A telomere-to-telomere gap-free genome of the new cultivar ‘Zhongtian No. 5,’ combined with pan-genome analysis, aids in exploration and genetic enhancement of red clover ( <i>Trifolium pratense</i> L.)

Abstract Trifolium pratense L. is a multifunctional crop of agronomic importance for forage, horticulture, and ecological restoration. However, the lack of a high-quality genome assembly and the limited representation of genetic diversity by a single reference have impeded its genetic research and molecular breeding. Here, we present the first telomere-to-telomere (T2T) gap-free genome for the diploid (2n = 2x = 14) cultivar T. pratense cv. ‘Zhongtian No. 5’ (TpraZt5), assembled through an integrated sequencing strategy. The 390.94 Mb assembly demonstrates high quality, with a base accuracy &amp;gt; 98.5%, 98.1% BUSCO completeness, a long terminal repeat assembly index of 25.65, and a contig N50 of 52.95 Mb. We annotated 35 971 protein-coding genes and found repeat sequences accounting for 59.6% of the genome. The assembly resolved all seven centromeres and 14 telomeres, providing unprecedented insight into these complex genomic regions. We further constructed a 480.76 Mb pan-genome by integrating two additional accessions, which classified genes into core (70.2%), dispensable (25.3%), and private (4.5%) sets. Comparative genomic analyses identified 606 species-specific genes in TpraZt5 and uncovered extensive structural variations. Functional investigations revealed four species-specific genes and six contracted genes associated with isoflavonoid biosynthesis, two expanded chlorophyll a-b binding proteins, and seven expanded auxin-related genes that may contribute to the high productivity of TpraZt5. Additionally, 44 Gypsy-type transposons within the zeatin biosynthesis pathway were identified as potential regulators of trifoliate leaf development. These genomic resources substantially improve structural annotation and functional characterization, providing vital tools for gene discovery and enhancing molecular breeding initiatives in red clover.