Screening of heat-tolerant germplasm and SV-GWAS analysis in watermelon at germination stage

【Objective】As global temperatures continue to rise, heat stress has become a primary environmental factor limiting sustainable crop production.. Seed germination, the initial stage of the plant life cycle, is highly sensitive to elevated temperatures. Its performance directly determines field emergence uniformity and early seedling establishment, ultimately impacting yield and quality. However, systematic research on heat tolerance at this critical stage remains insufficient, and the genetic loci regulating watermelon seed germination under heat stress are still unclear. This study aimed to establish an efficient evaluation and screening system for heat tolerance during watermelon germination, to identify superior heat-tolerant germplasm resources, and to pinpoint key genetic loci through Structural Variation- based Genome-Wide Association Study (SV- GWAS), thereby elucidating the genetic regulatory mechanisms of heat tolerance and providing a theoretical foundation and genetic resources for breeding heat-tolerant watermelon varieties.【Methods】Initially, to determine the optimal stress temperature for evaluating germination- stage heat tolerance, a temperature gradient using 33 ℃ as control and 42 ℃ and 45 ℃ as stress treatments was established. Based on the germination performance of a heat-tolerant line (8R098) and a heat-sensitive line (8R122) under these gradients and in accordance with existing lit-erature, 42 ℃ was selected as the standard stress temperature. Subsequently, 90 modern cultivated watermelon core accessions were selected; seeds from each accession were soaked in room-temperature distilled water for 24 h, then divided into a control group (5 seeds, 33 ℃ ) and a treatment group (15 seeds as 3 biological replicates, 42 ℃ ). Germination assays were conducted in an artificial climate chamber (16 h light/8 h dark, 70% relative humidity) using 90 mm Petri dishes lined with moist filter paper. Germination was defined as radicle emergence ≥1 mm. A multi-index evaluation system comprising Germination Percentage (GP), Germination Potential (GE), Germination Index (GI), Relative Germination Percentage (RGP), and Relative Germination Potential (RGE) was constructed. Correlation analysis and hierarchical clustering were performed for phenotypic evaluation and heat-tolerant germplasm screening. Finally, leveraging a previously constructed whole-genome SV map (30× sequencing depth) and the phenotypic data, a GWAS was conducted using the GAPIT package with four models (GLM, FarmCPU, BLINK, and MLMM), incorporating PCA and kinship matrices to control population structure. A stringent Bonferroni-corrected genome-wide significance threshold of -log10(P) ≥ 5 was applied to systematically identify SV loci significantly associated with heat tolerance during watermelon seed germination.【Results】Experimental validation confirmed 42 ℃ as the optimal stress temperature. Correlation analysis revealed highly significant positive correlations (r ＞ 0.966) among all evaluated indices, confirming their reliability for assessing germination-stage heat tolerance. Following data standardization, the optimal cluster number was determined to be three based on the silhouette coefficient and elbow method. K-means clustering clearly partitioned 88 accessions (two accessions with invalid RGP/RGE were excluded) into three phenotypically distinct groups: highly heat-tolerant (10 accessions), moderately heat-tolerant (10), and heat-sensitive (68), with extremely significant differences observed across all measured indicators among the groups. A comprehensive evaluation ultimately identified 11 elite heat-tolerant core accessions (8R001, 8R003, 8R005, 8R009, 8R015, 8R019, 8R097, 8R098, 8R099, 8R102, and 8R109). GWAS analysis identified a total of 55 SV loci significantly associated with heat tolerance at the genome-wide level, including 19 loci for GP, 20 for GE, 29 for GI, 10 for RGP, and 15 for RGE. The association signals were dispersed without major peaks, indicating that germination-stage heat tolerance was governed by multiple minor-effect loci. Notably, 55 high-confidence SV loci (e. g., SV5589, SV1590, SV7404, and SV7775) were reproducibly detected across multiple statistical models and traits, demonstrating robust and reliable associations. To further explore the functional relevance of these loci, we performed BLASTx annotation on all 55 significant SV loci. Among them, 12 loci yielded significant hits in the non- redundant protein database. Through BLASTp-based homology searches against Arabidopsis and rice protein databases combined with extensive literature validation, we successfully identified four high-confidence candidate genes located at or adjacent to four representative SV loci (SV1590, SV5589, SV7404, SV7775). These genes encode: (1) an HD-ZIP Ⅲ transcription factor (Cla97C02G047220) harboring SV1590 in its intron, which exhibits homology with Arabidopsis REVOLUTA (REV, At5G60690), a core component of the miR165/166-mediated heat tolerance pathway; (2) a PPR protein (Cla97C08G153120) with SV5589 located downstream, showing high homology to a rice PPR protein (XP_015632183. 1) closely related to the functionally validated heat tolerance gene OsPPR8; (3) an HSP20 chaperone (Cla97C10G198670) with SV7404 downstream, sharing homology with Arabidopsis AT5G51440 (HSP23. 5-M), a member of the small heat shock protein family essential for thermotolerance; and (4) a DEAD-box RNA helicase (Cla97C11G214970) carrying SV7775 in its intron, displaying 68. 53% identity with Arabidopsis RH20 (AT1G35530), whose family members LOS4 and STRS1/STRS2 have been experimentally demonstrated to participate in heat stress responses. The orthologs of all four candidate genes in model plants have been repeatedly validated as functional regulators of heat tolerance, involving diverse mechanisms such as small RNA-mediated transcriptional reprogramming, mitochondrial RNA splicing, protein chaperone activity, and RNA metabolism.【Conclusion】This study successfully established a rapid, germination-stage-specific heat tolerance evaluation system using 42 ℃ as the optimal stress temperature and identified 11 promising heat-tolerant core accessions that can be directly utilized as elite parents in breeding programs. Through comprehensive SV-GWAS, we systematically dissected the polygenic genetic architecture underlying this trait and identified 55 significant SV loci. Most importantly, by integrating functional annotation, comparative genomics, and literature-based validation, we pinpointed four high-confidence candidate genes with well-documented roles in heat stress responses in model plants. These findings not only provide an efficient screening method and valuable germplasm resources for heat-tolerance breeding but also deliver prioritized gene targets for subsequent functional characterization and marker-assisted selection. The research outcomes lay a solid theoretical and material foundation for unraveling the molecular mechanisms of watermelon heat tolerance and for its genetic improvement through modern breeding strategies.