Haplotype-resolved telomere-to-telomere assembly and haplotype-aware annotation pipeline enable high-quality reannotation of three Citrus genomes

Abstract

Citrus species are economically and nutritionally vital, with their fruits cultivated globally. Despite the publication of multiple genomes for Citrus, high-quality assemblies that achieve both haplotype resolution and telomere-to-telomere (HR-T2T) continuity remain scarce-pummelo ( Citrus maxima) being a notable example. Compounded by limitations in gene annotation quality, these gaps hinder functional genomic research and genomics-assisted breeding. Here, we report the first high-quality HR–T2T genome assembly of pummelo, generated using PacBio HiFi and Oxford Nanopore sequencing. The two haplotype assemblies presented contig N50 values of 38.58 and 32.57 Mb, completeness scores of 99.36% and 99.66%, and nucleotide accuracies of 99.99994% and 99.99997%, respectively. We developed HapGene, a haplotype-aware annotation pipeline that integrates short-read RNA-Seq and long-read Iso-Seq data to enable unbiased annotation. Benchmarking showed HapGene captured 3% to 10% of genes missed or misannotated by conventional pipelines and reduces false haplotype-specific genes by 4- to 5-fold. Leveraging 380 Gb of newly sequenced and 2792 Gb of public transcriptomic data, we comprehensively annotated protein-coding and non-coding genes across three major Citrus crops (sweet orange, pummelo, and mandarin). This effort revealed 18 757–21 083 alternative splicing events, 1725–1853 resistance gene analogues, and 2392–3757 long intergenic RNAs (lincRNAs). Genomic and transcriptomic characterization of lincRNAs indicated their functional innovation (many associated with stress responses) in Citrus. Additionally, we revealed around one-third of genes exhibited tissue-specific allelic differential expression. Our work provides a critical genomic resource and analytical tool to advance Citrus genomic research, thereby driving progress in functional and evolutionary genomics while laying a robust foundation for precise genomics-assisted breeding.

Acknowledgements

The authors would like to thank Shengjun Liu and Qiang Xu (Huazhong Agricultural University) for providing the sweet orange and mandarin genome assembly. 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).

Author contributions

W.-B.J. and J.H. conceived and designed the project. W.-B.J. supervised the project. J.H. developed the annotation pipeline, performed genome annotation and expression analysis. P.-X.X., L.C., and L.T. performed data analysis. S.Z. and J.Y. provided plant materials. J.H., P.-X.X. and W.-B.J. wrote the manuscript. All authors read and approved the final manuscript.

Data availability

The genome assemblies, and PacBio HiFi long reads of pummelo have been deposited into National Genomics Data Center (NGDC) (BioProject: PRJCA035310) and China National GeneBank DataBase (BioProject: CNP0006728). All transcriptome sequencing data generated in this study have been deposited in the NGDC under the accession number PRJCA042681. Lists of public RNA-Seq data used in this study are included in Table S11. Gene annotation files (GFF3) and genome assemblies for the three Citrus genomes analyzed in this study are deposited in the Figshare database (https://doi.org/10.6084/m9.figshare.30016324.v3).

Conflicts of interest statement

The authors declare no competing interests.

Supplementary material

Supplementary material is available at Horticulture Research online.

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