Genetic analysis and QTL mapping of seed hardness trait in a soybean (Glycine max) recombinant inbred line (RIL) population

•Genetic basis of soybean seed hardness explored in a diverse RIL population.•Six QTLs identified, with qHS-2-1 consistently dominant in multiple years and locations.•Four candidate genes within qHS-2-1 interval revealed, including Glyma.02G307000.•GO enrichment analysis unveils functions related to calcium binding and pectin lyase activity.•Expression differences in candidate genes between parent varieties underscore their role. Seed hardness is a critical quality trait impacting both the suitability of soybeans for consumption and their processing. The primary objective of this study was to explore the genetic foundations underlying seed hardness in soybeans. A 234 recombinant inbred line (RIL) population was evaluated for seed hardness across three years (2015 in Gansu, 2016, and 2017 in Hainan). Notably, the parent varieties, Zhonghuang35 and Jindou21, displayed significant differences in seed hardness. Also, the RIL population exhibited a wide range of genetic variation in seed hardness, with coefficients of variation between 70.53 % and 94.94 %. The frequency distribution of this trait conformed to a relatively normal distribution, making it suitable for QTL analysis. Six QTLs associated with seed hardness were identified with three located on chromosome 2 and three on chromosome 16. The major QTL, qHS-2-1, consistently exhibited the highest percentage of PVE and LOD in Gansu 2015, Hainan 2016, and Hainan 2017, suggesting its central role in determining seed hardness. Further investigation revealed four genes within the qHS-2-1 interval potentially related to seed hardness. GO enrichment analysis provided insights into their functions, including factors such as Glyma.02G307000, a pectin lyase-like superfamily protein, which could influence seed hardness through its role in pectin lyase enzyme activity. Expression analysis of these candidate genes demonstrated significant differences between the two parent varieties, further highlighting their potential role in seed coat hardness. This study offers valuable insights into the genetic mechanisms governing soybean seed coat hardness, providing a foundation for future research and crop improvement efforts.