What question did this study set out to answer?

The study aims to create a high-quality haplotype-resolved genome assembly for pummelo and improve gene annotation in Citrus species.

March 2, 2026Open Access

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

Key Points

The study aims to create a high-quality haplotype-resolved genome assembly for pummelo and improve gene annotation in Citrus species.
Generated pummelo genome using PacBio HiFi and Oxford Nanopore sequencing.
Developed HapGene, a haplotype-aware annotation pipeline integrating RNA-Seq and Iso-Seq data.
Annotated genomes of three significant Citrus crops using newly sequenced and public transcriptomic data.
Achieved N50 values of 38.58 Mb and 32.57 Mb for two haplotype assemblies.
Captured 3%-10% of genes missed by conventional annotation pipelines, reducing false haplotype-specific genes by 4-5-fold.
Revealed 18,757-21,083 alternative splicing events and thousands of long intergenic RNAs associated with stress responses.

Abstract

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 Mb 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%-10% of genes missed or misannotated by conventional pipelines and reduces false haplotype-specific genes by 4-5-fold. Leveraging 380 Gb of newly sequenced and 2,792 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, 1,725-1,853 resistance gene analogues, and 2,392-3,757 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.

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