Leveraging whole-genome re-sequencing for diversity, population structure, and a public mid-density genotyping enrichment panel in crimson clover ( <i>Trifolium incarnatum L</i> .) for breeding purposes

A bstract Crimson clover (Trifolium incarnatum L.) is an obligately outcrossing, cool-season annual legume valued for forage and cover cropping, yet genomic resources to support systematic improvement are limited. We performed the first and most comprehensive whole-genome resequencing (WGR) of global crimson clover germplasm to (i) characterize diversity and population structure and (ii) develop a public mid-density enrichment capture panel for breeding applications. A core set of 45 accessions sequenced at ∼50X generated 5.84 million variants, while 149 additional accessions sequenced at ∼2.54X yielded 17.05 million variants. After stringent filtering, we retained 542,790 high-confidence SNPs from the high-coverage dataset and ∼2.4 million from the low-pass cohort. Population analyses (PCA, ADMIXTURE) revealed compact clustering of cultivars, broader dispersion of wild and uncertain-status accessions, and low overall differentiation (FST = 0.0105) with excess heterozygosity (FIS = -0.0592), consistent with obligate outcrossing. Guided by these resources, we designed a 28,913-SNP TWIST hybrid-capture panel enriched for genic regions and evenly distributed across seven chromosomes. This panel is being deployed within Auburn University’s crimson clover breeding program to support population improvement and cultivar development. The resulting genomic resources provide a reproducible, mid-density genotyping platform for trait discovery, predictive breeding, and diversity monitoring. Together, these advances bring crimson clover genomic resources on par with other legumes such as soybean ( Glycine max (L.) Merr .) and alfalfa ( Medicago sativa ), establishing a robust foundation for genomics-assisted improvement of this key cover and forage crop in U.S. sustainable agriculture. CORE IDEAS Whole-genome re-sequencing of 194 crimson clover accessions revealed &gt;21 M variants. High-confidence SNP catalogs from 50X and 2X data enable cost-effective genotyping. Genetic diversity is weakly structured, with cultivars clustering narrowly by origin. A 28,913 SNP enrichment panel delivers uniform genome coverage and &gt;75% genic content. These genomic tools accelerate GWAS, genomic selection, and breeding innovation.