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DNA Barcoding and Phylogenetic Assessment of Family Lamiaceae from Pakistan Based on Plastid and Nuclear Sequence Data

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DNA Barcoding and Phylogenetic Assessment of Family Lamiaceae from Pakistan Based on Plastid and Nuclear Sequence Data DNA Barcoding and Phylogenetic Assessment of Family Lamiaceae from Pakistan based on Plastid and Nuclear Sequence Data. By Nadia Batool Zahra Department of Biotechnology Faculty of Biological Sciences Quaid-i-Azam University Islamabad 2017 DNA Barcoding and Phylogenetic Assessment of Family Lamiaceae from Pakistan based on Plastid and Nuclear Sequence Data. A thesis submitted in the partial fulfillment of requirements for the degree of Doctor of Philosophy In Plant Biotechnology By Nadia Batool Zahra Department of Biotechnology Faculty of Biological Sciences Quaid-i-Azam University Islamabad 2017 DEDICATED TO MY LOVING PARENTS For their support & prayers CONTENTS Acknowledgements i Index of Figures iii Index of Tables v List of Abbreviations vi Abstract vii Chapter 1 Introduction 1-31 1.1 Family Lamiaceae 1 1.2 Distribution 1 1.3 Medicinal and Economic Importance 2 1.4 Systematics 3 1.4.1 Subfamily Symphorematoideae Briq. 4 1.4.2 Subfamily Viticoideae Briq. 8 1.4.3 Subfamily Ajugoideae Kostel. 10 1.4.4 Subfamily Prostantheroideae Luerss. 12 1.4.5 Subfamily Scutellarioideae (Dumort.) Caruel 13 1.4.6 Subfamily Lamioideae Harley 14 1.4.7 Subfamily Nepetoideae (Dumort.) Luerss. 18 1.5 Molecular phylogenetics 20 1.5.1 Plastid and nuclear regions in angiosperm phylogeny 21 1.6 DNA Barcoding: A tool for standardization of herbal 22 medicinal products (HMPs) 23 1.6.1 What is a DNA Barcode? 1.6.2 Plastid and nuclear markers for DNA barcoding in plants 24 Maturase K gene (matK) 1.6.2.1 Ribulose-1,5-bisphosphate carboxylase/oxygenase large 25 subunit gene (rbcL) 1.6.2.2 trnH-psbA intergenic spacer 26 1.6.2.3 Internal transcribed spacer (ITS) 26 1.6.2.4 Challenges in DNA barcoding of medicinal plants/herbal 27 products 1.7 Research Objectives 31 Chapter 2 Materials & Methods 33-55 2.1 Section I: DNA Barcoding of herbal medicinal products 33 (HMPs) of Lamiaceae from Pakistan. 2.1.1 Sample collection from herbal markets/industry 33 2.1.2 DNA extraction from HMPs 33 2.1.2.1 Standard 2 X CTAB method 33 2.1.2.2 DNA isolation kit 38 2.1.3 Polymerase chain reaction (PCR) 38 2.1.3.1 PCR conditions for rbcL complete gene amplification 39 2.1.3.2 PCR conditions for matK partial gene amplification 39 2.1.3.2 PCR conditions for trnH-psbA spacer amplification 39 2.1.4 Purification of amplified products 40 2.1.5 DNA sequencing of barcoding loci 40 2.1.6 Nucleotide sequence data analysis 40 2.1.6.1 BLAST analysis 40 2.2 Section II: Phylogenetic utility of cpDNA rbcL, matK and 43 trnH-psbA regions 2.2.1 Sequence alignment and super matrix assembly 43 2.2.2 Data set construction 43 2.2.3 Phylogenetic analysis by RAxML method 43 2.3 Section III: Molecular phylogenetics of Lamiaceae based 44 on plastid (trnL-trnF) and nuclear (ITS) markers 2.3.1 Taxon sampling from wild and herbarium 44 2.3.2 DNA extraction from wild and herbarium specimen’s 44 2.3.2.1 2 X CTAB protocol 44 2.3.2.2 DNA extraction from herbarium samples 50 2.3.3 PCR 51 2.3.3.1 PCR parameters for trnL-trnF region 51 2.3.3.2 PCR parameters for ITS region 51 2.3.4 Purification of amplified products and DNA sequencing 51 2.3.5 Nucleotide sequence data analysis 54 2.3.5.1 Sequence alignment and super matrix assembly 54 2.3.5.2 Data set construction 54 2.3.6 Phylogenetic analysis 54 2.3.6.1 RAxML 55 2.3.6.2 Bayesian Inference (BI) 55 Chapter 3 Results 56-102 3.1 Section I: DNA Barcoding of herbal medicinal products 56 (HMPs) of Lamiaceae from Pakistan. 3.1.1 DNA extraction from HMPs and PCR of rbcL, matK and 56 trnH-psbA regions 3.1.2 Purification, sequencing and data analysis 56 3.1.3 BLAST analysis 56 3.1.3.1 rbcL gene analysis 58 3.1.3.2 matK nucleotide sequence analysis 62 3.1.3.3 trnH-psbA spacer region analysis 62 3.2 Section II: Evaluation of phylogenetic utility of cpDNA 65 rbcL, matK and trnH-psbA regions 3.3 Section III: Molecular phylogenetics of Lamiaceae based 79 on plastid (trnL-trnF) and nuclear (ITS) markers 3.3.1 DNA extraction and PCR 79 3.3.2 Purification, sequencing and data analysis 79 3.3.3 Phylogenetic analysis based on plastid trnL-trnF region 79 3.3.4 Phylogenetic analysis based on nuclear ITS region 92 Chapter 4 Discussion 103 4.1 Section I: DNA Barcoding of herbal medicinal products 103 (HMPs) of Lamiaceae from Pakistan. 4.1.1 Difficulty in DNA extraction and amplification of HMPs 103 4.1.2 Insufficient reference sequence data 104 4.1.3 Pros and cons of rbcL, matK and trnH-psbA regions 105 4.1.4 Challenges, improvements and regional recommendations for 105 DNA barcoding of HMPs 4.2 Section II: Evaluation of phylogenetic utility of cpDNA 109 rbcL, matK and trnH-psbA regions 4.3 Section III: Molecular phylogenetics of Lamiaceae based 110 on plastid (trnL-trnF) and nuclear (ITS) markers 4.3.1 Viticoideae 111 4.3.2 Scutellarioideae 112 4.3.3 Ajugoideae 112 4.3.4 Prostantheroideae 113 4.3.5 Symphorematoideae 113 4.3.6 Nepetoideae 114 4.3.7 Lamioideae 116 4.4 Conclusion 117 4.5 Future Recommendations 119 Publications 121 Literature Cited 122 ACKNOWLEDGEMENTS All praise to Allah Almighty, the most beneficent, the most merciful, who gave me strength and enabled me to undertake and execute this research task. I feel highly privileged in taking opportunity to express my deep sense of gratitude to my supervisor Dr. Zabta Shinwari, Professor, Department of Biotechnology. He was my greatest strength and my supreme mentor who not only supervised my work but also polished every aspect of my personality. I am thankful to him for his inspiration, reassurance and counseling from time to time and for his scholastic guidance and valuable suggestions throughout the study. I wish to express my heartfelt appreciation to Dr. Melanie Schori and Prof. Dr. Allan Showalter who were a source of guidance for completion of my research work at Environmental and Plant Biology Lab, Ohio University, Ohio, US. I would also like to pay my cordial thanks to Dr. Evgeny Mavrodiev, Prof. Dr. Douglas Soltis and Prof. Dr. Pamela Soltis for their cooperation, guidance, support and valuable advices they offered for completion of my research at Laboratory of Molecular Systematics & Evolutionary Genetics, University of Florida, Florida, US. It gives me great pleasure to express my gratitude to Higher Education Commission of Pakistan, for providing Indigenous and IRSIP scholarships. I would like to extend my deepest appreciation to those people, who helped me in one way or other in planning and executing this research work and writing up this thesis manuscript. I am obliged to Prof. Dr Wasim Ahmad, Dean, Biological Sciences and Dr. Muhammad Naeem, Chairman, Department of Biotechnology, Faculty of Biological Sciences, Quaid-i- Azam University, Islamabad for extending the research facilities of the department to accomplish this work. I am grateful to Faculty Department of Biotechnology, Quaid-i-Azam University, Islamabad for their help and support. Many thanks to Dr Muhammad Iqbal, Dr Mushtaq Ahmad, Dr Zafar Mahmood, Dr Anjum Parveen, Dr Tariq Mahmood, Dr Muhammad Ali and Mr Abdul Majid for extending their valuable advices and providing research material support. Sincere thanks to all my senior and junior lab fellows who were part of this journey at Molecular Sysytematics & Applied Ethnobotany Laboratory, Quaid-i- Azam University, Islamabad. My earnest gratitude to Dr Anwar Nasim for his continuous moral support and encouragement. Special thanks to Sohail Irshad and Muhammad Bilal Khan who happily extended their assistance at every instance. Solemn gratitude to my parents and my siblings who deserve special mention for their inseparable support and prayers. They have always been my source of strength and love. It wouldn’t have been this bearable if I didn’t have them in my life. Thank you for your unconditional support with my studies. Thank you for giving me a chance to prove and improve myself through all walks of my life. Nadia Zahra INDEX OF FIGURES Figure Title Page Fig 1.1 Clasification of family Lamiaceae proposed by Harley & colleagues 5 (2004). Fig 1.2 Flow chart representation of general transportation chain involved in 31 medicinal plants business in Pakistan. Fig 1.3 A double helix to show the relationship between classical taxonomy 32 and molecular database. Fig 2.1 Local herb store, Abpara, Islamabad, Pakistan. 34 Fig 2.2 Visit to herbal pharmaceutical industry, Pakistan. 34 Fig 2.3 Photograph showing the usual packaging of HMPs in plastic bags 35 purchased from Pansar stores. Fig 2.4 Field visit to Northern regions of Pakistan for collecting Lamiaceae 45 species. Fig 2.5 Showing locations of primers used to amplify different regions of 53 plastid (trnL-trnF) genome. Fig 2.6 Showing locations of primers used to amplify different regions of 53 nuclear (ITS) genome. Fig 3.1 Visualizations of extracted genomic DNA and amplified products of 57 DNA barcoding loci. Fig 3.2 Performance/Percentage success of DNA barcoding loci for 64 Lamiaceae. Fig 3.3 DNA barcodes of 32 HMPs belonging to Lamiaceae, collected from 64 local herbal stores and herbal pharmaceutical industry. Fig 3.4 The RAxML phylogenetic tree based on rbcL gene sequences of 66 Lamiaceae. Fig 3.5 The RAxML phylogenetic tree based on matK gene sequences of 70 Lamiaceae.
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