Genetic Architecture of Angular Leaf Spot Resistance in Cultivated Strawberry Shaped by Epistasis and Genotype-by-Environment Interactions

Abstract Angular leaf spot (ALS), caused by Xanthomonas fragariae, is a bacterial disease that limits strawberry (Fragaria ananassa) productivity worldwide. Although major resistance loci have been identified in wild Fragaria species, their introgression into elite germplasm remains constrained by linkage drag and inconsistent inheritance. To dissect the genetic architecture of ALS resistance, we evaluated a diverse panel of n = 241 greenhouse-tested and n = 468 field-tested strawberry accessions representing elite cultivars, breeding materials, and heirloom varieties of cultivated strawberry (F. ananassa). Plants were evaluated under controlled inoculations and natural field infection using a standardized 1–5 ordinal scale and genotyped with the FanaSNP 50K Affymetrix Axiom® SNP array. Broad-sense heritability was moderate (H² = 0.45), whereas narrow-sense heritability was low (h² = 0.10), reflecting the contribution of non-additive genetic effects such as dominance and epistasis. Genome-wide association studies identified moderate effect loci on chromosomes 1D and 2D, explaining 5% and 18% of phenotypic variance, respectively. A segregating F1 population validated the 2D locus, confirmed by co-localization with the GWAS signal. Two-dimensional QTL analysis in this F1 population revealed a significant epistatic interaction between locus on chromosome 2D and an additional locus on chromosome 6B, collectively explaining 15% of the phenotypic variance. Together, these results demonstrate that non-additive effects in these populations control ALS resistance. Understanding this complexity provides a foundation for developing elite cultivars with durable resistance, such as ‘UCD Royal Royce’. Our findings underscore the need for predictive breeding strategies that capture epistatic and environmental variance to accelerate genetic gain for ALS resistance in strawberry. Figures Supplementary Figure S1. Field symptom scores for angular leaf spot. Representative field images illustrating the 1–5 disease severity scale used in open-field assessments (1 = no visible symptoms; 2 = few small lesions; 3 = moderate lesions; 4 = extensive, coalescing lesions; 5 = severe, widespread lesions with chlorosis/necrosis). Supplementary Figure S2. Field scoring in the F₁ validation population. (A) Histogram of plant-level disease severity scores (1–5), showing quantitative segregation with most plants scoring 2–3 and few at the extremes. (B) Representative field photo used for scoring calibration, showing characteristic angular leaf spot lesions on a plant with intermediate severity (score 3). Files Supplemental File S1. The physical positions of 50K and 850K Axiom array SNPs corroborated by genetic mapping. SNPs were physically anchored to the 'Camarosa' (FaCA1; \cite{edger2019origin}; https://phytozome-next.jgi.doe.gov/info/Fxananassa_v1_0_a1) and ‘UCD Royal Royce’ (FaRR1; https://phytozome-next.jgi.doe.gov/info/FxananassaRoyalRoyce_v1_0) genomes in silico. Chromosomes were numbered using the nomenclature of \cite{hardigan2020genome} and cross-referenced to the nomenclature of \cite{edger2019origin}. This database includes the physical positions of SNPs identified by BLAST in the 'Camarosa' and 'Royal Royce' genomes, DNA sequences of the SNP probes, chromosome assignments and physical positions of SNPs corroborated by genetic mapping, and associated information. Supplemental File S2. Pedigree of the F1 mapping population. This file provides the detailed pedigree structure for the F1 family derived from the cross between the susceptible parent 19C164P045 and the resistant parent 21A137P047. The pedigree includes all known ancestral lines contributing to each parent, enabling tracking of shared founders, inheritance patterns, and potential sources of allelic variation relevant to ALS resistance Supplemental File S3. Two-dimensional QTL analysis and significant epistatic interactions. This file summarizes the results of the two-dimensional genome scan used to detect pairwise QTL interactions underlying ALS resistance. Supplemental File S4. Raw phenotypic data for the greenhouse diversity panel. This file contains the unprocessed disease severity scores collected from the greenhouse evaluation of the cultivated strawberry diversity panel. Data include plant identifiers, block assignments, tagged leaf scores at 7, 14, and 28 days post inoculation, and corresponding severity values. Supplemental File S5. Raw phenotypic data from the field panel evaluation. This file provides the field disease severity scores for the cultivated strawberry diversity field panel. Supplemental File S6. Estimated marginal means and AUDPS-AUDPC metrics for the greenhouse panel.This file reports the estimated marginal means (EMMs) generated from mixed-model analyses of disease severity across the greenhouse evaluation, along with area under the disease progress stairs (AUDPS) and area under the disease progress curve (AUDPC) values for each genotype. These summary statistics were used to quantify disease progression, compare genotypic performance, and support GWAS and multivariate analyses. Supplemental File S7. Genotypic data for the greenhouse diversity panel. This file includes the genotype matrix used for GWAS and multivariate analyses. SNPs are coded as (1) for the reference allele, (0) for heterozygotes, and (-1) for the alternative allele. Supplemental File S8. Genotypic data for the field panel. This file contains the filtered SNP genotype matrix used for field-based GWAS and validation analyses. Marker data are coded as (1) for the reference allele, (0) for heterozygotes, and (-1) for the alternative allele. Supplemental File S9. Genotype and phenotype data for the F1 mapping population with the calculated genetic map. This file integrates the SNP genotype matrix, disease severity phenotypes, and the final genetic map for the F1 population derived from 19C164P045 × 21A137P047. Map positions, linkage groups, marker orders, and inter-marker distances are included. These data support QTL mapping, epistasis analysis, and comparison with GWAS-identified loci. Supplemental File S10. The full SNP-level results from all association analyses conducted in this study. The file includes three sheets: Sheet 1 – Greenhouse MV-GWAS panel - SNP-level GWAS results for the greenhouse panel, including the marker name, the SNP position relative to the reference genome, and the p-value. Sheet 2 – Field GWAS panel - SNP-level GWAS results for the field panel, reported with the same information: marker name, genomic position, and p-values for each trait. Sheet 3 – F1 QTL panel - SNP-level results from the QTL analysis of the F1 mapping population, including the scored allele, marker position, and statistical significance values for each trait.