An Optimised Protocol for Fluorescent-dUTP Based SSR Genotyping and its Application to Genetic Mapping in Eucalyptus

Integration of fluorescent-dUTP in polymerase chain reaction (PCR) appears to be a sound method for fluorescence labelling of amplicons in genotyping with simple sequence repeats (SSRs) using an automated sequence analyser. However, the method has not been explored in terms of performance optimisation and cost control. In this paper, we optimised the protocol for fluorescent-dUTP based SSR genotyping in a case study with Eucalyptus. A combination of low dNTP concentration (25 μM each) in PCR reaction and a touchdown PCR programme contributed to increase dramatically the fluorescent intensity of SSR amplicons, thereby facilitating accurate and multiplexed scoring of SSR alleles. The usefulness of the optimised protocol was demonstrated in its application to genetic mapping of SSR loci onto E. urophylla and E. tereticornis linkage maps constructed previously. The protocol optimised here would provide a reliable and economical assay for sequencer-based SSR genotyping in a wide range of biological applications.