STUDYING DNA POLYMORPHISMS IN GENUS GOSSYPIUM AND THEIR IMPLICATIONS IN COTTON BREEDING By NABILA TABBASAM 2016 NATIONAL INSTITUTE FOR BIOTECHNOLOGY & GENETIC ENGINEERING (NIBGE), FAISALABAD, PAKISTAN QUAID-I-AZAM UNIVERSITY, ISLAMABAD, PAKISTAN STUDYING DNA POLYMORPHISMS IN GENUS GOSSYPIUM AND THEIR IMPLICATIONS IN COTTON BREEDING A dissertation submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy In Biotechnology By Nabila Tabbasam 2016 NATIONAL INSTITUTE FOR BIOTECHNOLOGY & GENETIC ENGINEERING (NIBGE), FAISALABAD, PAKISTAN QUAID-I-AZAM UNIVERSITY, ISLAMABAD, PAKISTAN Acknowledgement ACKNOWLEDGEMENT In the name of Allah, the most Beneficent and the Merciful. I bow my head, with all the humility and modesty, before Allah Almighty, the creator, the most supreme whose mercy enabled me to accomplish this task and bestowed me with success. May Allah shower His countless salutation upon His all Prophets including Muhammad (PBUH), His last messenger, who is the fountain of knowledge and guidance for the salvation of mankind in this world and in the hereafter. This gives me the privilege to acknowledge few of those people whose sincere help, guidance and prayers enabled me to accomplish my research work in a congenial and serene environment. First of all, I would like to pay attributes to all Directors (Drs. Yusuf Zafar, Zafar Mehmood Khalid, Sohail Hameed and Shahid Mansoor, SI) of the National Institute of Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan for providing me congenial environment for pursuing for my PhD. I must express my sincere regards to my supervisor Dr.Mehboob-Ur-Rahman (Pride of Performance) for his continuous guidance and patience throughout my study. I am greatly obliged to my co-supervisor Dr.Yusuf Zafar for his helpful suggestions without which it would not have been possible to begin the present to a successful end. I am thankful to the Director cotton CCRI Multan for providing us the leaves of Gossypium species. I am also grateful to late Prof J. McD. Stewart (University of Arkansas, Fayetteville, Arkansas, USA) for providing us the genomic DNA of G. hirsutum ‘‘yucatanense’’, G. capitis-viridis, G. longicalyx and G. australe. I too acknowledge Prof Andrew H Paterson (PGML, UGA, USA) for providing me research facilities in his labs for carrying out a part of my PhD thesis research work. I am also grateful to my lab fellows Dr.Tayyaba Shaheen, Mr. M Atif Iqbal and Mr. Zaman as well as field staff members, Mr. Shehbaz Shabbir, Mr.Wasif Ali and Mr. Muhammad Farooq for their support and cooperation. Funds for this research work were largely utilised through a project entitled “DNA- based genetic characterization of cotton Germplasm (Component-1, NIBGE, Faisalabad) financed through ALP-PARC program. I am also thankful to HEC Islamabad Pakistan for providing funds for doing some lab experiments in PGML, University of Georgia USA. I owe a heartfelt debt of gratitude to my parents, who endured all the strains and stress during the course of my study and whose hearts were beating with prayers for my success. I am also indebted and grateful to my brothers and sisters who always remembered me in their prayers. I am especially thankful to my sister Sonia Tabbasam whose help in take caring of my kids made me able to complete this work. I gratefully acknowledge the encouragement and support from my husband Mr. Fakhar Z. Wahla (Director Admin, BARDC, PARC, Quetta), whose love, support, encouragement, patience and praying made this work possible. I am also thankful to my in Laws for their encouragement and good wishes during my studies. Last but not the least are my loving kids Ch. Shehram Fakhar and Haleema Zaman, their tiny hands were always raised in prayers for my success. I am thankful to Allah Almighty, without His help it was impossible to complete this research work. Ms. Nabila Tabbasam I Dedicated to my family List of publications LIST OF PUBLICATIONS 1. Tabbasam, N., Zafar, Y. and Rahman, M., 2014. Pros and cons of using genomic SSRs and EST-SSRs for resolving phylogeny of the Genus Gossypium. Plant Systemetic and Crop Evol. DOI 10.1007/s00606-013-0891-x 2. Rahman, M., Shaheen, T., Tabbasam, N., Iqbal, M., A., Ashraf, M., Zafar, Y. and Paterson, A. H. 2012. Cotton genetic resources. A review. Agron Sustain Dev 32: 419-432. 3. Rahman, M., Asif, M., Shaheen, T., Tabbasam, N., Zafar, Y. and Paterson, A. H., 2011. Marker-assisted breeding in Higher Plants. Sustain Agric Review 6: 39-76. 4. Saeed, M., Guo, W., Ullah, I., Tabbasam, N., Zafar, Y., Rahman, M. and Zhang, T., 2011. QTL mapping for physiology, yield and plant architecture traits in cotton (Gossypium hirsutum L.) grown under well-watered versus water-stress conditions. Electronic Journal of Biotechnology, ISSN 0717-3458, DOI: 10.2225 Vol 14-Issue 3-fulltext-3 5. Lin, L., Pierce, G. J., Bowers, J. E., Estill, J. C., Compton, R. O., Rainville, L. K., Kim, C., Lemke, C., Rong, J., Tang, H., Wang, X., Braidotti, M., Chen, A. H., Chicola, K., Collura, K., Epps, E., Golser, W., Grover, C., Ingles, J., Karunakaran, S., Kudrna, D., Olive, J., Tabbasam, N., Um, E., Wissotski, M., Yu, Y., Zuccolo, A., Rahman, M., Peterson, D. G., Wing, R. A., Wendel, J. F. and Paterson, A. H., 2010. A draft physical map of a D-genome cotton species (Gossypium raimondii). BMC genomics, 11:395. 6. Rahman, M., Yasmin,T.,Tabbasam,N.,Ullah,I.,Asif, M. and Zafar, Y.,2008. Studying the extent of genetic diversity among Gossypium arboreum L. genotypes/cultivars using DNA fingerprinting. Genetic Resour Crop Evol, 55: 331-339. 7. Tabbasam, N., Zafar, Y., Rahman, M. and Paterson, A.H., 2012. BAC derived new SSRs for use in cotton (Gossypium spp.) improvement. Submitted in Pak J Bot. II Table of contents TABLE OF CONTENTS Chapter Topics Pages Title page Declaration Dedication Acknowledgement I List of Publications II Table of Contents III List of Figure VIII List of Table X Abstract XII 1 Introduction 16 1.1 Cotton 16 1.2 Evolution of genome size in cotton 17 1.3 Cotton and Arabidopsis 19 1.4 Need for the improvement of cotton genomic resources 20 1.5 Marker systems for genetic mapping 21 1.5.1 Morphological and Biochemical markers 21 1.5.2 DNA based markers 21 1.5.2.1 Advantages of DNA-based markers over other markers 21 1.5.2.2 Hybridization based markers 22 1.5.2.3 PCR-based DNA markers 22 1.5.2.3.1 PCR-based arbitrarily primed techniques 23 1.5.2.3.2 Random amplified polymorphic DNA (RAPD) 23 1.5.2.3.3 Amplified fragment length polymorphism (AFLP) 24 1.5.2.3.4 Cleaved amplified fragment length polymorphism 25 1.5.2.3.5 Microsatellites or Simple sequence repeats (SSRs) 26 1.6 Transcriptome analysis of cotton fiber development 29 1.6.1 Cotton fiber 29 1.6.2 Fiber Properties 29 1.6.2.1 Fiber length 29 1.6.2.2 Fiber strength 30 1.6.2.3 Micronaire 31 1.6.3 Fiber Development 32 1.6.3.1 Transcriptomes of a developing fiber 33 1.6.3.2 Comparison of diploid and tetraploid genomes regarding fiber 34 genes 1.6.3.3 Similarities between Arabidopsis trichome and cotton seed hair 34 development 1.7 Cotton fiber gene identification through differential display 35 1.8 Development of new SSRs 35 1.9 Molecular linkage maps in cotton 36 1.9.1 Construction of linkage maps 36 1.9.2 Cotton linkage maps 37 1.10 Implications of genomic tools for cotton improvement 43 1.11 Objectives 44 2 Materials and Methods 46 2.1 Genetic diversity and relationship of diploid and tetraploid cotton 46 III Table of contents species using EST-SSR and gSSRs 2.1.1 Plant material 46 2.1.2 Extraction of Genomic DNA 48 2.1.2.1 DNA purification 49 2.1.2.2 Quantification of genomic DNA 49 2.1.3 Simple sequence repeats (SSR) analysis 49 2.1.3.1 Polymerase chain reaction (PCR) 50 2.1.4 Electrophoresis of amplified products 51 2.1.5 Data scoring and statistical analysis 51 2.2 Identification of differentially expressed genes from normal (fiber 52 producing) and mutant (fiberless) ovules of Gossypium hirsutum during fiber elongation stage 2.2.1 Plant Material and RNA extraction 52 2.2.2 Synthesis of the first strand cDNA and Differential Display RT- 53 PCR DDRT-PCR) 2.2.3 DDRT-PCR 53 2.2.4 Electrophoresis of amplified products 56 2.2.5 Isolation, Reamplification and Confirmation of Differentially 56 Expressed Transcripts 2.2.5.1 Elution protocol from agarose gel 56 2.2.6 DNA Sequencing 57 2.2.7 Data Analysis 57 2.3 Isolation of fiber related partial sequences from Gossypium 57 arboreum at different developmental stages and phylogenetic study of translation elongation factor-1 gamma gene across the genomes of cotton 2.3.1 Plant material 57 2.3.2 RNA extraction at different developmental stages of cotton fibers 58 2.3.3 Synthesis of 1st strand cDNA 58 2.3.4 Gene specific primer designing 58 2.3.4.1 Criteria for primer designing 59 2.3.5 Polymerase chain reaction 60 2.3.6 Agarose gel electrophoresis of amplified products 61 2.3.7 DNA fragments elution protocol from agarose gel 61 2.3.7.1 Ligation of fragments 61 2.3.7.2 Heat shock Cells Preparation of E .coli 62 2.3.7.3 Transformation in E. coli by heat shock 62 2.3.7.4 Plasmid isolation from E. coli 63 2.3.7.5 Restriction reaction 63 2.3.8 DNA Sequencing 64 2.3.8.1 Sequence analysis 64 2.3.9 BAC library screening 64 2.3.9.1 Filter preparation, probe designing and hybridization 64 2.3.9.2 BAC clone sequencing 65 2.3.9.3 Sequencing reaction 65 2.3.9.4 Clean up protocol 67 2.3.9.5 Run on “ABI 377” DNA sequencer 67 2.3.9.6 Analysis of sequences 67 IV Table of contents 2.4 Development of BAC-gSSRs from Gossypium raimondii for use 67 in cotton