Genetic and chemical diversity in Stevia rebaudiana Bertoni using microsatellite markers and HPLC analysis

Stevia rebaudiana Bertoni (2n=22) belonging to genus Stevia and family Asteraceae, is indigenous to South America but its social and economic potential has generated worldwide interest in its species. It is a medicinal and perennial plant and its important bioactive compounds present antibacterial, antifungal, anti-inflammatory, antiviral, anti-yeast, cardiotonic, and diuretic properties. Furthermore, its leaf tissues contain sweet steviol glycosides (mainly stevioside and rebaudioside A) that have proven to be a viable and more efficient alternative to other sweeteners. Genetic and/or environmental factors, such as season, altitude, radiation and soil nutrition, affect metabolic production. Thus, the efficient characterization of the germplasm and chemistry of these plants is extremely relevant. Currently, few genetic studies on this species are reported and, in most cases, use markers with various limitations such as RAPD, AFLP and ISSRs. In this study, a rapid and efficient protocol was tested to evaluate the genetic diversity of six S. rebaudiana greenhouse-grown cultivars, collected in July, using five microsatellite markers, including two functionally involved in steviol biosynthesis. Optimization of DNA extraction yielded an average DNA yield of 60 mu g g-1 of the fresh leaf where the purity was 1.82 and allowed an easy DNA amplification. Primers set was fluorescently labeled with 6-FAM and the amplification fragments generated reproducible bands and electropherograms profiles. In parallel the quantitative profile in stevioside and rebaudioside A was determined for each sample, using water extracts, purified, by SPE, by normal phase HPLC. Stevioside and rebaudioside A contents varied between 3.85 and 7.92% and 0.23-2.00%, respectively. The present study concluded that the genetic and chemical characterization of the S. rebaudiana plants is feasible by microsatellite and HPLC and is important for the selection and conservation of S. rebaudiana cultivars.