Flooding stress poses a significant challenge to soybean cultivation, impacting plant growth, development, and ultimately yield. In this study, we investigated the responses of two distinct soybean cultivars: flooding-tolerant Nanxiadou 38 (ND38) and flooding-sensitive Nanxiadou 45 (ND45). To achieve this, healthy seedlings were cultivated with the water surface consistently maintained at 5 cm above the soil surface. Our objective was to elucidate the physiological and molecular adaptations of the two cultivars. Under flooding stress, seedlings of both cultivars exhibited significant dwarfing and a notable decrease in root length. While there were no significant differences in the dry weight of aboveground shoots, the dry weight of underground shoots in ND38 was strikingly decreased following flooding. Additionally, total chlorophyll content decreased significantly following flooding stress, indicating impaired photosynthetic performance of the cultivars. Moreover, malondialdehyde (MDA) levels increased significantly after flooding, particularly in the ND45 cultivar, suggesting heightened oxidative stress. Expression analysis of methylation and demethylation genes indicated that MET1 and DME play crucial roles in response to flooding stress in soybeans. Meanwhile, analysis of the hemoglobin family (GLBs), aquaporin family (AQPs), glycolytic pathway-related genes, and NAC transcription factor-related genes identified GLB1-1 and GLB1-2, GLB2-2, PIP2-6, PIP2-7, TIP2-2, TIP4-1, TIP5-1, Gm02G222400 (fructose-bisphosphate aldolase), Gm19G017200 (glucose-6-phosphate isomerase), and Gm04G213900 (alcohol dehydrogenase 1) as key contributors to flooding tolerance in both soybean cultivars. These findings provide crucial insights into the physiological and molecular mechanisms underlying flooding tolerance in soybeans, which could guide future molecular breeding strategies for the development of flooding-tolerant soybean cultivars.

Stem lodging significantly reduces soybean yield stability, particularly under dense planting and intercropping systems. Stem breaking strength is a key component of lodging resistance, but its genetic basis remains incompletely understood. In this study, an F₂ population consisting of 167 individuals derived from a cross between nanxiadou25 (NXD25, high stem breaking strength) and Shiyuehuang (SYH, low stem breaking strength) was analyzed using bulked segregant analysis with whole-genome resequencing (BSA-Seq) to identify loci associated with stem breaking strength. The trait showed broad quantitative variation in the F₂ population, ranging from 20.1 to 673.7 N. Two extreme bulks were constructed using 30 plants with the highest values and 30 plants with the lowest values. QTL-seq detected 21 candidate intervals at the 95% confidence level, among which, three major loci on Chr07, Chr13, and Chr16 exceeded the 99% threshold and were designated qBR7.2, qBR13.1, and qBR16.1. By integrating large-effect SNP/InDel variation, marker development, RNA-seq profiling, and qRT-PCR validation, nine candidate genes were retained for further study, and three marker-linked genes were highlighted as high-priority candidates. RNA-seq identified 9617 differentially expressed genes between the two parents. In addition, three co-dominant InDel markers, Chr07_01, Chr13_17, and Chr16_83, showed phenotype-consistent polymorphism in extreme F₂ individuals. These findings provide valuable loci, candidate genes, and molecular markers for soybean lodging-resistance breeding.