Citation: Asif, S.; Kim, E.-G.; Jang, Y.-H.; Jan, R.; Kim, N.; Asaf, S.; Lubna; Farooq, M.; Kim, K.-M. Identification of the OsCML4 Gene in Rice Related to Salt Stress Using QTL Analysis. Plants 2022, 11, 2467. https://doi.org/10.3390/ plants11192467 Academic Editor: Fengxia Liu Received: 19 August 2022 Accepted: 19 September 2022 Published: 21 September 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). plants Article Identification of the OsCML4 Gene in Rice Related to Salt Stress Using QTL Analysis Saleem Asif 1,† , Eun-Gyeong Kim 1,† , Yoon-Hee Jang 1,† , Rahmatullah Jan 1,2 , Nari Kim 1 , Sajjad Asaf 3 , Lubna 4 , Muhammad Farooq 1 and Kyung-Min Kim 1,2, * 1 Department of Applied Biosciences, Graduate School, Kyungpook National University, Daegu 41566, Korea 2 Coastal Agriculture Research Institute, Kyungpook National University, Daegu 41566, Korea 3 Natural and Medical Science Research Center, University of Nizwa, Nizwa 616, Oman 4 Department of Botany, Garden Campus, Abdul Wali Khan University, Mardan 23200, Pakistan * Correspondence: kkm@knu.ac.kr † These authors contributed equally to this work. Abstract: Soil salinity is a major abiotic stress that causes disastrous losses in crop yields. To identify favorable alleles that enhance the salinity resistance of rice (Oryza sativa L.) crops, a set of 120 Cheongcheong Nagdong double haploid (CNDH) lines derived from a cross between the Indica variety Cheongcheong and the Japonica variety Nagdong were used. A total of 23 QTLs for 8 different traits related to salinity resistance on chromosomes 1–3 and 5–12 were identified at the seedling stage. A QTL related to the salt injury score (SIS), qSIS-3b, had an LOD score of six within the interval RM3525–RM15904 on chromosome 3, and a phenotypic variation of 31% was further examined for the candidate genes. Among all the CNDH populations, five resistant lines (CNDH 27, CNDH 34-1, CNDH 64, CNDH 78, and CNDH 112), five susceptible lines (CNDH 52-1, CNDH 67, CNDH 69, CNDH 109, and CNDH 110), and the parent lines Cheongcheong and Nagdong were selected for relative gene expression analysis. Among all the genes, two candidate genes were highly upregulated in resistant lines, including the auxin-responsive protein IAA13 (Os03g0742900) and the calmodulin-like protein 4 (Os03g0743500-1). The calmodulin-like protein 4 (Os03g0743500-1) showed a higher expression in all the resistant lines than in the susceptible lines and a high similarity with other species in sequence alignment and phylogenetic tree, and it also showed a protein–protein interaction with other important proteins. The genes identified in our study will provide new genetic resources for improving salt resistance in rice using molecular breeding strategies in the future. Keywords: QTL; salinity; Cheongcheong Nagdong double haploid; calmodulin-like protein; rice seedling 1. Introduction Rice (Oryza sativa L.) is one of the most important cereal crops and is a basic component of diet and source of energy for more than 2.7 billion people on a daily basis [1]. It is a semiaquatic crop and a major source of food among cereal crops for more than one-half of the world’s population [2]. Besides its economic importance, it is rich in genetic diversity in the form of thousands of landraces and progenitor species. Various biotic and abiotic stresses limit its products worldwide, among which abiotic stress alone causes more than 50% of the total yield losses [3]. Soil salinity is the critical abiotic stress that limits rice production and is a major problem for rice-based farming systems in many rice-producing areas of the world [3]. At low salinity concentrations, yields are slightly changing—or not at all—but at high concentrations, yields can become zero and even sometimes result in plant death [4]. Approximately 20% of irrigated and 8% of non-irrigated agricultural land is affected by salinity [5]. It has been estimated that by 2050, salinity will affect more than 50% of arable land [6] for various reasons, including low precipitation, high surface evaporation, weathering of native rocks, irrigation with saline water, and poor cultural practices. All soils contain salts, and all irrigation waters, whether from canals or underground pumping, Plants 2022, 11, 2467. https://doi.org/10.3390/plants11192467 https://www.mdpi.com/journal/plants