A gene-based pangenome reveals genomic divergence and altitudinal differentiation in allotetraploid quinoa

Quinoa (Chenopodium quinoa Willd.) is an allotetraploid crop with extraordinary environmental adaptability. To explore its genetic diversity, evolutionary dynamics, and altitudinal differentiation, we constructed a gene-based pangenome using 11 genomes (7 highland and 4 lowland accessions), comprising 51,298 orthologous groups with 19,653 core pan-genes, suggesting a near-closed pangenome architecture within the sampled accessions. Evolutionary analysis revealed that core pan-genes experienced the strongest purifying selection, whereas private pan-genes exhibited the weakest purifying selection, though the difference was not statistically significant compared to the other three pan-gene sets. Subgenomic asymmetry analysis showed that subgenome A retained fewer single-copy genes than subgenome B, with dosage-sensitive two-copy genes enriched in biotic stress-related domains and tandemly duplicated genes associated with abiotic stress adaptation. Presence/absence variation (PAV) analysis identified 7,477 orthologous groups enriched in highland accessions and 3,549 enriched in lowland accessions. We caution that these counts may be substantially inflated by the uneven sample size (7 highland vs. 4 lowland accessions) and should be interpreted as descriptive enrichment patterns rather than definitive evidence of ecotype divergence. Highland-enriched genes were enriched in transposon-related families and FAR1 domains potentially enhancing genomic plasticity and light signaling, whereas lowland-enriched genes were enriched in nitrogen metabolism and defense-related families. Copy number variation of betalain biosynthetic genes (CqCYP76AD, CqDODA, CqDOPA5-GT) was not the primary driver of seed coat color divergence between highland and lowland ecotypes. Four positively selected genes, including NADH-quinone oxidoreductase and MT-A70, harbor population-specific amino acid substitutions that may contribute to differential adaptation, potentially optimizing photosynthetic metabolism, RNA epigenetic modification, and protein homeostasis. These findings provide insights into the genomic basis of quinoa's environmental adaptation and altitudinal differentiation, offering a genomic resource for molecular breeding.