Chromosome-Specific Expansion and Diversification of the Thionin Gene Family in Barley

Thionins are cysteine-rich peptides involved in plant defense. However, their genomic organization, evolutionary expansion, and potential function in barley remain unclear. Here, we integrated reference genome, pan-genome, and pan-transcriptome resources to systematically characterize the thionin gene family in barley. Fifty-six thionin genes were identified in the reference genome Morex V3, displaying pronounced chromosomal clustering and high sequence conservation consistent with extensive tandem duplication. Promoter analysis of these genes revealed enrichment of cis-acting elements associated with stress- and hormone-related signaling pathways, suggesting a potential role for thionins in biotic stress responses, including aphid defense, as suggested by previous studies. Analysis of 20 barley genotypes revealed substantial copy number variation, particularly on chromosomes 6H and 7H, indicating dynamic population-level expansion. Sequence-based clustering grouped thionins into ten clusters and five singletons, with major clusters corresponding to specific chromosomes. Integration of pan-transcriptome data showed that transcriptional activity was largely confined to four major clusters. Aphid infestation of four genotypes featuring copy number variation in chr 6H thionin genes resulted in a strong induction of thionin gene expression, with more pronounced responses during poor-host interactions. Aphid-induced expression tended to increase with thionin gene number, however, this no correlation was observed regarding basal gene expression levels. Together, these findings indicate that the thionin gene family in barley has undergone species-specific expansion driven by tandem duplication and contributes to genotype-dependent aphid defense responses.