Abstract
Background: Small heat shock proteins (sHsps), particularly Hsp20 family members, are pivotal for plant thermotolerance and abiotic stress adaptation. However, their evolutionary dynamics and functional roles in Lycium barbarum (goji berry), a commercially significant stress-tolerant crop, remain uncharacterized. This study aims to comprehensively identify LbHsp20 genes, delineate their evolutionary patterns, and decipher their regulatory mechanisms under heat stress to accelerate molecular breeding of resilient cultivars. Methods: Forty-three LbHsp20 genes were identified from the goji genome using HMMER and BLASTP. Phylogenetic relationships were reconstructed via MEGA-X (maximum likelihood, 1000 bootstraps), while conserved motifs and domains were annotated using MEME Suite and InterProScan. Promoter cis-elements were predicted via PlantCARE. Heat-responsive expression profiles of candidate genes were validated by qRT-PCR in two contrasting lines (N7 and 1402) under 42 °C treatment. Results: The LbHsp20 family clustered into 14 subfamilies, predominantly cytoplasmic (subfamilies I-VII). Chromosomal mapping revealed a tandem duplication hotspot on Chr4 (12 genes) and absence on Chr9, suggesting lineage-specific gene loss. All proteins retained the conserved α-crystallin domain (ACD), with 19 members harboring the ScHsp26-like ACD variant. Promoters were enriched in stress-responsive elements (HSE, ABRE, MYC). Heat stress induced significant upregulation (>15-fold in LbHsp17.6A and LbHsp18.3) in N7, whereas 1402 showed weaker induction (<5-fold). Subfamily specific divergence was observed, with cytoplasmic subfamily I genes exhibiting the strongest heat responsiveness. Conclusions: This study unveils the evolutionary conservation and functional diversification of LbHsp20 genes in L. barbarum. The tandem duplication-driven expansion on Chr4 and subfamily specific expression patterns underpin their roles in thermotolerance. These findings establish a foundation for engineering climate-resilient goji varieties.