Development and deployment of a public mid-density genotyping panel for crimson clover (Trifolium incarnatum L.) based on whole-genome re-sequencing of diverse germplasm.

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 whole-genome re-sequencing of global crimson clover germplasm to (i) develop a public mid-density enrichment capture panel for breeding applications and (ii) characterized genetic diversity among the sampled genotypes to validate the representativeness of the germplasm for panel design. From 45 crimson clover accessions, we sequenced 194 genotypes: 45 at ∼50X, generating 5.84 million variants and 149 at ∼2.54X, yielding 17.05 million variants. After filtering, we retained 542,790 high-confidence single-nucleotide polymorphisms (SNPs) from the high-coverage dataset and ∼2.4 million from the low-pass cohort, respectively. From these resources, we designed a 28,867-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. Genetic diversity analyses (principal component analysis, ADMIXTURE) revealed compact clustering of cultivars, broader dispersion of wild and uncertain-status accessions, and low overall differentiation (fixation index [FST] = 0.0105) with excess heterozygosity (inbreeding coefficient [FIS] = -0.0592), consistent with a high level of outcrossing. 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 L.), establishing a robust foundation for genomics-assisted improvement of this key cover and forage crop in US sustainable agriculture.