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

Castillo, M. P.; Oyebode, O. G.; Talag, J.; Bunting, V.; Kaur, N.; Doran, P.; Barry, K.; Schmutz, J.; Schlautman, B.; Humphries, A.; Ghamkhar, K.; Moore, V.; Rios, E.; Harkess, A.; Wolfe, M.

doi:10.64898/2026.01.26.701603

bioRxiv · 10.64898/2026.01.26.701603

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

Castillo, M. P.·Oyebode, O. G.·Talag, J.·Bunting, V.·Kaur, N.·Doran, P.·Barry, K.·Schmutz, J.·Schlautman, B.·Humphries, A.·Ghamkhar, K.·Moore, V.·Rios, E.·Harkess, A.·Wolfe, M.

View original source ↗Export metadata ↓

Abstract

AO_SCPLOWBSTRACTC_SCPLOWCrimson 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 Universitys 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 IDEASO_LIWhole-genome re-sequencing of 194 crimson clover accessions revealed >21 M variants. C_LIO_LIHigh-confidence SNP catalogs from 50X and 2X data enable cost-effective genotyping. C_LIO_LIGenetic diversity is weakly structured, with cultivars clustering narrowly by origin. C_LIO_LIA 28,913 SNP enrichment panel delivers uniform genome coverage and >75% genic content. C_LIO_LIThese genomic tools accelerate GWAS, genomic selection, and breeding innovation. C_LI

Explore related subjects

genomics Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Castillo, M. P., Oyebode, O. G., Talag, J., Bunting, V., Kaur, N., Doran, P., Barry, K., Schmutz, J., Schlautman, B., Humphries, A., Ghamkhar, K., Moore, V., Rios, E., Harkess, A., Wolfe, M.. 2026-01-28. 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. https://doi.org/10.64898/2026.01.26.701603

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

RNA isoform-resolved multiplexed sequencing with bioorthogonal barcoding

RNA isoform dysregulation drives disease pathogenesis and is the target of FDA-approved splice-switching therapeutics. However, multiplexed sequencing methods discard splice junction information because only 3' termini are barcoded and counted. Here, we repurpose acylation and click chemistries to conjugate bioorthogonal barcodes (bobcodes) directly onto multiple internal positions along cellular RNAs. Bobcoded RNAs from multiple samples are pooled for multiplexed cDNA synthesis, during which reverse transcriptase switches from each RNA template onto its tethered bobcode with greater than 99% accuracy in species mixing experiments. Bobcode attachment intervals set cDNA insert sizes without a library fragmentation step, and priming with poly(dT) or random hexamers selects between 3'-end counting and full-length isoform capture. A bioorthogonal barcode-sequencing (BOB-seq v0.1) drug screen identifies transcriptome-wide on- and off-target RNA splicing effects and outperforms existing multiplexing RNA sequencing methods in workflow simplicity, sample-to-sample variability, and barcoding accuracy. Bobcodes add isoform resolution to scalable multiplexed RNA sequencing.

genomics↗

bioRxiv2026

Integrative Nanopore and Illumina sequencing reveals age-associated tRNA modification and CCA-tail dynamics in yeast

Aging is characterized by a progressive loss of proteostasis. Transfer RNAs (tRNAs) are essential regulators of translation, yet their dynamics during aging remain poorly understood due to challenges in sequencing highly modified RNAs. Here we present a benchmarked Nanopore direct RNA sequencing (RNA004 chemistry) resource that profiles the Saccharomyces cerevisiae tRNAome during replicative aging at single-molecule resolution. Using in vitro transcribed tRNA controls, we establish modification detection thresholds and validate key findings with orthogonal Illumina sequencing. While overall tRNA abundance remains largely stable, our resource reveals age-associated terminal A cleavage at the 3' CCA tail of mature tRNAs, targeted T-loop and anticodon modification changes, and single-molecule evidence of modification co-occurrence. This dataset provides a resource for exploring tRNA regulation, translation fidelity, and longevity.

genomics↗

bioRxiv2026

A hydrogen-producing mitochondrion in an anaerobic eukaryotrophic rhizarian

Diverse eukaryotes thrive under low oxygen conditions, in part through highly modified mitochondrion-related organelles (MROs) that use alternate metabolic pathways to support ATP production and cofactor recycling. Anaerobic lifestyles have evolved repeatedly across the eukaryotic tree of life, each providing an independent opportunity to understand how eukaryotes adapt to life in low oxygen conditions. Here, we use single-cell transcriptomics to reconstruct the MRO metabolism of PCE SSF, a benthic eukaryotrophic flagellate and the first cultivated representative of Novel Clade 12 (NC12; Rhizaria), an independently anaerobic rhizarian lineage. PCE SSF possesses an anaerobic hydrogen-producing mitochondrion capable of hydrogenosome-type substrate-level phosphorylation. It also retains a nearly complete but likely branched tricarboxylic acid pathway that lacks citrate synthase and malate dehydrogenase. The function of citrate synthase may instead be fulfilled by the typically cytosolic ATP citrate lyase, previously reported in this context only in the anaerobic cercozoan, Brevimastigomonas motovehiculus. Unlike B. motovehiculus, however, PCE SSF retains only Complex II and the NuoE/NuoF subunits of the electron transport chain and lacks a mitochondrial genome. Together, these features indicate an atypical and reduced mitochondrial metabolism, highlighting the diversity of evolutionary solutions to anaerobic energy metabolism in eukaryotes.

genomics↗