Introduction Rye (Secale cereale L.) is a cereal crop well adapted to marginal environments, where traditional populations represent valuable reservoirs of genetic diversity. In the Aosta Valley (NW Italy), several local rye populations have historically been maintained under small-scale farming systems, but their genetic structure has not yet been investigated using genome-wide approaches. Methods We performed a high-resolution population genomic analysis of twelve rye populations using a K-seq genotyping approach. A genome-wide SNP dataset was generated and used to assess genetic diversity and population structure through complementary multivariate, distance-based, and model-based analyses. Results Substantial genetic variation was detected within populations (HT = 0.354), together with a moderate level of genetic differentiation among populations (GST = 0.254). Populations collected in the 1980s showed consistently higher genetic diversity and differentiation (HT = 0.392; GST = 0.311) than populations collected in 2000 (HT = 0.306; GST = 0.180), suggesting a reduction in genetic variability in more recent germplasm. Multivariate and clustering analyses identified distinct genetic groups and clusters of closely related populations, revealing a complex genetic structure shaped by both differentiation and admixture. Discussion These findings provide an updated view of the genetic architecture of Aosta Valley rye populations and underline the importance of conserving genetically diverse and differentiated populations. More broadly, the study demonstrates the utility of K-seq for generating informative SNP datasets and investigating population structure in rye, supporting its application to the characterization and management of plant genetic resources.
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