Genome wide association studies reveal candidate genes for salt tolerance in safflower (Carthamus tinctorius L.) at seedling stage

Fawad Ali¹Obaid Ullah Shah²Muhammad Azhar Nadeem³Muhammad Tanveer Altaf^4*Arif Ali⁵Mian Arif⁶Emre Aksoy⁷Faheem Shehzad Baloch⁸

• ¹Hainan University, Haikou, China
• ²Shanghai Jiao Tong University, Shanghai, China
• ³Mersin University, Mersin, Türkiye
• ⁴Recep Tayyip Erdoğan University, Pazar, Türkiye
• ⁵Quaid-I-Azam University, Islamabad, Pakistan
• ⁶Pakistan Institute of Engineering and Applied Sciences, Faisalabad, Pakistan
• ⁷Middle East Technical University, Ankara, Türkiye
• ⁸Jeju National University, Jeju, Republic of Korea

Safflower productivity is hindered by soil salinity, making the identification of genetic markers essential for breeding resilient cultivars. Despite the substantial yield losses caused by salt stress, research on parental genotypes and candidate genes associated with salt tolerance remains limited. A pot experiment with 94 safflower genotypes exposed to four sodium chloride (NaCl) concentrations at the seedling stage explored salt tolerance genetics. Results showed significant variability among genotypes, NaCl treatments, and their interactions for most traits, except biological yield (BY) and fresh shoot weight (FSW). Traits showed reductions from 8% (number of leaves) to 76% (dry root weight) under NaCl stress. Broad sense heritability ranged from 17% to 97%. Correlation analysis revealed positive associations among traits, except FSW and BY. PCA grouped genotypes into three distinct clusters. Using stress tolerance indices (> 0.65) and superior performance above the population mean, three top-performing safflower genotypes were identified. A genome-wide association study (GWAS) revealed 322 marker-trait associations (MTAs), distributed as follows: 34 for BY, 25 for dry root weight (DRW), 44 for dry shoot weight (DSW), 48 for fresh root weight (FRW), 46 for FSW, 60 for number of leaves (NL), 47 for plant height (PH), and 18 for root length (RL). Gene annotation revealed key candidates influencing salinity tolerance, including PLA1, APK4, GINT1, TPLATE, UL13M, SPP2, FRF3, AT1G33770, AT5G01610, DTX50, and RAF1. These genes regulate sulfation of secondary metabolites, chloroplast development, site-specific cell wall modifications, sucrose biosynthesis, and calcium signaling, as well as the functions of hypothetical proteins or proteins with unknown roles. Validating these candidate genes, in silico transcriptomics showed significant upregulation of PLA1, SPS2, and DTX50, alongside downregulation of APK4, GINT1, TPLATE, UL13M, FRF3, AT1G33770, AT5G01610, and RAF1 under salinity. These findings highlight the top-performing genotypes for salt-tolerant cultivar development and warrant further functional studies on the identified candidate genes to gain a deeper understanding of their mechanisms under salt stress.