To assess the genetic diversity of the primary Qingke cultivars and establish their unique genetic profiles, 837 barley simple sequence repeat (SSR) primers were screened across 12 cultivars. The selection process involved the utilization of polyacrylamide gel electrophoresis and capillary electrophoresis technology, to identify primers exhibiting desirable characteristics, such as polymorphism, stability, and reproducibility. Subsequently, we analyzed the genetic diversity of the primary Qingke cultivars to for DNA fingerprints. A total of 18 pairs of SSR markers were selected as the optimal markers for constructing fingerprints of major Qingke cultivars. These included 83 observed alleles (Na), ranging from there to 11, with an average of 4.61 per pair. Notably, Bmag0496 and Scssr04163 exhibited higher allelic diversity, with 11 and 8 loci, respectively. The polymorphism information content (PIC) ranged from 0.36 to 0.74, with an average of 0.52. The expected heterozygosity (He) ranged from 0.4031 to 0.7682, with an average of 0.59, and the observed heterozygosity (Ho) varied between 0.13 and 0.67, with an average of 0.32. The outcomes obtained through phylogenetic tree analysis, population structure assessment and principal component analysis demonstrated that the primary Qingke cultivars could be classified into three distinct groups: group I primarily originated from Xizang and Qinghai provinces; group II mainly consisted of cultivars from Yunnan and Heilongjiang provinces; and group III predominantly comprised cultivars originating from Qinghai and Gansu provinces. Interestingly, the Sichuan cultivars were distributed across all three groups without any clear tendency toward a specific cluster or subgroup. These findings indicated that the genetic distance among Qingke cultivars was significantly correlated with geographic location but not exclusively determined by it. The construction of DNA fingerprints for the primary Qingke cultivars used these identified sets of SSR primers (18 pairs) laid a solid foundation for cultivar identification, conservation and utilization efforts related to this crop.
