Soil salinization constitutes a pressing global concern, posing a significant threat to crop growth and food production (Zhai et al., 2023). Recent statistics reveal that approximately 23% of cultivated land worldwide is affected by soil salinization, with 1.1 billion hectares of global land area afflicted by this issue. China is not immune to this challenge, with a saline soil area encompassing 36.9 million hectares, a substantial 10% of the global saline soil extent, and accounting for 5% of China’s total available land (Zhao et al., 2022). China’s saline soil total area is 36.9 million hm², accounting for 10% of the global saline soil, accounting for 5% of the country’s available land area (Zhao et al., 2022). This issue manifests diversely across regions, including coastal saline soil and sea mud along the eastern coast, salt-affected soil in the Huang-Huai-Hai Plain, saline soil in the northeast plain, salt-impacted soil in the northwest inland areas, and desert salt soil in Qinghai and Xinjiang (Mao et al., 2020). In response to this critical concern, ensuring food security has prompted state initiatives aimed at optimizing available land resources through systematic planning of saline-alkali land, the selection of salt-tolerant crops for soil amelioration, and the preservation of cultivated land areas (Luan et al., 2023).

Soybean, a member of the legume family and the Papilionoideae stands as a pivotal cash crop, oilseed, and edible plant protein source in China. It also plays a crucial role as an industrial raw material (Li, 2011; Meng et al., 2021; Lu et al., 2023; Wang et al., 2023). Moreover, soybean holds a unique distinction as the cornerstone of Sino-U.S. agricultural trade relations, drawing significant attention from researchers. Because of saline land on soybean yield of serious damage to make our country’s soybean production. Therefore, we cultivate salt-tolerant high yield soybean varieties of this work is particularly important.

Up to now, 1536 QTLs associated with salt tolerance have been reported, generally located on chromosomes 2, 3, 6, 8, 9, 12, 13, 14, and 17.(SoyBase.org) Two different materials were used to locate the QTL of soybean salt tolerance, and one major QT of salt tolerance was detected on chromosome 3 (Hamwieh et al., 2011). A total of 21 QTLs were identified, including 4 QTLs related to relative germination rate, 8 QTLs related to relative imbibition rate, and 9 QTLs related to relative germination index (Teng et al., 2022). Based on the analysis of 549 soybean materials, 11 ERF genes were upregulated, among which the ERF158^H1, ERF166^H2, and ERF170^H1 haplotypes were excellent allelic variants, which significantly promoted soybean salt tolerance(Gao et al., 2023). In this study, 257 soybean cultivars with 135 SSR markers were used to perform epistatic association mapping for salt tolerance.A total of 83 QTL-by-environment (QE) interactions for salt tolerance index were detected (Zhang et al., 2014). In the study, a population of 184 recombinant inbred lines (RILs) was utilized to map quantitative trait loci (QTLs) related to salt tolerance. A major QTL related to salt tolerance at the soybean germination stage named qST-8 was closely linked with the marker Sat_162 and detected on chromosome 8 (Yu et al., 2019).

Genome-wide association studies (GWAS) have proven to be a potent tool for investigating complex quantitative traits (Huang et al., 2012). The rapid advancements in modern molecular biology technology have further bolstered the application of molecular marker techniques in various crop breeding domains, including wheat (Kun Dziayi Turhan et al., 2021), rice (He et al., 2023), maize (Ma et al., 2023), sorghum (Gao et al., 2023), rapeseed (Xiao et al., 2023), and other molecular breeding arenas. Soybean exhibits a multitude of complex quantitative traits, often under the control of multiple genes and influenced by both genotype and environmental factors (Kun Dziayi Turhan et al., 2021). Notably, Liang Tengyue et al. (Liang et al., 2023) conducted a genome-wide association analysis on 395 soybean germplasm resources using the GAPIT tool, identifying nine SNPs closely linked to single plant grain weight under low phosphorus conditions. Yang Hao et al. (Yang et al., 2023) conducted a study in the Sichuan-Chongqing region employing 135 SSR markers and 107,081 effective SNP markers for genotyping from 227 soybean varieties and detected 51 and 70 site significantly associated with fertility traits through comprehensive whole-genome association analysis. Compared with the seedling stage, research on the correlation analysis of salt tolerance at the germination stage of soybean has just begun.

The KASP (kompetitive allele-specific PCR) molecular marker is a new SNP typing method based on allele-specific amplification and high-sensitivity fluorescence detection. KASP is characterized by low cost and high throughput, and the accurate double-allele genotyping of SNP and InDel sites through specific matching of primer terminal bases. The method is widely used in molecular marker-assisted selection of rice, wheat, soybean, and other crops (Ertiro et al., 2015; Tian et al., 2018; Cheon et al., 2020; Jiang et al., 2021).

In this study, 283 soybean germplasm resources were used as materials. Under simulated NaCl salt stress, the germination rate, germination energy, and germination index at the germination stage were used as the screening parameters. The integration of genome-wide association analysis (GWAS) allowed us to identify pivotal site associated with soybean salt tolerance in a high-throughput manner. We further harnessed this knowledge to develop KASP markers, leveraging the significant association SNPs to facilitate early selection in the quest for salt-tolerant soybean breeding. This approach significantly alleviates the workload associated with soybean breeding efforts, expediting progress and advancing the field of soybean salt-tolerant molecular breeding. Our findings represent a valuable reference for future research on soybean salt tolerance and the selection and breeding of novel, salt-tolerant soybean varieties.

A natural soybean population containingused 283 representative soybean germplasms, including 52 landraces, 212 cultivars, and 19 wild soybeans, was used in this study. To identify an optimal stress concentration for evaluating salt tolerance in soybean germination, eight randomly selected varieties from our study’s test materials underwent a preliminary germination assay. Each variety was subjected to three replicate tests, with a concentration gradient spanning 0, 30, 60, 90, 120, 150, and 180 mM NaCl. Our test results revealed that at a concentration of 150 mmol/L NaCl, the germination rate and other measured parameters exhibited noticeable inhibition. Further increase of the NaCl concentration to 180 mM/L elicited significant disparities in the germination rate, germination energy, and germination index of the materials. Consequently, we identified 180 mmol/L NaCl as the optimal stress concentration for our subsequent experiments.

The germination test was carried out in a dedicated germination room, using a 3×4 grid layout of small squares, with each grid covered by 25 g vermiculite. 50 healthy, full, and pest-free seeds of the same size were used for each condition. 90 ml 0mM and 180 mM Nacl solutions were used for the stress treatment. The seeds were spread on the vermiculite and watered with the treatment solution then covered with 3-4 layers of filter paper soaked with treatment solution. The count of germination seeds was recorded every 24 hours for 7-8 days. This protocol was conducted with three biological replicates per material. Utilizing an established formula, the relative salt damage rate for each germination parameter, including germination rate, germination energy, and germination index, was calculated.

Several key parameters were calculated to assess soybean germination under salt stress conditions, providing insights into salt tolerance:

Germination Rate (GR): The germination rate, expressed as a percentage, was calculated using the formula: GR (%) = (Nt/N) × 100, where Nt represents the number of seeds germinated per grid on day t, and N represents the total number of seeds per grid for testing (unit: %) (Wang et al., 2019).

Germination Index (GI): The germination index was determined using the formula: GI = ∑Gt/Dt, where Gt represents the number of seeds germinated per grid on day t, and Dt signifies the number of days in the germination test up to day (Wang et al., 2019).

Germination Energy (GE): Germination energy, also expressed as a percentage, was calculated as GE (%) = N3/N × 100, where N3 represents the number of seeds germinated per grid on the 3rd day, and N represents the total number of seeds per grid for testing (unit: %) (Wang et al., 2019).

Relative Salt Damage Index (ST): The relative salt damage index was derived using the formula: ST = S/C. Here, C represents the control germination rate, germination index, and germination energy, while S signifies the germination rate, germination index, and germination energy under salt treatment (Wang et al., 2019).

Generalized heritability h 2 = σ g 2 / ( σ g 2 + σ g e 2 n + σ 2 / n r ) , where σ g 2   ( g = 1 , 2 , 3…264 ) is the genotype variance of the test material, σ g e 2   ( e = 1 , 2 ) is the variance of the interaction between the genotype and the environment of the test material, σ 2 is the error variance, n is the number of environments, and r is the number of replicates(Yuan et al., 2023).

The research and development of salt-tolerant soybeans for saline-alkali soybean production and the expansion of planting areas through various strategies are pivotal steps in addressing the issue of insufficient soybean production capacity in China. These efforts bear significance for China’s food security. Several studies have contributed to our understanding of salt tolerance mechanisms during soybean germination, shedding light on the physiological changes that occur under salt stress. Hao Xuefeng et al. examined salt tolerance and the salt tolerance mechanism in soybean seeds during germination and found changes in parameters such as SOD, POD, and MDA in the radicle germ in response to increasing NaCl concentrations. This research underscores the existence of specific salt tolerance mechanisms and associated physiological changes during germination (Hao et al., 2013).

Some studies have identified the impact of salt stress on organelle formation, including chloroplasts and endoplasmic reticulum, resulting in varying degrees of influence on growth traits such as root length, hypocotyl length, and lateral root numbers. Ultimately, this process inhibited soybean germination (Liao et al., 2013). demonstrated that high-concentration NaCl stress significantly impeded water absorption in soybean seeds, leading to reduced amylase and protease activity, further elucidating the complexities of salt stress on germination (Xu et al., 2017). Kan found that 22 SSR markers and 11 related QTL sites were closely linked to salt tolerance in soybean germination, and localized on chromosomes 2, 7, 8, 10, 17, and 18 (Kan et al., 2016). research identified a total of 31 salt-tolerant-related QTLs through linkage analysis of ST-IR, ST-GI, ST-GE and ST-GR during the germination stage of an NJIKY population, mainly distributed on chromosomes 1, 2, 7, 8, 10, 15, 17 and 18 (Zhang et al., 2018). Furthermore, Kan used a natural population of 191 local soybean varieties and 1356 SNP markers to perform genome-wide association analysis. Their work identified five candidate genes closely linked to salt tolerance during soybean germination (Kan et al., 2015).

In our pursuit of identifying outstanding salt-tolerant soybean varieties and enhancing soybean yield, this study conducted a comprehensive analysis of germination traits, including germination rate, germination energy, germination index, and their relative values, across a diverse set of 283 soybean germplasm resources subjected to salt stress at the germination stage. Our investigation revealed that the germination traits within this population exhibited a rich and continuous distribution. At the same time, using the high-density SNP physical map combined with phenotype and genotype data for genome-wide association analysis, a total of 1841 SNP sites significantly associated with soybean germination stage were detected on chromosomes 2, 5, 6, 9, and 20. Notably, the loci located on chromosome 5 were repeatedly detected in 2 environments, and the genetic variation explainable by the GWAS signal reached 14.00%, marking it as a prominent genetic locus. MAP kinase 9 may be an effector gene for this site.In the same chromosomal interval as the results of other researchers, there may be allelic variation of the same QTL.Chromosomes 2 and 20 have also been confirmed by previous studies (Zhang, 2014), and the sites associated with chromosome 6, 9 are two new research intervals, which are of great significance for future studies, The related genes of this site should be explored and studied.

Furthermore, sequence comparison of genes within the remaining three significant correlation sites allowed us to predict 12 candidate genes closely linked to the regulation of salt tolerance during the germination stage of soybeans. These candidate genes play roles in the coordination of cellular responses, the regulation of osmotic stress, the attenuation of oxidative stress, the clearance of reactive oxygen species (ROS), and the management of heavy metal ion transport. Collectively, these genes are vital components in plant development, stress tolerance, and the maintenance of normal growth, immune response, and tolerance to abiotic and biotic stresses.

Our findings contribute valuable genetic resources and a solid theoretical foundation for the breeding of salt-tolerant soybeans. They represent a critical step towards addressing the challenges of saline-alkali soybean production and increasing soybean yield, thereby bolstering food security.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

WJ: Writing – original draft, Writing – review & editing. MZ: Writing – review & editing. HZ: Writing – review & editing. XL: Writing – review & editing. WZ: Writing – review & editing. QW: Writing – review & editing. JQ: Writing – review & editing. DX: Writing – review & editing. HC: Writing – review & editing. CS: Writing – review & editing.