Development of simple sequence repeat (SSR) markers in Eucalyptus from amplified inter-simple sequence repeats (ISSR)

Eucalyptus spp. are widely used in exotic plantations. Since many of these trees are derived from vegetative propagation, the routine identification of clones has become increasingly important. The most widely used molecular based method for fingerprinting these clones is by random amplified polymorphic DNAs (RAPDs). Although this technique is useful, its results are not very repeatable, especially between laboratories. The aim of this study was to develop microsatellite markers that are highly repeatable, and to investigate their value in Eucalyptus fingerprinting. Typically, this process involves the expensive procedure of constructing an enriched genomic library. However, we used an intersimple sequence repeat (ISSR) polymerase chain reaction (PCR)-based enrichment technique for microsatellite-rich regions. With this relatively inexpensive method, microsatellite-rich regions were amplified directly from genomic DNA, after which PCR products were cloned and sequenced. From these microsatellite-rich sequences, primer sets were constructed to amplify mono-, di-, tri-, hexa-and nona-nucleotide repeats. These markers were all inherited in a Mendelian fashion in the progeny of a test cross between two Eucalyptus grandis trees. The primer sets developed were also able to amplify the corresponding microsatellite loci from five different Eucalyptus spp., namely E. grandis, E. nitens, E. globulus, E. camaldulensis and E. urophylla.