Red clover (Trifolium pratense L.) is a globally important temperate forage legume. Its symbiosis with soil-borne rhizobia enables nitrogen fixation, and its ability to produce quality forage under diverse soil conditions enhances pasture productivity, particularly during water deficits. With increasing climate-related stresses, harnessing adaptive traits absent in current cultivars is critical. Genebanks conserve diverse red clover germplasm, providing genetic variation for agronomic and adaptive traits. In this study, we introgressed novel germplasm into locally-adapted cultivars to track the inheritance of allelic variants using genotyping-by-sequencing (GBS). Multi-location, multi-year trials evaluated half-sib families two generations removed from the exotic germplasm (Gen 2) against local cultivars. Several Gen 2 populations matched or outperformed local cultivars, and exhibited a moderate family mean heritability (h² > 0.40) for most traits. Integrating genomic, phenotypic, and environmental data, 77 bioclimatic-associated SNPs were identified, of which 35 SNPs and 27 associated genes were significantly linked to trait expression. By using the original germplasm (Gen 0) as a training population and the half-sib families derived (Gen 2) as a validation population, genomic prediction models were developed to calculate prediction accuracies for key agronomic traits. Growth and plot density traits showed high predictive abilities and the highest prediction accuracies across generations. This study demonstrates a route by which genetic diversity from genebanks can be successfully incorporated into local populations, enabling evaluation and selection of key traits. The identified molecular markers and genomic prediction models provide a pathway to efficiently develop climate adaptive red clover cultivars.

Allotetraploid white clover formed during the last glaciation through hybridisation of two European diploid progenitors from restricted niches: one coastal, the other alpine. Here, we examine which hybridisation-derived molecular events may have underpinned white clover’s post-glacial niche expansion. We compared the transcriptomic frost responses of white clovers (inbred line and an alpine-adapted ecotype), extant descendants of its progenitor species and a resynthesised white clover neopolyploid to identify genes that were exclusively frost-induced in the alpine progenitor and its derived subgenomes. From these analyses we identified galactinol synthase, the rate-limiting enzyme in biosynthesis of the cryoprotectant raffinose, and found that the extant descendants of the alpine progenitor as well as the neopolyploid white clover rapidly accumulated significantly more galactinol and raffinose than the coastal progenitor under cold stress. The frost-induced galactinol synthase expression and rapid raffinose accumulation derived from the alpine progenitor likely provided an advantage during early post-glacial colonisation for white clover compared to its coastal progenitor.