Red clover (Trifolium pratense L.) supports livestock feed, green manure systems, ecological restoration, and sustainable landscapes. Its nitrogen-fixing capacity can reduce dependence on synthetic fertilizers, while its protein-rich biomass and isoflavonoids enhance its agricultural and nutraceutical value. However, red clover is highly heterozygous and strictly outcrossing, making it difficult to develop stable inbred lines and contiguous reference genomes. Earlier assemblies left unresolved gaps, repetitive regions, telomeres, and centromeres, while single-reference genomes could not fully capture genetic diversity within the species. Based on these challenges, in-depth research is needed to build high-quality genome and pan-genome resources for red clover improvement.
Researchers from the Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences, together with collaborators from the Laboratory of Quality & Safety Risk Assessment for Livestock Products, Ministry of Agriculture and Rural Affairs, and Yunnan Vocational College of Agriculture, published (DOI: 10.1093/hr/uhag013) the study in Horticulture Research on January 13, 2026. The study generated a gap-free genome and pan-genome framework for red clover, revealing genomic features associated with five-leaflet morphology, biomass production, and isoflavonoid biosynthesis.
Using Oxford Nanopore Technologies (ONT), PacBio high-fidelity (HiFi) sequencing, chromatin conformation capture (Hi-C) sequencing, and BGI sequencing data, the team assembled a 390.94 megabase (Mb) genome across seven pseudochromosomes, matching the haploid chromosome number of red clover. The assembly reached a contig N50 of 52.95 Mb, showed 98.1% Benchmarking Universal Single-Copy Orthologs (BUSCO) completeness, achieved a long terminal repeat assembly index (LAI) of 25.65, and resolved all seven centromeres and 14 telomeres. Genome annotation identified 35,971 protein-coding genes and showed that repetitive sequences accounted for 59.6% of the genome, with long terminal repeat retrotransposons (LTR-RTs) forming a major component. Comparative analyses found expanded gene families related to chlorophyll a–b-binding proteins and auxin signaling, including Aux/IAA proteins and small auxin-up RNA (SAUR) proteins, suggesting possible links to improved photosynthesis, growth regulation, biomass, and seed yield. The team also constructed a 480.76 Mb pan-genome using two additional red clover accessions, classifying genes into core, dispensable, and private groups. ‘Zhongtian No. 5’ contained 606 private gene families, including four candidate genes involved in isoflavonoid biosynthesis: rna-Tpr13038.1, rna-Tpr13040.1, rna-Tpr16090.1, and rna-Tpr16092.1. Structural variation (SV) analysis further identified 44 Gypsy-type transposons within the zeatin biosynthesis pathway, pointing to a possible regulatory route for leaf morphogenesis.
The authors said the genome offers more than a cleaner reference sequence; it gives researchers a way to see biological regions that were previously difficult to resolve. They said the integration of gap-free assembly and pan-genome comparison brings telomeres, centromeres, transposons, and structural variants into view, helping connect genome structure with traits that matter in breeding. They said these findings provide a practical starting point for explaining why ‘Zhongtian No. 5’ differs from ordinary red clover in leaf form, productivity, and metabolite profile, while also opening new routes for genome-guided improvement.
The new genomic resources could support marker development, gene discovery, and precision breeding in red clover. Candidate genes linked to isoflavonoid biosynthesis may guide the development of varieties with improved nutritional or bioactive value, while auxin- and photosynthesis-related genes may help breeders select lines with higher biomass and seed productivity. The discovery of transposon-associated variation in the zeatin pathway also provides a useful clue for studying multifoliolate leaves, a trait with both ornamental and productivity potential. Future work should functionally validate these candidates and expand the pan-genome with broader germplasm, including wild relatives, to capture more useful diversity.
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References
DOI
10.1093/hr/uhag013
Original Source URL
https://doi.org/10.1093/hr/uhag013
Funding information
This research was funded by National Natural Science Foundation of China (32401292), the Youth Innovation Program of Chinese Academy of Agricultural Sciences (Y2023QC31), Lanzhou Young Scientific & Technological Talent Program (2024-QN-4), the Natural Science Foundation of Gansu Province (25JRRA452, 22JR5RA040), Fundamental Research Funds for the Central Public-interest Scientific Institution (1610322025004), and the Science and Technology Innovation Program of Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences (CAAS-LMY-04).
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.