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

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This study performed the first comprehensive whole-genome resequencing of global crimson clover germplasm to characterize genetic diversity and population structure and to develop a public mid-density enrichment genotyping panel for breeding. The authors generated variant datasets from 45 accessions sequenced at ~50X (5.84 million variants) and 149 additional accessions sequenced at ~2.54X (17.05 million variants), then applied stringent filtering to retain 542,790 high-confidence SNPs from the high-coverage set and ~2.4 million from the low-pass cohort. Population analyses using PCA and ADMIXTURE showed compact clustering of cultivars, broader dispersion of wild/uncertain-status accessions, low differentiation (FST = 0.0105), and excess heterozygosity consistent with obligate outcrossing, while the paper’s main limitation is that much of the diversity inference relies on a mixed-depth resequencing design with different variant counts per cohort. Based on these resources, they designed a 28,913-SNP TWIST hybrid-capture panel targeting genic regions and evenly distributed across seven chromosomes. The paper does not explicitly discuss endometriosis or adenomyosis; it was included in the corpus via a keyword match in the upstream search index.

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Abstract

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 >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 >75% genic content. These genomic tools accelerate GWAS, genomic selection, and breeding innovation.
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Abstract 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 >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 >75% genic content. These genomic tools accelerate GWAS, genomic selection, and breeding innovation. Competing Interest Statement The authors have declared no competing interest. Footnotes Marnin Wolfe mdw0092{at}auburn.edu ABBREVIATIONS - WGR - whole-genome re-sequencing - LD - linkage disequilibrium - PYT - Preliminary Yield Trial - MAF - minor allele frequency - PCA - principal component analysis - SNP - single-nucleotide polymorphism - SPT - Space Plant Trial - BLUP - Best Linear Unbiased Predictor - AU - Auburn University - UF - University of Florida - PYT - Preliminary Yield Trial - DR2 - Dosage coefficient of determination (Beagle imputation accuracy metric) - AUHPC - Auburn University High Performance Computing - JGI - Joint Genome Institute - WGS - Whole genome sequencing.

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