Legume Genomics and Genetics 2026, Vol.17, No.1, 14-31 http://cropscipublisher.com/index.php/lgg 22 phenotype (Nzepang et al., 2025). In red clover, RNA-seq of genotypes with contrasting biological nitrogen fixation efficiency cataloged 491 differentially expressed genes and revealed enrichment of tandemly and dispersedly duplicated genes, which, together with co-expression network analysis, provided a prioritized list of candidates whose functions can be tested in breeding and biotechnology programs. Targeted reverse-genetic approaches, particularly CRISPR/Cas-based genome editing, have dramatically accelerated functional dissection of SNF genes in both model and crop legumes. In Lotus japonicus, CRISPR/Cas9 was used to efficiently disrupt the symbiosis receptor kinase SYMRK and three leghemoglobin genes (LjLb1–3), with biallelic and multi-gene knockouts showing strong nodulation and nitrogen fixation defects, thus validating their essential roles and demonstrating the feasibility of multiplex editing in nodules (Wang et al., 2016). Subsequent work using CRISPR/Cas9-generated triple leghemoglobin mutants combined with physiological, biochemical, ultrastructural, and RNA-seq analyses revealed synergistic functions of leghemoglobins in maintaining optimal nitrogen fixation and preventing premature nodule senescence (Figure 2). Figure 2 Epigenetic regulation of nitrogen fixation in legumes (Adopted from Wang et al., 2016) 6 Case Study: Comparative Genomic Analysis of Key Nitrogen Fixation Genes in Soybean 6.1 Comparison of nitrogen fixation genes in soybean and model legumes Comparative genomic studies across legumes indicate that nitrogen fixation (NF) genes are generally highly conserved, despite extensive polyploidization and genome duplication in some species, including soybean. Analysis of 20 NF-related genes in six legumes (Medicago truncatula, Cicer arietinum, Lotus japonicus, Cajanus cajan, Phaseolus vulgaris and Glycine max) showed that orthologs of core nodulation and symbiotic signaling genes are present in all species, with relatively low non-synonymous substitution rates, supporting strong purifying selection on this gene set. Ks-based analyses clearly resolved the ancient genome duplications shared across legumes and the more recent whole-genome duplication (WGD) unique to soybean, which generated numerous NF-related paralogs. This WGD-derived expansion is also evident for several large transcription factor families implicated in nodulation and symbiosis, such as GATA, MYB, bZIP, BES1 and ZF-HD, where soybean contains substantially more members than non-polyploid model legumes (Rizwan et al., 2025). Many of these TFs show tissue-specific or nodule-enriched expression and are co-expressed with canonical symbiosis genes, suggesting that soybean has diversified regulatory components around a relatively conserved NF core (Leng et al., 2025).
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