Abstract:

Objective: With the highly heterozygous hybrid poplar ‘84K’ as a model, this study aims to characterize the divergence of alleles in the IQD gene family from three dimensions of systematic evolution, protein structure, and expression regulation, in order to provide new insights into the genetic basis of complex traits in forest trees and the potential functional complementarity between alleles. Method: The sequence heterozygosity of PagIQD alleles was calculated, combined with systematic evolutionary analysis and chromosome localization, to determine the gene distribution and evolutionary relationship. Protein physicochemical properties, transmembrane structure, and subcellular localization were predicted. Motifs feature analysis, 3D structure modeling, and calmodulin-binding were conducted. The differences in allele protein characteristics were compared. The tissue and stress response patterns of allele-specific expression (ASE) were analyzed and its expression synergy was assessed by integrating cis-element analysis with RNA-seq data. Allele-dependent genome editing efficiency was further evaluated based on sequence divergence. Result: Phylogenetically, the number of PagIQD gene family members in 84K showed a slight contraction, and the average heterozygosity of coding regions, full-length genes, proteins, and promoters sequences were all higher than the genome-wide level (2.1%). Several IQD genes in 84K did not align perfectly with their chromosomal positions in the reference Populus alba genome. Allelic pairs displayed six distinct phylogenetic branching patterns, suggesting these alleles may originate from different ancestors origins or represent retention of ancient polymorphisms. At the protein level, some allele pairs differed markedly in physicochemical properties, especially in stability and hydrophobicity, and individual alleles showed discrepancies in predicted transmembrane regions and subcellular localization. Approximately 14% of allele pairs varied in motif number or arrangement, and some exhibited differences in secondary structure proportions and IQ67-domain spatial conformation, which may influence their calmodulin (CaM) binding mode and binding energy. In terms of expression regulation, there were significant differences in cis-element and transcription factor binding sites between alleles, resulting in widespread allele-specific expression, with about 88.5% of gene pairs showing biased expression in at least one tissue, and strong tissue specificity. Under stress, allelic pairs often showed different or even opposite responses in terms of responsiveness. Integrated analysis revealed three interaction patterns between alleles: synergistic response, expression buffering, and homeostasis maintenance. Sequence divergence also strongly affected the design and predicted efficiency of allele-specific genome-editing targets. Conclusion: This study demonstrates that multi-dimensional allelic divergence is a pervasive feature in the highly heterozygous genome of 84K. The results highlight the necessity of treating alleles as potentially distinct functional units, and two alleles should be considered simultaneously in functional analysis, expression analysis, and gene editing design of high heterozygous forest trees, which is of great significance for accurately analyzing the genetic mechanism of forest traits and guiding genetic improvement.

Key words: high genomic heterozygosity, IQD gene family, allele-specific expression, functional divergence, genome editing