Scientists build the first gap-free genome of the Chinese holly

The Chinese holly, Ilex cornuta, has long been prized in gardens across East Asia for its glossy, spine-tipped leaves, bright red berries and remarkable tolerance of heat, drought and poor soils. It is also a plant of medicinal interest, with extracts studied for their bioactive compounds. Yet despite its horticultural and pharmacological importance, scientists have struggled to read its genome with any real precision, because no high-quality reference existed for the species or, indeed, for the entire holly genus Ilex. That gap has now been closed. A research team led by Peng Zhou and Min Zhang of the Jiangsu Academy of Forestry in Nanjing, working with colleagues at Nanjing Forestry University and Beijing Compass Agritechnology, has published the first telomere-to-telomere, gap-free genome assembly of I. cornuta in BMC Plant Biology.

Telomere-to-telomere, or T2T, assembly represents the gold standard of modern genomics. Conventional genome assemblies leave behind hundreds or even thousands of unresolved stretches known as gaps, typically in regions crammed with repetitive DNA such as centromeres, ribosomal RNA gene clusters and other satellite-rich zones. These gaps matter. They hide genes, obscure structural variation and make it impossible to say exactly how many chromosomes a species truly carries. A T2T assembly eliminates those holes entirely, reading each chromosome as one continuous thread from the telomere at one end to the telomere at the other. Achieving this for a plant genome is notoriously difficult, since plant genomes are often loaded with repetitive transposable elements that confound sequencing algorithms.

The team overcame the challenge by combining three complementary sequencing strategies. Pacific Biosciences HiFi reads provided highly accurate long stretches of DNA, Oxford Nanopore ultra-long reads extended those stretches across the most stubborn repetitive regions, and Hi-C, a technique that captures the physical contacts between chromosomes inside the cell nucleus, was used to order and orient the sequences into full-length chromosomes. The final assembly spans roughly 890.50 megabase pairs and achieves a scaffold N50 of 44.26 megabases, meaning half of the assembled sequence resides in chromosomes longer than 44 million bases. The entire genome was organized into exactly 20 gap-free chromosomes, each capped by telomeres, with 40 telomeres and 20 putative centromere regions identified in total.

One of the most consequential results is the definitive resolution of the chromosome number of I. cornuta. Botanists have debated the basic chromosome number of this species for years, with conflicting counts circulating in the cytological literature. The new gap-free assembly shows unambiguously that the species carries 20 pairs of chromosomes, a diploid complement written as 2n = 2x = 40. Crucially, the team did not rely on sequencing data alone. An independent karyotype analysis of root-tip cells, in which chromosomes are stained and counted directly under the microscope, confirmed the genomic count. Agreement between the molecular assembly and classical cytogenetics settles a long-standing controversy and provides a firm foundation for evolutionary comparisons across the Aquifoliaceae family.

Genome quality was assessed with the metrics that the assembly community takes most seriously. The annotation, recovered using RNA sequencing data, predicted 35,437 protein-coding genes. Benchmarking with BUSCO, a test that searches for a set of universally conserved single-copy genes, returned a completeness score of 98.33 percent, indicating that almost the entire expected gene repertoire is present. The base-level accuracy of the assembly, expressed as a quality value or QV, reached 48.97, which corresponds to an error rate of roughly one mistake in every 79,000 bases. These figures place the Chinese holly assembly among the most accurate plant genomes published to date and demonstrate that the integration of HiFi, ultra-long and Hi-C data can deliver reference-grade sequences even for species with no prior genomic resources.

With the reference in hand, the researchers turned to evolutionary history. Comparative genomics against related plant species revealed two whole-genome duplication events in the ancestry of I. cornuta, ancient episodes in which the entire genetic complement was doubled and subsequently pared down through gene loss and divergence. Whole-genome duplications are a major engine of plant evolution, because duplicate genes are free to accumulate mutations and take on new functions. The analysis also uncovered lineage-specific expansions of gene families, with a pronounced enrichment in pathways governing secondary metabolism, the biochemical arsenal plants use to synthesize defensive compounds, pigments and medicinal molecules.

These expanded gene families are particularly intriguing given what the Chinese holly is known for. The species thrives under stresses that would defeat many ornamentals, and its tissues contain bioactive compounds of pharmaceutical interest. The authors are careful, however, to frame these expansions as candidate gene sets that could potentially contribute to environmental adaptability. Genomic evidence of expansion does not by itself prove function. Verifying the biological roles of these genes will require transcriptomic profiling across stress conditions and direct functional assays, such as gene knockout or overexpression experiments, work that the T2T reference now makes dramatically easier to design and interpret.

The significance of the assembly extends well beyond a single species. Ilex is a large and ecologically diverse genus of more than 400 evergreen and deciduous trees and shrubs distributed across temperate and tropical regions, and it had never before had a gap-free reference genome of any kind. The I. cornuta assembly therefore serves as a genomic anchor for the entire genus, enabling researchers to map other holly species against a complete, contiguous blueprint, reconstruct chromosome evolution within the family, and pinpoint the genetic basis of traits ranging from sex determination, a famously thorny question in hollies, to fruit color and cold hardiness.

For breeders, the practical implications are immediate. Ornamental holly breeding has traditionally depended on slow phenotypic selection and conventional crossing, approaches that are poorly suited to traits controlled by many genes or by structural variants buried in repetitive DNA. A telomere-to-telomere reference changes that calculus. It allows genome-wide association studies, marker-assisted selection and eventually genome editing to be pursued with confidence that the underlying sequence is complete and correctly ordered. Genes involved in stress tolerance, secondary metabolite production and ornamental quality can now be located precisely, tracked in breeding populations and, in time, targeted directly.

The work also stands as a showcase of how far genome science has traveled. Little more than a decade ago, completing a plant chromosome end to end was deemed unreachable; today, a coordinated pipeline of three sequencing technologies can deliver a gap-free reference for a species whose genome had never been assembled before. The Chinese holly now joins the small but growing roster of plants with fully finished genomes, and the resource, deposited and openly described by the Nanjing-led team, is freely available to researchers worldwide. For a genus that decorates gardens, supports traditional medicine and endures some of the toughest growing conditions in its native range, the holly has finally been given a complete instruction manual, and scientists are only beginning to read it.

Subject of Research: Telomere-to-telomere gap-free genome assembly of the Chinese holly, Ilex cornuta

Article Title: Telomere-to-telomere, gap-free genome assembly of the Chinese holly, Ilex cornuta

Article References: Zhou, P., Xu, K., Gong, W., Li, F., Sun, H., Zhang, Q., & Zhang, M. (2026). Telomere-to-telomere, gap-free genome assembly of the Chinese holly, Ilex cornuta. BMC Plant Biology. https://doi.org/10.1186/s12870-026-10103-w

Image Credits: AI Generated

DOI: 10.1186/s12870-026-10103-w

Keywords: Ilex cornuta, Chinese holly, telomere-to-telomere, genome assembly, centromere, chromosome number, whole-genome duplication, comparative genomics, PacBio HiFi, Oxford Nanopore, Hi-C, molecular breeding