Genome-wide analysis of bHLH transcription factor family reveals their involvement in kernel development and biotic stress responses in Chinese chestnut

Rongchen Li¹Liyun Huang¹Xiaolu Huang¹Jianming Liao¹Xiaojuan Wei¹Junji Li¹Guangyu Zeng¹Zhuogong Shi²Zhiheng Zhao^1*

• ¹Guangxi Forestry Research Institute, Nanning, China
• ²Zhaotong University, Zhaotong, Yunnan, China

The basic helix-loop-helix (bHLH) transcription factor family plays critical roles in plant growth, development, and stress responses. In this study, 124 non-redundant bHLH genes (CmbHLHs) were identified from the Castanea mollissima genome.Phylogenetic analysis grouped these genes into eight subfamilies, consistent with conserved gene structures and motif compositions, particularly the highly conserved motif-1 and motif-2 that constitute the canonical bHLH domain. Chromosomal mapping revealed non-random distribution, with several tandem duplications suggesting gene expansion events. Cis-element analysis indicated that CmbHLH promoters are enriched in elements responsive to abiotic and biotic stresses, hormone signaling, and developmental regulation. Co-expression network analysis identified hub genes, such as CmbHLH82 and CmbHLH57, potentially regulating key developmental processes. Moreover, transcriptomic data under Dryocosmus kuriphilus infestation showed that most CmbHLHs were downregulated, including CmbHLH63 and CmbHLH51, indicating potential roles in insect resistance. qRT-PCR validation of five representative CmbHLHs confirmed their dynamic expression across seed developmental stages. Collectively, this study provides a comprehensive overview of the CmbHLH gene family and lays the foundation for future functional studies on chestnut development and defense mechanisms.