Evaluation and Association Analysis of Cowpea Salt Tolerance Waltram Second Ravelombola University of Arkansas, Fayetteville
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University of Arkansas, Fayetteville ScholarWorks@UARK Theses and Dissertations 5-2017 Evaluation and Association Analysis of Cowpea Salt Tolerance Waltram Second Ravelombola University of Arkansas, Fayetteville Follow this and additional works at: http://scholarworks.uark.edu/etd Part of the Agronomy and Crop Sciences Commons, Horticulture Commons, and the Plant Biology Commons Recommended Citation Ravelombola, Waltram Second, "Evaluation and Association Analysis of Cowpea Salt Tolerance" (2017). Theses and Dissertations. 1966. http://scholarworks.uark.edu/etd/1966 This Thesis is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of ScholarWorks@UARK. For more information, please contact [email protected], [email protected]. Evaluation and Association Analysis of Cowpea Salt Tolerance A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Cell and Molecular Biology by Waltram Second Ravelombola University of Antananarivo Master of Science in Agricultural Engineering, 2013 May 2017 University of Arkansas This thesis is approved for recommendation to the Graduate Council Dr. Ainong Shi Thesis Director Dr. John Reuben Clark Dr. Pengyin Chen Committee member Committee member Dr. Vibha Srivastava Committee member Abstract Cowpea [Vigna unguiculata (L.) Walp.] (2n=2x=22) is a leguminous crop providing inexpensive protein for human consumption that can be grown worldwide. Salinity is one of the major threats to cowpea production, particularly in semi-arid regions of Africa. Salinity significantly affects seed germination and decreases pod yield in cowpea. However, little has been done to provide farmers salt-tolerant cowpea cultivars. Knowledge on the genetics of cowpea salt tolerance is very limited. This study aimed to:(1) evaluate cowpea salt tolerance at germination stage, (2) evaluate cowpea salt tolerance at seedling stage, (3) conduct an association study for cowpea salt tolerance at germination and seedling stages and to identify SNP markers associated with salt tolerance in cowpea. A total of 151 cowpea genotypes at germination stage and 203 cowpea genotypes at seedling stage were evaluated in this research. Association analysis was performed on 116 genotypes at germination stage and 155 genotypes at seedling stage. The results indicated that: (1) substantial variability in salt tolerance was found among the tested cowpea genotypes at both germination and seedling stages; (2) three SNPs, Scaffold87490_622, Scaffold87490_630, and C35017374_128 were highly associated with salt tolerance at germination stage; (3) seven SNPs Scaffold93827_270, Scaffold68489_600, Scaffold87490_633, Scaffold87490_640, Scaffold82042_3387, C35069468_1916, and Scaffold93942_1089 were found to be associated with salt tolerance at seedling stage, and (4) PI582422, 09-529, PI293584, and PI582570 were highly salt tolerant at germination stage, and PI293570, PI582812, PI582856, PI180014, PI257463, 09-175, 09-529, PI666260, I582402, and PI582340 were highly salt tolerant at seedling stage. This research will have practical applications in cowpea breeding and genetics. The salt tolerant lines could be used as parents for breeding programs and the SNP markers could be used as a tools in cowpea molecular breeding through marker-assisted selection. ©2017 by Waltram Second Ravelombola All Rights Reserved Acknowledgments I would like to thank the Fulbright Program for providing me a scholarship, which allows me to complete a Master of Science in Cell and Molecular Biology. I am also grateful to Drs. Ainong Shi and Yuejin Weng for their precious help, guidance, and full support not only for academic and research purposes, but also for social skills. I would like also to thank my committee members, Dr. John Reuben Clark, Dr. Pengyin Chen, and Dr. Vibha Srivastava for their suggestions, and academic and research support. I am also grateful to Douglas Rhoads (Head of CEMB Program) and Dr. Wayne Mackay (Head of the Horticulture Department) for letting me participate in their programs. I would like also to thank all the lab members of the Vegetable Breeding and Genetics Program of the University of Arkansas. I would like also to thank my dad, my mom, and my sister for their full support, assistance, and unconditional love. Dedication This thesis is dedicated to: My dad: Second Modeste Velombola My mom: Francine Razanamalala My sister: Francia Seconde Ravelombola Table of Contents Chapter 1. Introduction................................................................................................................ 1 Cowpea ........................................................................................................................................... 1 Effects of salinity on cowpea .......................................................................................................... 2 Major mechanisms of salt tolerance in plant .................................................................................. 3 Ion selectivity .............................................................................................................................. 3 Ion accumulation ......................................................................................................................... 4 Osmotic adjustment ..................................................................................................................... 4 Organic solutes ............................................................................................................................ 5 Growth regulation ....................................................................................................................... 5 Genetics and breeding for salt tolerance ......................................................................................... 5 Molecular breeding in plant ............................................................................................................ 6 DNA marker technology and next generation sequencing .......................................................... 6 QTL and association mapping..................................................................................................... 8 Rationale and significance ............................................................................................................ 11 References ..................................................................................................................................... 12 Chapter 2. Evaluation of Salt Tolerance at Germination Stage in Cowpea (Vigna unguiculata (L.) Walp)................................................................................................................ 18 Abstract ......................................................................................................................................... 18 Introduction ................................................................................................................................... 19 Materials and Methods .................................................................................................................. 22 Plant Materials ........................................................................................................................... 22 Determination of optimal seed germination temperature and salt concentration ...................... 22 Germination conditions ............................................................................................................. 23 Measurements ............................................................................................................................ 23 Experimental design .................................................................................................................. 24 Data analysis ............................................................................................................................. 25 Results and Discussion ................................................................................................................. 26 Optimal concentration for the assessment of cowpea salt tolerance at germination stage ....... 26 Germination at non-salt stress and salt stress conditions .......................................................... 27 Absolute decrease and inhibition index .................................................................................... 28 Relative salt tolerance and salt tolerance index ........................................................................ 29 Analysis by geographical location ............................................................................................ 30 Broad sense heritability ............................................................................................................. 31 Correlation between the parameters .......................................................................................... 31 Cluster analysis ......................................................................................................................... 32 Conclusion .................................................................................................................................... 32 References ..................................................................................................................................... 33 Chapter 3. Evaluation of Salt tolerance at Seedling Stage