ORIGINAL ARTICLE Genome‐wide association analysis of salinity responsive traits in Medicago truncatula Yun Kang 1 | Ivone Torres‐Jerez 1 | Zewei An 2 | Veronica Greve 3 | David Huhman 1 | Nicholas Krom 1 | Yuehua Cui 4 | Michael Udvardi 1 1 Noble Research Institute, Ardmore, Oklahoma 73401 2 State Center for Rubber Breeding and Rubber Research Institute, Danzhou, Hainan 571700, China 3 College of Biological Sciences, University of Minnesota, Huntsville, Alabama 35806 4 Department of Statistics and Probability, Michigan State University, East Lansing, Michigan 48824 Correspondence Michael Udvardi, Noble Research Institute, Ardmore, OK 73401. Email: mudvardi@noble.org Present Address Veronica Greve, Hudson Alpha Institute for Biotechnology, Huntsville, Alabama 35806. Funding information The Samuel Roberts Noble Foundation Abstract Salinity stress is an important cause of crop yield loss in many parts of the world. Here, we performed genome‐wide association studies of salinity‐stress responsive traits in 132 HapMap genotypes of the model legume Medicago truncatula. Plants grown in soil were sub- jected to a step‐wise increase in NaCl concentration, from 0 through 0.5% and 1.0% to 1.5%, and the following traits were measured: vigor, shoot biomass, shoot water content, leaf chlo- rophyll content, leaf size, and leaf and root concentrations of proline and major ions (Na + , Cl - , K + , Ca 2+ , etc.). Genome‐wide association studies were carried out using 2.5 million single nucleotide polymorphisms, and 12 genomic regions associated with at least four traits each were identified. Transcript‐level analysis of the top eight candidate genes in five extreme genotypes revealed association between salinity tolerance and transcript‐level changes for seven of the genes, encoding a vacuolar H + ‐ATPase, two transcription factors, two proteins involved in vesicle trafficking, one peroxidase, and a protein of unknown function. Earlier functional studies on putative orthologues of two of the top eight genes (a vacuolar H + ‐ ATPase and a peroxidase) demonstrated their involvement in plant salinity tolerance. KEYWORDS GWAS, legume, Medicago truncatula, proline, salinity, SNP, vesicle trafficking 1 | INTRODUCTION Salinity is an important abiotic stress that restricts crop distribution and reduces agricultural yield. It is estimated that over 6% of the world's total land area is affected by excess salts (Smajgl et al., 2015), and approximately 20% of arable land in more than 100 countries is affected by salinity (Sairam & Tyagi, 2004). Increasing salinity tolerance in crops will help to ensure food, feed, and industrial feedstock production on salt‐affected land. The fundamental mechanisms of how plants sense and respond to salinity stress in both glycophytes and halophytes have been studied extensively, but remain incompletely understood. Multiple transporters and channels such as the Na + /H + antiporter SOS1, the Na + /H + exchanger NHX, the high affinity potassium transporter HKT1, as well as nonselective cation channels, have been shown to play important roles in maintaining cellular and plant‐level ion homeostasis under salinity stress (Julkowska & Testerink, 2015; Keisham, Mukherjee, & Bhatla, 2018). In addition, important genes involved in the signal transduction pathways that respond to salinity have been identified, including calcium‐dependent protein kinases (CDPKs), calcineurin B‐like proteins (CBLs), CBL‐interacting protein kinases (CIPKs), and mitogen‐activated protein kinases (MAPKs) (Shabala, Wu, & Bose, 2015). To minimize the ionic stress caused by Na + and Cl - , cells exclude and/or remove these ions from their cytoplasm via transporters, which can result in osmotic stress. To alleviate such stress, plant cells synthesize compatible solutes such as proline, glycine betaine, and soluble sugars that help them to retain water when ion levels in the apoplast or -------------------------------------------------------------------------------------------------------------------------------- This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2018 The Authors. Plant, Cell & Environment Published by John Wiley & Sons Ltd Received: 12 July 2018 Accepted: 16 December 2018 DOI: 10.1111/pce.13508 Plant Cell Environ. 2019;42:1513–1531. wileyonlinelibrary.com/journal/pce 1513