Chromosome-level genome and organ-specific transcriptome of Alnus glutinosa uncover lineage-specific innovations in root nodule symbiosis

Alnus glutinosa is one of only three lineages within the order Fagales capable of establishing root nodule symbiosis (RNS). Although a fragmented genome assembly of A. glutinosa was previously available, its limited quality, combined with the lack of comprehensive transcriptomic resources has constrained in-depth comparative and functional genomic analyses. In this study, we present a 505Mb chromosome-level genome assembly of A. glutinosa, anchored to 14 pseudochromosomes, representing the most complete and high-quality genomic resource for this species to date. Whole-genome alignment and synonymous substitution rate (Ks) analysis confirm Alnus and Betula as sister genera with shared genomic architectures and evolutionary histories. Functional enrichment analyses of nodule-enhanced genes reveal significant associations with photosynthesis and sugar metabolism, while expanded gene families are enriched in terpenoid biosynthesis and malate transport pathways, likely critical to RNS in A. glutinosa. Phylogenetic analysis indicated that Alnus has retained non-symbiotic class 1 hemoglobin (nsHB1), but lost non-symbiotic class 2 hemoglobin (nsHB2), suggesting a lineage-specific adaptation in symbiotic oxygen regulation. Further comparative analysis of nsHB1 protein sequences across nodulating taxa highlights evolutionary patterns within the Alnus lineage. Through a targeted phylogenetic survey of known RNS-related genes, we identified presence-absence variation (PAV) in RPG and copy number variation (CNV) in AGO5, both of which may underlie Alnus-specific RNS adaptations. Weighted gene co-expression network analysis (WGCNA) identified a nodule-specific module (NSM) comprising 231 genes significantly enriched in sugar-related metabolic pathways. Notably, the bZIP ortholog shows conserved nodule-specific expression across species from Cucurbitales, Rosales, and Fabales, suggesting deep evolutionary conservation of within the nitrogen-fixing clade. Together, these findings provide a high-resolution view of Alnus-specific RNS adaptations and uncover conserved regulatory modules potentially critical for RNS.