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Identification of Dwarf Mistletoe Resistant Genes in Ziarat Junipers (Juniperus Excelsa M

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Identification of Dwarf Mistletoe Resistant Genes in Ziarat Junipers (Juniperus Excelsa M IDENTIFICATION OF DWARF MISTLETOE RESISTANT GENES IN ZIARAT JUNIPERS (JUNIPERUS EXCELSA M. BIEB) Ph. D. Thesis (Botany) Submitted by HUMAIRA ABDUL WAHID Ph. D. Scholar Department of Botany University of Balochistan Quetta IDENTIFICATION OF DWARF MISTLETOE RESISTANT GENES IN ZIARAT JUNIPERS (JUNIPERUS EXCELSA M. BIEB) Ph. D. Thesis (Botany) Supervised by Dr. Muhammad Younas Khan Barozai Associate Professor Department of Botany University of Balochistan Quetta Submitted by Humaira Abdul Wahid Ph. D. Scholar Department of Botany University of Balochistan Quetta IDENTIFICATION OF DWARF MISTLETOE RESISTANT GENES IN ZIARAT JUNIPERS (JUNIPERUS EXCELSA M. BIEB) Submitted by Humaira Abdul Wahid Ph. D. Scholar Department of Botany University of Balochistan Quetta IDENTIFICATION OF DWARF MISTLETOE RESISTANT GENES IN ZIARAT JUNIPERS (JUNIPERUS EXCELSA M. BIEB) Thesis submitted for the requirement of the degree of Doctor of Philosophy (Ph. D.) in Botany, University of Balochistan Quetta Supervised by Dr. Muhammad Younas Khan Barozai Associate Professor Department of Botany University of Balochistan Submitted by Humaira Abdul Wahid Ph. D. Scholar Department of Botany University of Balochistan Quetta CERTIFICATE This is to certify that Ms. Humaira Abdul Wahid who was registered in Doctor of Philosophy (Ph. D.) (Registration No. 1993/UB-2013/R-215) in Botany, Department of Botany, University of Balochistan, Quetta, under the supervision of Dr. Muhammad Younas Khan Barozai, has successfully completed her course work of eighteen (18) credit hours and research work under the title “Identification of Dwarf Mistletoe Resistant Genes in Ziarat junipers (Juniperus excelsa M. BIEB)”. She may be allowed to submit the thesis on the above cited topic to the University of Balochistan for the fulfillment of Ph. D. (Botany) degree. Dr. Muhammad Younas Khan Barozai Prof: Dr. Atta Muhammad Sarangzai Research Supervisor Chairperson Associate Professor Department of Botany, Department of Botany, University of Balochistan, Quetta University of Balochistan, Quetta Prof: Dr. Mudassir Asrar External Examiner Dean Faculty of Life Sciences University of Balochistan, Quetta I CERTIFICATE It is to certify that the said dissertation is based on my research work under the title “Identification of Dwarf Mistletoe Resistant Genes in Ziarat junipers (Juniperus excelsa M. BIEB)” and its results carried out by me. This is written and compiled by me. This research work has not been submitted for higher studies in any other institution. Humaira Abdul Wahid Ph. D. Scholar Department of Botany University of Balochistan, Quetta II ACKNOWLEDGEMENT First and foremost, I am thankful to Almighty Allah for giving me such contentment and vision for accomplishment to my research work and compilation of this Thesis. I would like to express my sincere gratitude to my advisor Dr. Muhammad Younas Khan Barozai, Associate Professor, Department of Botany, University of Balochistan, Quetta, for the continuous support of my Ph. D. study and related research, for his patience, motivation, and immense knowledge. His guidance helped me in all the time of research and writing of this thesis. I am highly thankful and acknowledge the financial support of the higher education commission (HEC) of Pakistan, Islamabad for this research project (HEC- NRPU Project 20-1867/R&D/11) I am also grateful to Mr. Yasir Hameed Ansari, Programmer, Directorate of Information Technology (DIT), University of Balochistan, Quetta, for his valuable help during this research. I wish to express my gratitude to Chairperson and all the faculty members, Department of Botany, University of Balochistan, Dean, Faculty of Life Sciences, University of Balochistan, Quetta, for their significant guidance, encouragement, helpful suggestions and full cooperation. I would like to thank my humble colleagues for their moral support and helpful suggestions. III DEDICATION I take pleasure in dedicating this thesis to my sweet and loving parents whose affection, love, encouragement and prays make me able to get such success and honor. This work is also dedicated to my sister (Nayyar Wahid) niece (Aleena Zahid) and nephew (Muhammad Sheharyar). I also dedicated this work to all those who believe in the richness of learning. IV INDEX CONTENTS PAGE Certificate I Acknowledgment III Abbreviations IX Abstract 2 1. Introduction 4 2. Review of literature 8 2.1. Parasitic Angiosperms 8 2.2. Mistletoes 8 2.2.1. Dwarf mistletoes 10 2.2.2. Dwarf mistletoe life cycle 12 2.2.3. Arceuthobium oxycedri 14 2.3. Junipers 15 2.3.1. Juniperus excelsa 17 2.3.2. Juniper forests of Balochistan 18 2.4. Differential gene expression 21 2.5. Suppression Subtractive Hybridization (SSH) 23 2.6. Next generation Sequencing (NGS) 27 2.6.1. Illumina based studies 28 2.7. Quality check (QC) analysis 28 2.8. BLAST (Basic Local Alignment Search Tool) 29 2.9. Gene Ontology (GO) 30 3. Materials and Methods 32 3.1. Samples collection 32 3.2. Total RNA isolation from infested and non-infested shoots 32 3.2.1. Reagents and solutions 32 3.2.2. Extraction procedure 34 3.2.3. Quantitative and Qualitative analysis 34 3.3. Isolation of mRNA from total RNA 34 3.4. Suppression Subtractive Hybridization (SSH) 35 3.4.1. Synthesis of cDNA 35 3.4.1.1. First-Strand cDNA Synthesis 35 3.4.1.2. Second-Strand cDNA Synthesis 36 V 3.4.2. Rsa I Digestion 36 3.4.3. Adaptor Ligation 36 3.4.4. First Hybridization 36 3.4.4.1. Second Hybridization 36 3.4.5. PCR Amplification 37 3.5. Next generation sequencing (NGS) 37 3.6. FastQC analysis 37 3.7. Raw data trimming 37 3.8. De novo assembly and analysis of DEGs and transcripts 37 3.9. Functional annotation 38 4. Results 40 4.1. Sample collection 40 4.2. Total RNA isolation and mRNA purification 40 4.2.1. Total RNA isolation 40 4.2.1.1. Agarose gel electrophoresis 41 4.2.2. Purification of mRNA 41 4.3. Suppression Subtractive Hybridization (SSH) 41 4.3.1. Agarose gel analysis of SSH PCR products 41 4.4. Next generation sequencing (NGS) 42 4.4.1. Sequenced raw Data 42 4.5. FastQC analysis of raw data 42 4.5.1. Per base sequence quality analysis 42 4.5.2. Per base N content analysis 43 4.5.3. Per sequence quality scores analysis 43 4.5.4. Sequence length distribution analysis 43 4.6. Trimmed and filtered raw data 43 4.7. FastQC analysis of trimmed and filtered raw data 44 4.7.1. Per base sequence quality analysis 44 4.7.2. Per base N content analysis 44 4.7.3. Per sequence quality scores analysis 44 4.7.4. Sequence length distribution analysis 44 4.8. De novo assembly and identification of differentially expressed genes 44 4.9. Functional Annotation of DEGs 45 4.10. Significant resistant genes 46 VI TABLES Table1. Total RNA extracted from the DMIS of Juniperus excelsa 48 Table 2. Total RNA extracted from the DMNIS of Juniperus excelsa 48 Table 3. Purification of mRNA from total RNA extracted from DMIS 48 Table 4. Purification of mRNA from total RNA extracted from DMNIS 48 Table 5. PCR product produced through SSH 49 Table 6. Sequenced raw data with GC, AT content and quality score 49 Table 7. Trimmed raw data with GC, AT content and quality score 49 Table 8. Differentially expressed transcripts and genes identified in infested and non-infested shoots of Juniperus excelsa 49 Table 9. Classification of Contigs identified in dwarf mistletoe infested shoots of Juniperus excelsa according to size 50 Table 10. Classification of Contigs identified in dwarf mistletoe non-infested shoots of Juniperus excelsa according to size 59 FIGURES Figure 1. Generalized life cycle of dwarf mistletoe 13 Figure 2. Flow chart for the identification of dwarf mistletoe resistant genes in Ziarat juniper by using SSH, NGS and bioinformatics algorithms 33 Figure 3a. Juniper tree with dwarf mistletoe (Arceuthobium oxycedri) 64 Figure 3b. Samples of DMIS and DMNIS of Juniper tree 65 Figure 4. Total RNA extracted from DMIS and DMNIS of Juniperus excelsa 65 Figure 5a. Agarose gel analysis of forward subtracted PCR products 66 Figure 5b. Agarose gel analysis of reverse subtracted PCR products 66 Figure 6. Per base sequence quality analysis of F-SSH sequenced raw data 67 Figure 7. Per base N content analysis of F-SSH sequenced raw data 68 Figure 8. Per sequence quality scores analysis of F-SSH sequenced raw data 69 Figure 9. Sequence length distribution analysis of F-SSH sequenced raw data 70 Figure 10. Per base sequence quality analysis of R-SSH sequenced raw data 71 Figure 11. Per base N content analysis of R-SSH sequenced raw data 72 Figure 12. Per sequence quality scores analysis of R-SSH sequenced raw data 73 Figure 13. Sequence length distribution analysis of R-SSH sequenced raw data 74 Figure 14. Per base sequence quality analysis of F-SSH trimmed raw data 75 Figure 15. Per base N content analysis of F-SSH trimmed raw data 76 Figure 16. Per sequence quality scores analysis of F-SSH trimmed raw data 77 VII Figure 17. Sequence length distribution analysis of F-SSH trimmed raw data 78 Figure 18. Per base sequence quality analysis of R-SSH trimmed raw data 79 Figure 19. Per base N content analysis of R-SSH trimmed raw data 80 Figure 20. Per sequence quality scores analysis of R-SSH trimmed raw data 81 Figure 21. Sequence length distribution analysis of R-SSH trimmed raw data 82 Figure 22. Classification of contigs identified in DMIS according to size 83 Figure 23. Classification of contigs identified in DMNIS according to size 84 Figure 24. Functional characterization of DEGs identified in DMIS of Juniperus excelsa 85 Figure 25. Functional characterization of DEGs identified in DMIS of Juniperus excelsa according to biological processes 86 Figure 26.
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