Highly heterozygous <i>Citrus changshan‐huyou</i> Y. B. Chang originated from ancient hybridization between mandarin and pummelo and displayed distinct tissue‐specific allelic imbalance

The genus Citrus is characterized by a reticulate evolutionary history with frequent hybridization, making it an intriguing subject for genome evolution investigation. Citrus changshan-huyou Y. B. Chang (Huyou) is a unique landrace first discovered in Zhejiang Province, China, with premium fruit quality. The evolutionary origin of Huyou has puzzled local botanists and growers. Here, we sequenced a 120-year-old "ancestral tree" of Huyou using PacBio long read and Hi-C sequencing and assembled two high-quality, haplotype-resolved genomes, HY1 and HY2. Huyou displayed a genome heterozygosity level at 3.07%, among the highest in published citrus genomes. Using a k-mer-based tracing approach, we explicitly resolved that HY1 genome contained 87.8% mandarin, 7.3% pummelo, and 0.2% citron origin, whereas HY2 had 85.0% pummelo, 2.9% mandarin, and 0.3% citron, implying a hybridization event between mandarin and pummelo. Phylogeny dating showed that HY1 (2.0 Mya) and HY2 (2.18 Mya) had diverged earlier than the split of Citrus clementina and Citrus reticulata and the split of Citrus grandis and Citrus maxima, respectively. We observed clear chromosomal recombination on chr8 and chr9 in HY1, which may have occurred after the ancestral hybridization. Further transcriptome analyses in six tissues revealed a strong allelic dominance of HY2 over HY1 in root tissue and moderately in stem, leaf, flower, and fruit. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses revealed that genes related to antioxidant biosynthesis and lipid metabolism were most significantly affected by allelic imbalance. This pioneering report of allelic imbalance in citrus species support Huyou as an interesting model to investigate genome evolution following distant hybridization.