Haplotype-level allelic characterization facilitates research on the functional, evolutionary and breeding-related features of extremely large and complex plant genomes. We report a 21.7-Gb chromosome-level haplotype-resolved assembly in Pinus densiflora. We found genome rearrangements involving translocations and inversions between chromosomes 1 and 3 of Pinus species and a proliferation of specific long terminal repeat (LTR) retrotransposons (LTR-RTs) in P. densiflora. Evolutionary analyses illustrated that tandem and LTR-RT-mediated duplications led to an increment of transcription factor (TF) genes in P. densiflora. The haplotype sequence comparison showed allelic imbalances, including presence–absence variations of genes (PAV genes) and their functional contributions to flowering and abiotic stress-related traits in P. densiflora. Allele-aware resequencing analysis revealed PAV gene diversity across P. densiflora accessions. Our study provides insights into key mechanisms underlying the evolution of genome structure, LTR-RTs and TFs within the Pinus lineage as well as allelic imbalances and diversity across P. densiflora.

单倍型水平等位基因表征有助于研究超大型和复杂植物基因组的功能、进化和育种相关特征。我们报道了 Pinus densiflora 中的 21.7 Gb 染色体水平单倍型分辨组装。我们发现基因组重排涉及松树物种 1 号和 3 号染色体之间的易位和倒位，以及 P. densiflora 中特异性长末端重复 （LTR） 反转录转座子 （LTR-RTs） 的增殖。进化分析表明，串联和 LTR-RT 介导的重复导致 P. densiflora 中转录因子 （TF） 基因的增加。单倍型序列比较显示等位基因不平衡，包括基因 （PAV 基因） 的存在-不存在变异及其对 P. densiflora 开花和非生物胁迫相关性状的功能贡献。等位基因感知重测序分析揭示了 P. densiflora 种质的 PAV 基因多样性。我们的研究提供了对 Pinus 谱系中基因组结构进化、LTR-RTs 和 TFs 以及 P. densiflora 的等位基因失衡和多样性的关键机制的见解。