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Cicer Arietinum L. DEVELOPMENT OF FUNCTIONAL MARKERS AND TRANSCRIPT MAP OF CHICKPEA (Cicer arietinum L.) Ms. Neha Gujaria DEVELOPMENT OF FUNCTIONAL MARKERS AND TRANSCRIPT MAP OF CHICKPEA (Cicer arietinum L.) A Thesis submitted for the degree of SUBMITTED BY Ms. Neha Gujaria CO- SUPERVISOR SUPERVISOR Dr. Rajeev K. Varshney Prof. Mangla Bhide DEPARTMENT OF ZOOLOGY SCHOOL OF BIOLOGICAL AND CHEMICAL SCIENCES DR. HARI SINGH GOUR UNIVERSITY SAGAR - 470 003 (M.P.) INDIA DECLARATION BY THE CANDIDATE (para 12b) DECLARATION I declare that the thesis entitled ―DEVELOPMENT OF FUNCTIONAL MARKERS AND TRANSCRIPT MAP OF CHICKPEA (Cicer arietinum L.)‖ is my own work conducted under the supervision of Dr. Mangla Bhide, Prof. & Head, Chairman BOS, Department of Zoology, School of Biological and Chemical Sciences, Dr. Harisingh Gour Vishwavidyalaya, Sagar (M.P.) and co- supervision of Dr. Rajeev Kumar Varshney, Director, Centre of Excellence in Genomics (CEG), International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad (A.P.) approved by Research Degree Committee. I have put in more than 200 days of attendance with the supervisor at the centre. I further declare that to the best of my knowledge the thesis does not contain any part of work, which has been submitted for the award of any other degree either in this University or in any other Universities without proper citation. Signature of Candidate (Neha Gujaria) Signature of the Co-supervisor Signature of the Supervisor (Rajeev Kumar Varshney) (Mangla Bhide) Signature of Head of the Department CERTIFICATE OF THE SUPERVISOR (para 12 C) CERTIFICATE This is to certify that the work entitled “DEVELOPMENT OF FUNCTIONAL MARKERS AND TRANSCRIPT MAP OF CHICKPEA (Cicer arietinum L.)” is a piece of research work done by Ms. Neha Gujaria under our guidance and supervision for the degree of Doctor of Philosophy of Dr. HARISINGH GOUR VISHWAVIDYALAYA, SAGAR (M.P.), INDIA. The candidate has put-in an attendance of more than 200 days with us. To the best of our knowledge and belief the thesis: (1) embodies the work of the candidate herself (2) has duly been completed (3) fulfills the requirements of the Ordinance relating to the Ph.D. Degree of the University and (4) is up to the standard both in respect of contents and language for being referred to the examiner. Signature of the Co-supervisor Signature of the Supervisor (Rajeev Kumar Varshney) (Mangla Bhide) Forwarded Signature of Head of the Department ACKNOWLEDGEMENT Endless compassion of Almighty turns my difficult task to a feeling of pleasant journey of my life. Emotions cannot be adequately expressed in words because emotions are transformed into a mere formality. Hence, my acknowledgements are many times more than what I am expressing here. I bow my head before my parents whose hardships, patience and perseverance today I stand has kept me going at all hard times. I have received a lot of ad-hoc response from many generous grandees in many quarters to those pain stacking days encompassing the research work. I wish to vow my genuflexion with deep sense of gratitude to my co-supervisor Dr. Rajeev Kumar Varshney, Director-Centre of Excellence in Genomics, Applied genomics,ICRISAT and Leader Theme 1- Comparative and Applied Genomics, Generation Challenge Programme, for his conscientious guidance, gracious, cordial and meticulous explications and encapsilative remarks towards the representation of this dissertation. His valuable suggestions brought a panacea for me dealing with this research work. I can truly say that whatever he has done for me is not possible for every advisor to do for his advisee. At this stage I feel pleasure to express my profound regards, indebtness and gratitude to my supervisor Dr (Prof) Mangla Bhide, , Prof. & Head, Chairperson BOS, Department of Zoology, School of Biological and Chemical Sciences, Dr. Harisingh Gour Vishwavidyalaya, Sagar (M.P.) for her expedient advice, debonair discussion, innovative ideas, abiding interest and invaluable support during this tenure of research work. A depth of gratitude is owed to Dr. William Dollente Dar, Director General and Dr. Dave Hoisington, Deputy Director General of the ICRISAT for giving me opportunity to work in one of the best labs of International standards. With respect, regards and immense pleasure, I wish to acknowledge and express sincere thanks from my heart to several scientists including Dr. Trushar Shah, Dr. Abhishek Rathore, Dr. Vivek Thakur, Dr. T Mahendar for the valuable suggestions. I render special thanks Dr. RR Mir, Visiting Scientist and Dr. Himabindu Kudupa for their help in finalizing the thesis. I feel immense pleasure to express my sincere thanks to Dr. Junichi Kashiwagi and Dr. L Krishnamurthy, Chcikpea Breeding for their help in conducting the research trials at Patancheru and Dr. H D Upadhyaya and Dr. P M Gaur for providing the genetic material used in the present study. With immense pleasure, I express my cordial thanks to Mrs. Seetha Kannan, Mrs. Manjula B, Mrs B Poornima Reddy, Mr. Prasad KDV, Mr. Murali Krishna Y and Mr. B Anjaiah, for their administrative help. It is my immense pleasure to render sincere regards to Mr. K Eshwar and Mr. G Somaraju for teaching me the laboratory techniques at the beginning of the research work.Words are less to express my gratitude to Mr. A Gafoor, Mr. R Samantula, Mr. T Veerendar from Applied Genomics Laboratory, ICRISAT for providing technical help. I place on record my heartfelt thanks to all my labmates and friends especially Abhishek, Anuja, Ashish, Preeti, Sarwer, Spurthi, Manish Pandey, Pavana, Lekha, Kim, Deepa, Monisha, Ramesh, Mayank, Gautmi, Swathi, P. Swathi, Gnanesh, Rachit, Usha, Manish Roorkiwal, Prasad KVSS, Yogendra, Ashutosh, Bhanuprakash, Reetu, Prasad, Pradeep. I also thank all the friends and staff at Department, School of Biological and Chemical Sciences, Dr. Harisingh Gour Vishwavidyalaya, Sagar (M.P.) for their kind help and co-operation. Assistance rendered by the members of Central Support Lab, Library and Learning System Unit at ICRISAT is gratefully acknowledged. Words would fail to express the depth of my feelings for my strength, my husband Manish for his all time support, constant encouragement, affectionate love and for always being there for me. I feel indebted to my grandparent in-law Mr. Om Prakash Verma for his constant support and encouragement throughout the tenure of research work. I seize the opportunity to express my heartiest sense of reverence, respect and affection to my parents Dr. S.C. Gujaria and Mrs.Vimla Gujaria, whose unending love, care, subdued spirit of co-operation, and timely encouragement throughout my research work and their support at every crucial moment of my life. I also express my heartiest sense of reverence, respect and affection to my brother Mr. Rajiv Gujaria, loving sister Mrs. Manvi and nephew Nishchay, who always supported and encouraged me during all the time. ABBREVIATIONS °C : degree Celsius μl : Microliter ABA : Abscisic acid AFLP : Amplified Fragment Length Polymorphism ANOVA : Analysis of Variance ASR : Abscisic acid Stress and Ripening BAC : Bacterial Artificial Chromosome BES : BAC-end Sequences BIBAC : Binary Bacterial Artificial Chromosome BLAST : Basic Local Alignment Search Tool bp : base pair CAPS : Cleaved Amplified Polymorphic Sequences C : complementary DNA CIM : Composite Interval Mapping cM : centiMorgan CTAB : Cetyl Trimethyl Ammonium Bromide DArT : Diversity Array Technology DIVEST : Diversity Estimator DNA : Deoxyribonucleic Acid DREB : Drought Responsive Element Binding proteins EMBL : European Molecular Biology Laboratory EST : Expressed Sequence Tag GCP : Generation Challenge Programme ICCM : ICRISAT Chickpea Microsatellites ICRISAT : International Crops research Institute for the Semi-Arid Tropics Indel : Insertion deletion ISSR : Inter Simple Sequence Repeats JCVI : J. Craig Venter Institute kbp : kilo base pairs LDW : Leaf Dry Weight LG : Linkage Group LOD : Logarithm of odds (base 10) MAS : Marker-Assisted Selection Mb : Million bases mM : milliMolar MSA : Multiple Sequence Alignment NCBI : National Center for Biotechnology Information ng : nanograms PAGE : Polyacrylamide Gel Electrophoresis PCR : Polymerase Chain Reaction PIC : Polymorphism Information Content QTL : Quantitative Trait Loci RAPD : Random Amplified Polymorphic DNA RD : Rooting Depth RDW : Root Dry Weight RFLP : Restricted Fragment Length Polymorphism RGA : Resistance Gene Analogues RIL : Recombinant Inbred Line RL : Root Length RLD : Root Length Density RSA : Root Surface Area RT : Root Dry Weight/Total Dry Weight ratio RV : Root Volume SCAR : Sequence Characterized Amplified Region SDW : Shoot Dry Weight SIM : Simple Interval Mapping SNP : Single Nucleotide Polymorphism SSR : Simple Sequence Repeats StDW : Stem Dry Weight STMS : Sequence Tagged Microsatellite Sites ABSTRACT To develop repertoire of genic molecular markers (GMMs) including single nucleotide polymorphism (SNP) and intron spanning region (ISR) based markers, in chickpea, the third food legume crop of the world and the first food legume crop of India. In first approach, Solexa 1 Gb was carried out on pooled RNA from all drought challenged root tissues of genotypes ICC 4958 and ICC 1882. 15.6 and 22.1 million reads were generated and aligned against chickpea transcriptome assembly. A total of 26,082 SNPs were identified between these two genotypes. In second approach for SNP discovery through allele re-sequencing, primer pairs were designed for 970 genes/ expressed sequence tags (ESTs)
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