Pan-Genome and Transcriptome-Guided Analysis Reveals Duplication-Driven Evolution and Candidate MYB-bHLH Modules...

Gene duplication and subsequent selection are central to genome evolution and transcription factor diversification, but the conservation and divergence of the basic helix-loop-helix (bHLH) family in pear remain unclear from a pan-genome perspective. Here, we performed a pan-genome and transcriptome-guided analysis across 15 pear genome assemblies, including Asian pear, European pear, and hybrid/haplotype assemblies. Genome-wide duplicated gene pairs were classified into different duplication types, and Ka, Ks, and Ka/Ks values were calculated to establish an evolutionary background for duplicated pear genes. Based on this framework, 3222 bHLH were identified and grouped into evolutionary clades and orthologous gene groups. The pear bHLH family contained conserved core members and variable dispensable members, indicating both functional conservation and genome diversification. Duplication and Ka/Ks analyses showed that WGD/segmental duplication contributed to bHLH expansion and that most duplicated PbrbHLH gene pairs were constrained by purifying selection. By integrating 17-tissue and fruit-development transcriptomes from three pear cultivars, 39 fruit-development-associated PbrbHLHs were selected. Co-expression analysis with 185 PbrMYBs identified candidate MYB-bHLH co-expression modules from the available pear fruit-development transcriptomes. These results provide an evolutionary framework for pear bHLH diversification and candidate regulatory modules for future functional studies.