The GmCYCLOPS paralogs regulate soybean nodulation and exhibit signatures during domestication

CYCLOPS functions as a central regulator in legume-rhizobia symbiosis, but its role in cultivated soybean remains incompletely characterized. Through homology-based sequence analysis using Lotus japonicus LjCYCLOPS as a query, two soybean orthologs were identified, GmCYCLOPS1 and GmCYCLOPS2, sharing 98.1% amino acid identity. Both genes exhibited root- and nodule-enriched expression, with peak induction at 12 h post-rhizobial inoculation. Subcellular localization assays showed that both GmCYCLOPS1 and GmCYCLOPS2 proteins are enriched in the nucleus but also present in the cytoplasm. Functional analyses using Agrobacterium rhizogenes-mediated hairy root transformation demonstrated that RNA interference targeting either paralog reduced expression of both genes and significantly decreased nodulation. Consistently, CRISPR/Cas9-mediated disruption of both GmCYCLOPS1 and GmCYCLOPS2 generated double mutants with severe nodulation defects, whereas overexpression of these paralogs enhanced nodule formation. Population genomic analysis of 1504 accessions (34 wild, 423 landraces, and 1047 cultivars) revealed that the GmCYCLOPS1 haplotype Hap10, absent in wild accessions, increased to 91% in landraces and 99% in cultivars, showing strong selection signatures and enrichment in Northeast China. Meanwhile, GmCYCLOPS2 exhibited more moderate and regionally structured haplotype diversity. Together, these results reveal that GmCYCLOPS1 and GmCYCLOPS2 act as core regulators of soybean nodulation and have undergone divergent selection and geographic differentiation during soybean domestication. This study characterizes the functional and evolutionary features of the GmCYCLOPS paralogs, providing valuable gene and haplotype resources to improve soybean nodulation traits.