Identificação molecular e criopreservação de microalgas verdes (chlorophyta) isoladas de águas continentais brasileiras

Abstract

Microalgae are photosynthetic unicellular organisms that have eukaryotic cell structure and occur in colonial or free forms. These organisms have been widely studied in the application of bioremediation and in biorefineries. Their biotechnological potential stands out in the production of biofuels and bioproducts, because they have features like high growth rate and high storage capacity of reserve substances, such as lipids and starch. Collections of genetic resources and breeding programs have as a prerequisite the identification and the maintenance of the organisms in an inactive metabolic state. Thus, two molecular markers, the chloroplastid gene rbcL and the ITS2 region of the nuclear ribosomal DNA were used as DNA barcodes for identification of microalgal strains collected from Brazilian continental waters, deposited in Embrapa’s Collection of Microorganisms and Microalgae Applied to Agroenergy and Biorefineries. For the genetic resources maintenance in an inactive metabolic state, it was applied the cryopreservation method combined with a slow cooling strategy, using the chemical compounds methanol and dimethyl sulfoxide (DMSO) as cryoprotectans agents. The ITS2 region could be amplified and sequenced successfully in 48 (94%) of the samples using a pair of universal primers available in the literature. On the other hand, new sets of primers had to be designed for the rbcL gene, which allowed the sequencing of 49 (96%) of the samples. Similar levels of nucleotide diversity were observed among the ITS2 (0.472) and rbcL (0.461) sequences, suggesting a similar potential for taxa discrimination. However, the results indicates that the ITS2 should be used as a primary marker, and rbcL as an auxiliary marker for the identification of green microalgae. Cryopreservation tests showed that it is possible to use this method for maintaining Brazilian continental microalgae in an inactive metabolic state using DMSO in 10% concentration as a cryoprotectant agent.