Using different bioinformatic criteria, the SUCEST database was used to mine for simple sequence repeat (SSR) markers. Among 42,189 clusters, 1,425 expressed sequence tag-simple sequence repeats (EST-SSRs) were identified in silico. Trinucleotide repeats were the most abundant SSRs detected. Of 212 primer pairs selected, based on repeat patterns of n≥8 for di-, tri-, tetra-and penta-nucleotide repeat motifs verified using 15 sugarcane (Saccharum spp.) genotypes and marker segregation using F1

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... ogenies of a cultivated sugarcane and Saccharum spontaneum, 191 loci were identified. All new EST-SSR loci detected a total of 1,529 markers ranging from 2 to 21 markers per locus, with an average of eight markers per locus. Observed polymorphism ranged from 0.12 to 0.93 with a mean of 0.74. A total of 426 and 333 markers were putatively identified as simplex in the cultivated sugarcane and S. spontaneum, respectively and corresponding to 2.23 and 1.74 markers per primer, respectively while 167 markers were identified as double-simplex markers, with 0.87 markers per primer. Cluster analysis revealed a high genetic similarity among the sugarcane (Saccharum spp.) breeding lines which could reduce the genetic gain in sugarcane breeding.