Stable loci and functional gene controlling pod shattering resistance across multiple environments under different genetic backgrounds in soybean.

Soybean is a globally important grain and oil crop, yet its production cannot meet the increasing demand. While pod shattering is a key domestication trait essential for seed dispersal in wild soybean, it has become a major constraint that severely limits yield improvement and mechanized harvesting in cultivated varieties. Consequently, discovering the stable genetic loci and causal genes underlying pod shattering resistance is of growing importance for modern agriculture production. In this study, the pod shattering resistance of 284 accessions was evaluated in three environments, and 1,715 stable associated SNPs and 132 InDels were consistently associated with the trait. To reduce the false-positive signals in GWAS, these associated variations were further validated in another natural population containing 539 accessions. The result showed that 98.54% (1,690/1,715) of the SNPs and 99.24% (131/132) of the InDels were successfully validated, indicating that these loci contribute to pod shattering resistance across diverse genetic backgrounds. Additionally, a causal gene, GmLRR16, showing significant expression differences between the sensitive and resistant varieties at five pod developmental stages, was identified within the verified stable genetic locus GmLRR16 was mainly expressed in pod. While its overexpression significantly decreased pod shattering resistance, its knockout or EMS‑induced mutation significantly increased resistance, confirming that GmLRR16 acts as a negative regulator of pod shattering resistance. The negative regulation mechanism of GmLRR16 was further dissected through its interaction with a previously reported negative regulation factor Pdh1. Moreover, 21 hydrogen bonds, seven salt bridges and a negative binding free energy were detected in GmLRR16-Pdh1 complex, suggesting that this interaction is a thermodynamically spontaneous binding process. Natural variation analysis of GmLRR16 identified a SNP (A-resistant/T-sensitive) in its promoter region strongly associated pod shattering characteristics. The frequency of the sensitive allele (T) was 61% in wild soybean but only 21% in cultivated soybean, suggesting that GmLRR16 may have undergone artificial selection during soybean domestication.