Genotyping systems for Eucalyptus based on tetra-, penta-, and hexanucleotide repeat EST microsatellites and their use for individual fingerprinting and assignment tests

Eucalypts are keystone species in their natural ranges and are extensively planted worldwide for high-quality woody biomass. A novel set of 21 polymorphic and interspecifically transferable microsatellite markers based on tetra-, penta- and hexanucleotide repeats were developed and tested for high-precision genotyping of species of Eucalyptus. These microsatellites were characterized in population samples of four species, Eucalyptus grandis, Eucalyptus globulus, Eucalyptus urophylla, and Eucalyptus camaldulensis, representing three phylogenetic sections of subgenus Symphyomyrtus. These markers provide a clear advantage for accurate allele calling due to their larger allele size difference. Two multiplexed microsatellite combinations, a 14-locus/four-dye and an 18-locus/five-dye set, analyzable in single lanes were designed, providing resolution and throughput analogous to those routinely used in human DNA profiling. This set of microsatellites was shown to have high resolution for clone fingerprinting, inter-individual genetic distance estimation, species distinction, and assignment of hybrid individuals to their most likely ancestral species. These systems will be particularly useful for comparative population genetics and molecular breeding applications that require consistent allele calling across different points in time or laboratories.