Genomic selection in timothy (Phleum pratense L.): a comprehensive evaluation of prediction models, multi-trait strategies, and forward validation across Norwegian environments

KEY MESSAGE: This study presents a comprehensive evaluation of genomic selection (GS) in timothy (Phleum pratense L.), comparing nine prediction models across yield and quality traits at two Norwegian locations. Forward validation with independent full-sib (FS2) families revealed a substantial generalization gap, highlighting the need for realistic accuracy assessment in polyploid forage breeding. Timothy (Phleum pratense L.) is the most important forage grass in Northern Europe, yet genomic selection has not been systematically evaluated in this hexaploid species. We assessed 889 FS2-families originating from biparental crosses among 49 cultivars/populations. The FS2-families were genotyped with 30,698 SNP markers derived from genotyping-by-sequencing (GBS) and field tested for three harvest years at a highland and a lowland continental location in Southern Norway. Nine genomic prediction models were compared for six yield traits (dry matter yield per cut and total) and six quality traits (protein, digestibility, and fiber fractions) across three cuts/year. Within-training cross-validation accuracies were moderate to high (mean r = 0.62), with Random Forest and SVR consistently outperforming GBLUP. However, forward validation using 213 independent FS2-families revealed dramatically lower accuracies (mean r = 0.16), with only 16 of 30 trait-dataset combinations reaching statistical significance (p < 0.05). Genomic heritabilities (GREML), estimated across environments, ranged from near zero for the quality traits to 0.55 for the yield traits. Multi-trait models improved accuracy by 3-5% over single-trait approaches, while FS2 families-by-environment interaction models with Random Forest achieved the highest within-training accuracy (mean r = 0.71). Marker density analysis showed accuracy plateauing at approximately 15000 SNPs. Genetic correlations among the yield component traits were estimated by multi-trait REML; correlations among the quality traits could not be estimated reliably because their genomic heritabilities were low. A multi-trait selection index identified top-performing FS2-families for further crossing recommendations. These results provide a benchmark for GS implementation in hexaploid timothy and emphasize that cross-validation substantially overestimates prediction accuracy for truly independent material.