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Eucalyptus is the second largest forestation species and the most important raw wood pulp tree in the world. Eucalyptus consists of many species including 522 species and 150 varieties. It’s easy to breed the excellent hybrids from the different eucalyptus species, and easy to breed the superior clones by vegetative propagation, however, their genetic relationship and species identification is very difficult.ISSR technique has been widely applied to many research aspects, such as tree germplasm identification, inheritance mapping, gene positioning, genetic diversity, evolution, phylogeny, molecular marker breeding and so on. ISSR technique will be used in more tree species and forestry fields by its advantages including high polymorphism, strong stability, simple technology and convenience.In this paper, four kinds (total 18 samples) of Eucalyptus, including E. camaldulensis, E. globulus, E. tereticornis and E. bark were used as the materials to analyze the parameters which will affect eucalyptus ISSR-PCR, including six factors:templet DNA, primer concentration, dNTPs, Mg2+ concentration, Taq DNA polymerase dosage, annealing temperature. The improved 1SSR-PCR reaction system (total 20μL) was established as:1 x Taq buffer,0.5 U Taq DNA polymerase,0.6μmol/L primer, 100μmol/L dNTPs,2.5 mmol/LMgCl2,20ng template DNA. The optimal ISSR-PCR cycle condition: 94℃3min; 94℃30sec,60℃45sec,72℃1min,5 cycles; 94℃30sec,58℃45sec,72℃1min,5 cycles; 94℃30sec,56℃45sec,72℃1min,5 cycles;94℃30sec,54℃45sec,72℃1min,10 cycles; 94℃30sec,52°45sec,72℃1min,20 cycles; 72℃10min, 4℃hold.According to the improved ISSR-PCR reaction system and amplification conditions,12 primers were screened from 100 ramdon primers, which can yield specific bands with moderate band numbers, clear background and well reproducibility.Total 120 specific and reproducible DNA bands were obtaied, of which 93 are polymorphic bands. Each primer can yield bands spaning 300 2500 bp, and the band number varies from 4 to 11 (average 7.75 and 77.5% polymorphism). Biosofteares including POPGENE 1.31, MVSP 32 and NTSYS-pc were used to analyze the obtained polymorphic bands, and hence to yield the genetic similarity coefficient of the four sepcies (18 samples) and map the related graphics.The result showed that the genetic similarity between the four kinds can be clear distincted easily, and that the genetic similarity between species is very high.In oder to develop STS markers from the ISSR-derived bands, some specific bands from ISSR-PCR of Eucalyptus were recoverd and cloned, followed by sequencing. According to the sequence information,19 pairs of primers were designed to amplify the Eucalyptus genomic DNA. The result showed that only 6 pairs of specific primers yield the interest bands. The result will provide a foundation for the development of STS markers for Eucalyptus.
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