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

ABSTRACT

Crimson clover (Trifolium incarnatum L.) is an obligately outcrossing, cool-season annual legume valued for forage 52 and cover cropping, yet genomic resources to support systematic improvement are limited. We performed the first and 53 most comprehensive whole-genome resequencing (WGR) of global crimson clover germplasm to (i) characterize 54 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 56 accessions sequenced at ~2.54X yielded 17.05 million variants. After stringent filtering, we retained 542,790 high57 confidence SNPs from the high-coverage dataset and ~2.4 million from the low-pass cohort. Population analyses 58 (PCA, ADMIXTURE) revealed compact clustering of cultivars, broader dispersion of wild and uncertain-status 59 accessions, and low overall differentiation (FST = 0.0105) with excess heterozygosity (FIS = -0.0592), consistent with 60 obligate outcrossing. Guided by these resources, we designed a 28,913-SNP TWIST hybrid-capture panel enriched 61 for genic regions and evenly distributed across seven chromosomes. This panel is being deployed within Auburn 62 University’s crimson clover breeding program to support population improvement and cultivar development. The 63 resulting genomic resources provide a reproducible, mid-density genotyping platform for trait discovery, predictive 64 breeding, and diversity monitoring. Together, these advances bring crimson clover genomic resources on par with 65 other legumes such as soybean (Glycine max (L.) Merr.) and alfalfa (Medicago sativa), establishing a robust 66 foundation for genomics-assisted improvement of this key cover and forage crop in U.S. sustainable agriculture.