Genome-wide identification, characterization, and expression analysis of the plant defensin (PDF) gene family in soybean

Plant defensins (PDFs) are small, cysteine-rich antimicrobial peptides that play central roles in plant innate immunity. Despite their importance, a systematic characterization of the PDF gene family in soybean (Glycine max) has been lacking. Here, we report a genome-wide identification and comprehensive characterization of 27 GlyPDF genes encoding 31 protein isoforms in the soybean Williams82 v4 genome, identified through combined BLASTP and Hidden Markov Model (HMM) profiling using the plant defensin domain (PF00304). Phylogenetic analysis classified the GlyPDF family into four subfamilies (GlyPDF1-4), with GlyPDF2 being the largest (11 genes, accounting for > 40% of members). The 27 genes are unevenly distributed across 13 of the 20 soybean chromosomes, with chromosome 8 harboring the highest number of loci (6 genes). Three tandem duplication clusters and 19 intra-genomic collinear pairs were identified, indicating that both tandem duplication and ancestral whole-genome duplication (WGD) have driven family expansion. Ka/Ks analysis confirmed purifying selection across all duplicated gene pairs (Ka/Ks < 1.0). Physicochemical characterization revealed that the majority of GlyPDF proteins are basic (pI > 7.0), consistent with their roles as cationic antimicrobial effectors. Promoter analysis identified enrichment of jasmonate-responsive elements (22/27 genes), abscisic acid-responsive elements (19/27), and salicylate-responsive elements (16/27), as well as WRKY-binding W-box motifs (14/27), suggesting multifaceted hormonal and stress regulation. Tissue-specific qRT-PCR revealed broad but variable expression across roots, stems, leaves, flowers, and pods. Upon inoculation with Fusarium oxysporum, 18 of 27 GlyPDF genes were differentially expressed, with GlyPDF2.7, GlyPDF2.8, GlyPDF2.9, and GlyPDF3.3 displaying the strongest induction kinetics. These findings provide a useful genetic resource for the functional characterization of soybean defensins and their potential application in breeding disease-resistant varieties.