Identification of the HSP20 Gene Family in L. barbarum and Their Contrasting Response to Heat Stress Between Two Varieties

<b>Background</b>: Small heat shock proteins (<i>sHsps</i>), particularly <i>Hsp20</i> family members, are pivotal for plant thermotolerance and abiotic stress adaptation. However, their evolutionary dynamics and functional roles in <i>Lycium barbarum</i> (goji berry), a commercially significant stress-tolerant crop, remain uncharacterized. This study aims to comprehensively identify <i>LbHsp20</i> genes, delineate their evolutionary patterns, and decipher their regulatory mechanisms under heat stress to accelerate molecular breeding of resilient cultivars. <b>Methods</b>: Forty-three <i>LbHsp20</i> 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. <b>Results</b>: The <i>LbHsp20</i> 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 <i>LbHsp17.6A</i> and <i>LbHsp18.3</i>) in N7, whereas 1402 showed weaker induction (<5-fold). Subfamily specific divergence was observed, with cytoplasmic subfamily I genes exhibiting the strongest heat responsiveness. <b>Conclusions</b>: This study unveils the evolutionary conservation and functional diversification of <i>LbHsp20</i> genes in <i>L. barbarum</i>. 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.