Legume Genomics and Genetics 2026, Vol.17, No.1, 14-31 http://cropscipublisher.com/index.php/lgg 24 predominantly purifying selection (Gou et al., 2025). For families such as GATA and MYB, integrative phylogenetic and expression analyses across nitrogen-fixing legumes revealed subgroups whose members are specifically co-expressed with NIN and nodulation markers in nodules, pointing to co-evolution of NIN-centered regulatory circuits with lineage-specific TF expansions (Leng et al., 2025; Xia et al., 2025). At the regulatory-network scale, GmNIN appears embedded within broader transcriptional hierarchies that have diversified through gene family expansion and regulatory rewiring. In soybean, independent component and co-expression analyses across 380 accessions identified modules enriched for transcription factors, including MYB, NAC, GATA, BES1 and clock genes, that are significantly associated with nitrogen fixation phenotypes such as nodule number, nodule weight and nitrogen fixation efficiency (Li et al., 2025). Many of these TFs show evidence of selection during domestication and improvement, whereas the core NF genes they regulate-including GmNIN orthologs-remain more conserved, suggesting that evolutionary tuning of GmNIN function in soybean has occurred largely through changes in dosage, cis-regulation and interaction partners rather than radical coding divergence. Comparative regulatory network analysis between G. max and M. truncatula during AMS and NFS further showed that while NIN-associated modules are shared, soybean has incorporated additional regulators linked to defense and hormonal signaling into these networks, implying that GmNIN operates within an expanded, soybean-specific regulatory context shaped by WGD and subsequent subfunctionalization (Wu et al., 2025). 6.3 Implications for molecular breeding to improve nitrogen fixation efficiency in soybean Insights from comparative and evolutionary genomics of nitrogen fixation genes in soybean have direct implications for molecular breeding. Genome-wide association and mapping studies have revealed that symbiotic nitrogen fixation (SNF) traits-including nodule number, nodule biomass, total N accumulation and percent N derived from the atmosphere-are highly polygenic and environmentally responsive, with many loci of small effect dispersed across the genome. QTLs and TWAS hits for SNF traits are particularly enriched in regions harboring transcription factors, signaling genes and hormone-related regulators rather than structural NF genes, consistent with the notion that variation in regulatory modules around conserved NF cores, such as GmNIN networks, underlies much of the phenotypic diversity in SNF. Integrative analyses combining GWAS, TWAS, eQTLs and ATAC-seq in soybean have further demonstrated that cis-regulatory variants affecting the expression of SNF-related TFs (e.g., GmLHY1b) in open chromatin regions can significantly impact nodulation and nitrogen fixation, providing concrete targets for marker-assisted and genomic selection. Functional genomic interventions highlight complementary strategies in which knowledge of NF gene evolution can guide precise editing to optimize nodulation and fixation efficiency without compromising yield. Gene editing of systemic nodulation regulators such as CLE receptors (e.g., ric1a/2a alleles) produced soybean lines with moderately increased nodule numbers, improved carbon–nitrogen balance, and higher grain yield and seed protein in multi-year field trials, demonstrating that fine-tuning nodulation control modules can improve both SNF and agronomic performance (Zhong et al., 2024). Similarly, CRISPR/Cas9 editing of the gibberellin receptor gene GmGID1-2 created semi-dwarf soybean lines with enhanced architecture, higher yield and improved nodulation, nitrogenase activity and plant N content, indicating that pleiotropic regulators connecting growth and symbiosis can be leveraged for sustainable yield gains (Tang et al., 2025). Reviews on soybean functional genomics and breeding emphasize that integrating such targeted editing of regulatory hubs (e.g., GmNIN-associated TFs, hormone receptors, clock genes) with genomic prediction using SNF-associated markers offers a promising route to develop cultivars with enhanced nitrogen fixation, optimized plant architecture and reduced fertilizer dependency under variable environments (Zhou et al., 2025). 7 Prospects for the Application of Comparative Genomics in Improving Nitrogen Fixation in Legume Crops 7.1 Development of molecular markers for nitrogen fixation efficiency Comparative genomics, coupled with high-throughput phenotyping, is rapidly transforming efforts to dissect and improve nitrogen fixation efficiency in legumes. Genome-wide association studies (GWAS) and diversity panels
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