Pan-Genome Analysis Reveals Evolutionary Dynamics and Functional Divergence of the NAC Gene Family in Soybean

Soybean (Glycine max) is an important model crop for studying plant functional genes, such as the NAC transcription factor (TF) gene family. The NAC transcription factor (TF) family is one of the largest plant-specific TF families and plays critical roles in plant growth, development, and stress responses. In this study, we performed a pan-genome-wide analysis of NAC genes using 29 soybean genomes. A total of 5051 NAC genes were identified and clustered into 245 orthologous gene groups (OGGs), including 58 core, 88 soft-core, 32 shell, and 67 cloud groups. Based on phylogenetic relationships, the representative NAC OGGs were assigned to 18 subfamilies, 17 of which contained soybean NAC genes. Gene duplication analysis indicated that whole-genome duplication (WGD)/segmental duplication was the predominant driver of NAC family expansion, accounting for 90.88% of duplication events. Approximately 39.30% of NAC genes carried at least one intact transposable element (TE) within 2 kb upstream or downstream regions. NAC genes with copy number variation (CNV) harbored more nearby TEs than non-CNV genes (1.54 vs. 1.31 TEs per gene), and dispensable NAC genes contained more nearby TEs than core NAC genes (1.59 vs. 1.33 TEs per gene). These results indicate a significant association between local TE abundance and NAC gene CNV or dispensability. Selection pressure analysis showed that dispensable NAC genes had higher Ka, Ks, and Ka/Ks values than core genes, suggesting relatively relaxed evolutionary constraints. Expression profiling across six tissues revealed distinct transcriptional patterns among NAC subfamilies. Structurally conserved subfamilies generally showed broader expression, whereas structurally divergent subfamilies displayed greater expression variability. Regulatory network and Gene Ontology (GO) enrichment analyses suggested that conserved subfamilies were mainly associated with stress responses, while divergent subfamilies were related to cell wall regulation, signal transduction, and ion homeostasis. Further analysis of Wm82 drought RNA-seq data prioritized several putative drought-responsive NAC candidates, including Glyma.16G043200, Glyma.06G248900, Glyma.07G050600, Glyma.12G206900, and Glyma.18G261300. Overall, these findings elucidate the mechanisms of expansion and the functional divergence of the NAC gene family at the soybean pan-genome level, providing a theoretical basis for understanding NAC gene evolution and facilitating future crop improvement.