A multiplex assay with 21 single nucleotide polymorphisms for commercial Eucalyptus clones identification

Abstract Eucalyptus is the most widely cultivated hardwoodacross tropical, sub-tropical and temperate regions worldwide. However, with successive generations of tissue culture and the rapid expansion of plantations, clone mislabeling and quality decline have become increasingly problematic. In this study, 21 highly polymorphic single nucleotide polymorphism (SNP) markers were identified and characterized across 58 Eucalyptusclones using SNaPshot technology. The polymorphic information content ranged from 0.064 to 0.375, observed heterozygosity from 0.068 to 0.703, and expected heterozygosity from 0.067 to 0.503. We further established a single-tube, multicolor fluorescent SNP-detection assay that enables rapid and accurate clone authentication. These findings can be useful for identifying fake or deranged clones, safeguarding germplasm resources, and supporting future genomic and breeding studies.