Citrus fruits are cultivated in more than 140 countries and are valued for their nutritional, medical, cosmetic, and economic importance. Yet citrus improvement is complicated by a tangled history of domestication, interspecific hybridization, and genetic introgression. Although more than 50 Citrus genome assemblies have been reported, many lack full haplotype resolution, telomere-to-telomere continuity, or accurate annotation of protein-coding and non-coding genes. Conventional annotation pipelines can also misread haplotype differences, generating false haplotype-specific genes. Due to these challenges, deeper research is needed into high-quality haplotype-resolved citrus genomes and annotation approaches that can distinguish true biological variation from technical artifacts.
The study was conducted by researchers from Huazhong Agricultural University, including the National Key Laboratory for Germplasm Innovation and Utilization of Horticultural Crops, Hubei Hongshan Laboratory, and the Hubei Key Laboratory of Agricultural Bioinformatics. Published (DOI: 10.1093/hr/uhag048) on February 28, 2026, in Horticulture Research, the article reports a high-quality haplotype-resolved telomere-to-telomere (HR-T2T) genome assembly of pummelo (Citrus maxima) and introduces HapGene for haplotype-aware genome annotation. The study focuses on three major citrus crops: sweet orange (Citrus sinensis), pummelo, and mandarin (Citrus reticulata).
Using PacBio high-fidelity (HiFi) and Oxford Nanopore Technologies (ONT) long-read sequencing, the team assembled two pummelo haplotypes with contig N50 values of 38.58 and 32.57 megabases (Mb), completeness scores of 99.36% and 99.66%, and nucleotide accuracies of 99.99994% and 99.99997%. The assembly exposed extensive haplotype variation, including single-nucleotide polymorphisms (SNPs), insertions and deletions (indels), and structural variations (SVs). HapGene then improved genome annotation by integrating short-read RNA sequencing (RNA-Seq) and long-read isoform sequencing (Iso-Seq) data while comparing the two haplotypes directly. Benchmarking showed that HapGene captured 3% to 10% of genes missed or misannotated by conventional pipelines and reduced false haplotype-specific genes by four- to five-fold. By combining 380 gigabases (Gb) of newly generated transcriptomic data with 2,792 Gb of public data, the researchers identified 18,757–21,083 alternative splicing (AS) events, 1,725–1,853 resistance gene analogues (RGAs), and 2,392–3,757 lincRNAs across the analyzed genomes.
The authors said the study turns a difficult citrus genome into a clearer working map. Instead of annotating each haplotype in isolation, they said HapGene reads the two inherited chromosome sets together, helping separate real biological differences from annotation noise. They said this matters because many agronomic traits, including stress tolerance, disease resistance, and fruit quality, may depend on allele-specific variation hidden in complex regions of the genome. With a more complete assembly and cleaner annotation, they said citrus researchers can move more confidently from sequence data to trait discovery.
The findings provide new tools for understanding citrus evolution, domestication, and crop improvement. A high-quality pummelo genome can help clarify how modern citrus hybrids, including sweet orange, inherited and reshaped ancestral genetic variation. Improved RGA annotation may support disease-resistance studies, while stress-responsive lincRNAs offer new clues to regulatory adaptation. The discovery that around one-third of genes show tissue-specific allelic differential expression also highlights the need for haplotype-aware breeding strategies. Expanding HR-T2T assemblies to more wild and cultivated Citrus germplasm could accelerate molecular marker development, targeted gene editing, and rational haplotype design for future citrus varieties.
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References
DOI
10.1093/hr/uhag048
Original Source URL
https://doi.org/10.1093/hr/uhag048
Funding information
This work was funded by the National Natural Science Fund for Excellent Young Scientists Fund Program (Overseas), the Young Scientist Fostering Funds for the National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops and the Fundamental Research Funds for the Central Universities, China (2662024SZ002).
About Horticulture Research
Horticulture Research is an open access journal of Nanjing Agricultural University and ranked number one in the Horticulture category of the Journal Citation Reports ™ from Clarivate, 2023. The journal is committed to publishing original research articles, reviews, perspectives, comments, correspondence articles and letters to the editor related to all major horticultural plants and disciplines, including biotechnology, breeding, cellular and molecular biology, evolution, genetics, inter-species interactions, physiology, and the origination and domestication of crops.