| A total of 100 SSR primers from University of British Columbia Biotechnology Lab (UBCBL) primer set UBC#9 was used for PCR amplification from a sample set of 11 genotypes including individuals of S. spontaneum, S. sinense, S. barberi, S. robustom, S. officinarum, and other wild relatives, Erianthus rockii, E. arundinaceus and Miscanthus floridulus. The PCR reactions were performed in 96-well microtiter plates using a Perkin Elmer 9600 thermocycler. Amplification products were resolved in 1.5% agarose gels and banding patterns compared among the 11 genotypes. Of the 100 primers tested, 32 produced clear banding patterns that could be easily scored. Nineteen of these 32 primers were designed to dinucleotide repeats, three to trinucleotide repeats, two to tetranucleotide repeats, three to pentanucleotide repeats. Four primers designed to 5抎egenerate motifs also yielded discrete repeats. Primers based on (AC)~ produced the highest level of polymorphism, followed by S抎egenerate motifs and (GT)~ motifs. All primers designed to (AG)~ motifs and 5 of six (TC)~ motifs gave polymorphic bands indicating that in Sugarcane, (AG)~ or (TC)~ is the most abundant. PCR amplification using ISSR primers produced 9.5 bands, on average, for species amongst genus Saccharum and 11.6 for related genera.A similarity matrix and a dendrogram were generated using the similarity coefficient with a gel analysis software, Bio1D~. The 11 Saccharum genotypes were clustered into two distinct groups. The first group is composed of one S. officinaruni clone, one S. barberi clone, three S. robustom clones, and two S. sinense clones. Another group is composed solely of one S. spontaneum clone.Fifteen S. spontanewn clones were amplified with a subset of 7 primers. Four S. offlcinarum, three F. arundinaceus and three hybrids that are descendants from Badila and F. arundinaceus were studied by 20 of the 32 primers. The characteristic features of the ISSR markers, i.e., repeatability, polymorphism, and easy handing, suggest their potential applicability to classification, phylogenetic analysis and construction of PCR-based genome maps in Sugarcane. |
