Abstract
Background/Objectives: Barley (Hordeum vulgare L.) is a major cereal crop in Kazakhstan; however, its productivity is frequently constrained by foliar diseases, particularly barley scald (BS) and net blotch (NB). Understanding the genetic diversity of barley germplasm and identifying resistance-associated alleles are essential for improving disease resistance in breeding programs. The objective of this study was to assess the genetic diversity and population structure in a collection of two-rowed spring barley accessions and to identify SSR alleles associated with resistance to BS and NB. Methods: A total of 86 two-rowed spring barley accessions were genotyped using 14 SSR markers. Phenotypic evaluation for BS and NB resistance was conducted under natural infection conditions across two environments in southeastern and southern Kazakhstan. Genetic diversity and population structure were analyzed using Neighbor-Joining (NJ) clustering, Principal Coordinate Analysis (PCoA), and STRUCTURE. Marker–trait associations were evaluated using MLM method. Results: Phenotypic assessments revealed significant environment-dependent variation in disease severity for both BS and NB. Population structure analyses consistently identified distinct genetic clusters within the collection. Seven significant (p < 0.05) allele–trait associations were detected. The Bmac209 176 bp allele exhibited the strongest association with NB severity at KRIAPG. Among the identified markers, Bmag206 246 bp was uniquely associated with reduced NB infection, whereas Bmag206 252 bp, Bmag613 176 bp, and HvLEU 186 bp were linked with higher susceptibility to NB and BS. Conclusions: The identified resistance- and susceptibility-associated SSR alleles provide useful diagnostic markers for marker-assisted selection and support the potential of allele pyramiding for developing barley cultivars with combined resistance to BS and NB. This study establishes a genetic framework to enhance barley disease resistance in Central Asian breeding programs.