata, Dr. Naoki Tani for sharing his valuable experience and some papers by him and also members of the Genetics Laboratory for being unbelievably helpful during the total DNA extraction. I greatly acknowledge the Ministry of Agriculture and Agro-Based Industry, Malaysia (Science Fund No: 05-01-03-SF0199 & 05-03-10-SF1031) and postgraduate research fund (PS228/2009C) for funding this project. Besides, I also appreciate the University of Malaya Fellowship Scheme for its financial support. Special thanks to my fellow lab mates for their friendship, encouragement, support and care. My gratitude also goes to my friends whom I share my joys and grieves with. Last but not least, I want to dedicate this thesis to my family for their lifetime of love and unconditional support. v PUBLICATION AND PRESENTATION Paper publication: Ang, C. C., Lee, S. L., Lee, C. T., Tnah, L. H., Zakaria, R. M., & Ng, C. C. (2011). Isolation and characterization of microsatellite loci in an endangered palm, Johannesteijsmannia lanceolata (Arecaceae). American Journal of Botany, 98(5), e117- e119. Poster presentation: Ang Cheng Choon, Ng Ching Ching, Lee Soon Leong, Lee Chai Ting, Tnah Lee Hong and Rozainah Mohamad Zakaria, 2010. Isolation and characterization of microsatellite loci of Johannesteijsmannia lanceolata (Arecaceae). My1bio conference 2010: Accelerating biotechnology through innovative research, 30-31 Oct 2010, Berjaya Times Square Hotel, Kuala Lumpur, Malaysia. (National) vi TABLE OF CONTENTS ABSTRACT i ABSTRAK iii ACKNOWLEDGEMENTS v PUBLICATION AND PRESENTATION vi TABLE OF CONTENTS vii LIST OF FIGURES x LIST OF TABLES xi LIST OF SYMBOLS AND ABBREVIATIONS xii CHAPTER 1 INTRODUCTION 1 2 LITERATURE REVIEW 4 2.1 The genus, Johannesteijsmmania 4 2.1.1 Biology and Ecology 6 2.1.2 Distribution 6 2.2 Johannesteijsmannia lanceolata 7 2.3 Genetic markers 9 2.3.1 DNA-based molecular markers 9 2.3.1.1 Restriction Fragment Length Polymorphism, RFLP 11 2.3.1.2 Random Amplified polymorphic DNA, RAPD 11 2.3.1.3 Amplified Fragment Length Polymorphism, AFLP 12 2.3.1.4 Single Nucleotide Polymorphism, SNP 12 2.3.1.5 Expressed Sequences Tagged, EST 13 2.3.1.6 Microsatellites 13 2.3.1.6.1 Classification of microsatellites 14 2.3.1.6.2 Mutation mechanisms of microsatellites 14 vii 2.4 Applications of microsatellite markers to plant 16 population analysis 2.4.1 Population structure assessment 16 2.4.2 Parentage and paternity analysis 17 2.4.3 Gene flow 17 2.5 Development of microsatellites 18 2.6 Cross-species amplification 19 3 MATERIALS AND METHODS 21 3.1 Leaf samples collection 21 3.2 Total genomic DNA extraction 21 3.3 Construction of CT-enriched library 22 3.3.1 Digestion of genomic DNA with restriction enzyme 22 3.3.2 Ligation of DNA fragments with cassettes 23 3.3.3 PCR amplification to confirm ligation 24 3.3.4 Selective hybridization using biotinylated (CT)15 25 3.3.5 Digestion of cassettes 27 3.3.6 Ligation of CT-enriched DNA into plasmid 27 3.3.7 Transformation of recombinant plasmids into 28 competent cells 3.4 Amplification of circular DNA 28 3.4.1 DNA sequencing and analysis of sequence data 28 3.5 Primer design 29 3.6 Primer screening and PCR amplification 30 3.7 Fragment analysis 30 3.8 Characterization of microsatellite loci 30 3.9 Analysis of inheritance 31 3.10 Cross-species amplification 32 viii 4 RESULTS 33 4.1 Construction of CT-enriched library 33 4.2 DNA sequencing of positive clones 38 4.3 Primer design
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