| Summary: | The genus <i>Salix</i>, comprising some 400–500 species, is important in various alluvial or wet habitats of the northern hemisphere. It is a promising crop for applications such as biomass production, biofuels, or environmental projects. Clear species delimitation is crucial in ecology, biotechnology, and horticulture. DArTseq markers, a genome-wide technique, were tested for species and hybrid identification. A total of 179 willow samples were analysed, including six species of <i>Salix</i> subgen. <i>Salix</i> and four species of <i>Salix</i> subgen. <i>Vetrix</i>, including those used in biomass crop production, representing important European taxa. Identification of species-specific markers, clustering analyses (principal coordinate analysis, neighbor-joining) and Bayesian methods (Structure) unambiguously identified putative hybrids. In addition to demonstrating the high efficiency of DArT-seq markers in identifying willow hybrids, we also opened-up new questions about hybridisation processes and systematics. We detected unidirectional hybridisation between <i>S. alba</i> and <i>S. fragilis</i>, forming backcross hybrids, and we rejected the hypothesis that <i>S. fragilis</i> does not occur naturally in Europe. Further, the isolated position of <i>Salix triandra</i> within the genus was confirmed.
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