Characterization of new polymorphic functional markers for sugarcane.

Expressed sequence tags (ESTs) offer the opportunity to exploit single, low-copy, conserved sequence motifs for the development of simple sequence repeats (SSRs). The authors have examined the Sugarcane Expressed Sequence Tag database for the presence of SSRs. To test the utility of EST-derived SSR markers, a total of 342 EST-SSRs, which represent a subset of over 2005 SSR-containing sequences that were located in the sugarcane EST database, could be designed from the nonredundant SSR-positive ESTs for possible use as potential genic markers. These EST-SSR markers were used to screen 18 sugarcane (Saccharum spp.) varieties. A high proportion (65.5%) of the above EST-SSRs, which gave amplified fragments of foreseen size, detected polymorphism. The number of alleles ranged from 2 to 24 with an average of 7.55 alleles per locus, while polymorphism information content values ranged from 0.16 to 0.94, with an average of 0.73. The ability of each set of EST-SSR markers to discriminate between varieties was generally higher than the polymorphism information content analysis. When tested for functionality, 82.1% of these 224 EST-SSRs were found to be functional, showing homology to known genes. As the EST-SSRs are within the expressed portion of the genome, they are likely to be associated to a particular gene of interest, improving their utility for genetic mapping; identification of quantitative trait loci, and comparative genomics studies of sugarcane. The development of new EST-SSR markers will have important implications for the genetic analysis and exploitation of the genetic resources of sugarcane and related species and will provide a more direct estimate of functional diversity.

Transformation and expression of a gene for herbicide resistance in a Brazilian sugarcane.

Embryogenic calli of the Brazilian sugarcane (Saccharum officinarum L.) genotype SP80-180 were transformed with two plasmids containing genes coding for neomycin phosphotransferase (neo) and phosphinotrycin acetyltransferase (bar), by particle bombardment using an apparatus developed at Copersucar Technology Center. Transformed plants were initially selected on culture medium containing Geneticin, and resistance was confirmed by localized application of a kanamycin solution to leaves of hardened plants at the nursery. A commercial formulation of ammonium gluphosinate was sprayed twice on these antibiotic-resistant plants. The resistant plants were considered co-transformed, and Southern analysis confirmed stable integration of both bar and neo genes. In addition, phosphinotrycin acetyltransferase expression was supported by RT-PCR analysis and neomycin phosphotransferase presence was demonstrated by western blotting. Similar analyses were also performed with micropropagated transformants after three cycles of subculture.