Physiological and Genetic Deciphering of Water, Salinity and Relative Humidity Stress in Chickpea (Cicer arietinum L.) A THESIS submitted for the award of the Degree of DOCTOR OF PHILOSOPHY in PLANT BIOTECHNOLOGY Submitted by R Pushpavalli Department of Plant Science Center of Excellence in Life Sciences Bharathidasan University Tiruchirappalli - 620 024 Tamil Nadu, India 2015 Physiological and Genetic Deciphering of Water, Salinity and Relative Humidity Stress in Chickpea (Cicer arietinum L.) A THESIS submitted to Bharathidasan University for the award of the Degree of DOCTOR OF PHILOSOPHY in PLANT BIOTECHNOLOGY by R Pushpavalli (Ref no: 45115/Ph.D.1/Plant Biotechnology/ P.T/April 2011/ Date 29.03.2011) Under the guidance of Dr M V Rao Honorary Professor Department of Plant Science Bharathidasan University Tiruchirappalli, Tamil Nadu & Under the co-guidance of Dr Vincent Vadez Principal Investigator- Crop physiology lab Assistant Director-Dryland Cereals International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) Patancheru-502324, Telangana Department of Plant Science Center of Excellence in Life Sciences Bharathidasan University Tiruchirappalli - 620 024 Tamil Nadu India 2015 This thesis is dedicated to My respectful father Thiru S Raju who consistently believed, supported and inspired me during this journey. Acknowledgements I would like to acknowledge my supervisor Dr Vincent Vadez from International Crops Research Institute of Semi-arid Tropics for his guidance, fatherly care and invaluable support throughout this study. Sincere thanks to Dr M V Rao, Research Supervisor, for his guidance and help at Bharathidasan University during this study. I am extremely grateful to Dr Neil Clifford Turner, University of Western Australia for his deep insights while writing each and every chapter and for making me to understand the results in much clear manner. My heartfelt thanks to Dr Mainassara Zaman Allah, Dr Rajeev Varshney, Dr Mahendar Thudi, Dr Pooran M Gaur, Dr L Krishnamurthy from ICRISAT and Dr Timothy Colmer, Dr Kadambot HM Siddique from UWA for their great support during various point of this study. Special thanks to Mr Jengiah, Mr Gafoor (Genotyping Service Lab) at ICRISAT and Mr John Quealy (UWA) for their technical assistance in salinity experiments. At this point of time, I would like to acknowledge the World Bank for IFAR grant, United Nations Food and Agriculture Organization, Crawford fund (Australia), Australia India strategic fund for their financial support to conduct this study and facilitating my attendance to international conferences. I like to extend my thanks to students and staffs at Crop Physiology Laboratory, staffs at Genomics service laboratory, library, and Controlled Environmental Facility for their support during this study. I would like to thank my friends Ms Krithika A, Mr Praneeth Kumar M, Dr Sunita Choudhary, Ms Deepa J, Dr Spurthi Nayak, Mr Purushothaman R, Ms Aparna Kakera, Mr Sivasakthi S, Ms Tharanya M, Ms Rajani T, Dr Abirami Ramalingam, Ms Vidhya Ramesh, Ms Roma Das, Ms Priyanka, Ms Usha, Ms Madhuparni Sen, Mr Lakshminarayana, Dr Jana Kholova, Mr Srikanth, Ms Dharani S, Ms Karthiga, Mr Habeeb Pasha, Ms Madina Diacombu, Mr Sanago, Ms Susan Medina Mary, Mr William Nelson, Mr Omar H, Mr Belko N, Ms Esti Leiber P, Ms Helene Marrou, Ms Francesca Beggi, Ms Esther, Ms Manasa, Dr Govindraj, Dr Vetriventhan, Dr Seetharaman, Mr Anand, Ms Ruth Wangari, Ms Clementine, Mr Seyni B, Ms Halime, Ms Rose, Ms Emilie, Mr Surendra Kumar Meena, Mr Bassiro, Mr Anirban, Mr Mathieu, Mr Simon, Mr Vikram, Mr Rakesh, Mr Bulama, Ms Mareme Belko, and all colleagues at Crop physiology lab, ICRISAT from bottom of my heart for their love, support, critics and sharing sorrow and joy with me during the tenure of this study. Thanks to Ms Rekha Baddam for her assistance in statistical analysis and Mr Prabhakar K for his support in administrative activities. Warm thanks to many Isha volunteers, my extended family at Hyderabad for their gentleness and hospitality towards me during my stay. My thanks to several temporary farm labourers who helped me while conducting this study. I wish to thank Dr Jayabalan N- Professor, Dr Viswanathan MB- Professor, Dr Ranjitha Kumari BD- Professor, Dr Lakshmi Prabha A- Associate Professor, Dr Sathiyabama M- Asst. Professor and Dr Sivakumar SR-Asst. Professor, Dept. of Plant Science, Bharathidasan University. My sincere thanks to Dr A. S. Rao, Professor, Bharathidasan University for his valuable comments and critical views that helped me in improving this thesis. I also would like to extend my thanks to Ms Revathi Lakshmi S, Mr Parthibhan S, Mr Ahamed Sherif N, Ms Senbagalakshmi P, Mr Muthukumar M, Mr Muthukrishnan S, Mr Thaniarasu R, Mr Baradwaj RG, Dr Sandhya Rani T, Mr Rakesh Kumar Verma, Mr Santhosh Kumar VV my lab mates for their warm support during my stay at Bharathidasan University. No words are enough to thank my family for their trust, care, support and understanding without which it is not easy for me complete this study. I bow down to Dhyanalinga and Linga Bhairavi for the endless grace on me. Place: Tiruchirappalli Date: DECLARATION I hereby declare that the work embodied in this thesis has been originally carried out by me under the guidance of Dr M V Rao, Honorary Professor, Department of Plant Science, Bharathidasan University, Tiruchirappalli- 620024, Tamil Nadu, India and co-guidance of Dr Vincent Vadez, Principal Investigator, Crop physiology laboratory, Assistant Director, Dryland Cereals, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, Telangana, India. I, further, assure that the work presented in this thesis, to the best of my knowledge and belief, is original except as acknowledged in the text. I declare that I have not submitted this material, either in whole or in part, for a degree, diploma, associateship or fellowship at this or any other institution. Place: Tiruchirappalli Signature of the candidate Date: (R Pushpavalli) CONTENTS Chapter No. Chapter Page No. Abstract 1 General Introduction 1 2 General Review of literature 5 3 Effect of Water stress on flowering and seed set 16 4 Effect of Salinity on phenology and yield parameters 37 5 Quantitative trait loci mapping for salinity tolerance 56 6 Effect of Atmospheric drought imposed by low relative 77 humidity on yield and pollen germination Summary and Conclusions 95 References Appendices ABBREVIATIONS -1 : Per % : Percentage < : Lesser than > : Greater than ABA : Abscisic Acid ADM : Aboveground Dry Matter ADP : Adenine Di Phosphate ANOVA : Analysis of Variance BGM : Botrytis Gray Mold BLAST : Basic Local Alignment Search Tool C. reticulatum L. : Cicer reticulatum L. Ca2+ : Calcium ion CaLG : Cicer arietinum Linkage Group CaM : Cicer arietinum Marker CEC : Cation Exchange Capacity CKAM : Kaspar Marker Cl- : Chloride ion cM : centi Morgan Corp. : Corporation DABS : Decolorized Aniline Blue Solution DAS : Days After Sowing DF : Days to Flower DM : Days to Maturity DTI : Drought Tolerance Index EC : Electrical Conductivity et al : Et alia (and others) Et : Ethylene FC : Field Capacity FTSW : Fraction of Transpirable Water G : Gram GO : Gene Ontology H2 : Heritability HI : Harvest Index i.e : That is ICCM : ICRISAT Chickpea Marker ICRISAT : International Crops Research Institute for the Semi-Arid Tropics ID : Identification IST : Indian Standard Time JA : Jasmonic Acid K+ : Potassium ion kg : Kilogram kPa : Kilo Pascal LSD : Least Significant Difference L : Litre M : Meter MAB : Marker Assisted Breeding Mha : Million Hectare mM : milliMolar mM : milliMeter Mt : Million tonnes Na+ : Sodium ion Na2PO4 : Sodium Di Phosphate NaCl : Sodium Chloride NTR : Normalized Transpiration Rate PCR : Polymerase Chain Reaction PVE : Phenotypic Variation Explained Px : Number of Pixels for a given Temperature Pxt : Total Number of Pixels for Range of Temperatures QTL : Quantitative Trait Loci RBD : Randomized Block Design RH : Relative Humidity RILs : Recombinant Inbred lines ROS : Reactive Oxygen Species S : Sensitive SNP : Single Nucleotide Polymorphism SPS : Sucrose Phosphate Synthase SS : Sucrose Synthase SSR : Simple Sequence Repeats t : Tonne T : Tolerant TILLING : Targeting Induced Local Lesions IN Genome Tr : Transpiration Rate TTSW : Total Transpirable Soil Water UK : United Kingdom UniProt KB : The UniProt Knowledge Base VPD : Vapor Pressure Deficit WS : Water Stressed wt. : Weight WW : Well Watered × : Cross / : Per Abstract Chickpea (Cicer arietinum L.), an important cool-season, food legume crop, is known to be sensitive to several abiotic stresses: drought, salinity and heat. The yield losses caused by these stresses are accounted to 6.4 million tonnes (t)/ year on global production. To improve any existing cultivar and harness the genetic regions involved in the tolerance it is important to understand the genetic and physiological mechanisms that underlie any tolerance. The objectives of this study were to (i) understanding the effect of either water deficit or salt stress on the reproductive biology of genotypes know to contrast for either salt or drought stress and (ii) construction of genetic map and identification of QTLs and candidate genes for salinity tolerance in 188 RILs derived from the ICCV 2 × JG 11 cross. In the water deficit study conducted in two consecutive years, ten genotypes with contrasting yields under terminal drought stress in the field were exposed to a gradual, but similar, water stress in the glasshouse. Nine parameters related