Abstract
INTRODUCTION: The basic helix-loop-helix (bHLH) transcription factor family plays crucial roles in plant growth, development, and responses to environmental stresses. However, a systematic characterization of bHLH genes in Castanea mollissima has been lacking. METHODS: We performed a genome-wide identification of bHLH genes in C. mollissima, followed by phylogenetic, structural, motif, chromosomal distribution, and cis-element analyses. Co-expression network analysis and transcriptomic profiling under Dryocosmus kuriphilus infestation were conducted, and representative genes were validated using qRT-PCR across seed developmental stages. RESULTS: A total of 124 non-redundant bHLH genes (CmbHLHs) were identified and classified into eight subfamilies, consistent with conserved gene structures and motif compositions, particularly motif-1 and motif-2 of the canonical bHLH domain. Chromosomal mapping revealed non-random distribution and tandem duplication events. Promoter analysis indicated enrichment of cis-elements responsive to stress, hormone signaling, and developmental regulation. Co-expression analysis highlighted hub genes, including CmbHLH82 and CmbHLH57, with potential regulatory functions. Transcriptomic data showed that most CmbHLHs were downregulated under D. kuriphilus infestation, notably CmbHLH63 and CmbHLH51. qRT-PCR confirmed the dynamic expression of five selected genes during seed development. DISCUSSION: These findings provide the first comprehensive overview of the bHLH gene family in Chinese chestnut. The results highlight candidate genes potentially involved in development and insect resistance, thereby laying a foundation for future functional and breeding studies.