DNA extraction and RAPD markers to assess the genetic similarity among Gelidium sesquipedale (Rhodophyta) populations

A simple method developed for genomic DNA isolation from fungus was tested on the red alga, Gelidium sesquipedale (Clem.) Born et Thur., which is commercially exploited for its high sulfated polysaccharide (agar) content. This method is faster, cheaper, and less toxic than conventional phenol/chloroform methods. Random amplified polymorphic DNA (RAPD) amplifications were performed successfully without the necessity of purifying the DNA. RAPD markers were used to investigate the genetic similarity among three natural populations of G. sesquipedale from southern Portugal. Bulked-genomic DNA samples of 15 different individuals were made in each population. These can be conceived of as a sample of the population DNA. Of the 62 primers screened, 41 produced bands and 22 revealed polymorphisms. Genetic similarities among populations were high. Populations that are further away from each other have the lowest similarity coefficients, whereas the intermediate Ingrina population, located on the south coast, showed higher genetic similarity with the Odeceixe population located on the southwest coast, than with the Sao Rafael southern population. This suggests a higher genetic flow between Odeceixe and Ingrina or the result may be a founder effect in the sense that the species has propagated from the east coast to the south coast of Portugal. We conclude that the use of this isolation method with RAPD analysis is appropriate to characterize the genetic variability of this commercial species along its geographical distribution. Large sample sizes can be screened at a relatively low cost. Finding genetic markers for commercial populations of C. sesquipedale may be of industrial interest.

Charged pullulan derivatives for the development of nanocarriers by polyelectrolyte complexation

Pullulan, a neutral polysaccharide, was chemically modified in order to obtain two charged derivatives: reaction with SO3(.)DMF complex afforded a sulfate derivative (SP), while reaction with glycidyltrimethylammonium chloride gave a quaternary ammonium salt (AP). The presence of the charged groups was confirmed by FTIR. Assessment of the positions where the reaction took place was based on (1)H- and (13)C NMR (COSY, HSQC-TOCSY, HSQC-DEPT, and HMBC) experiments. Estimation of the degree of substitution (DS) was made from elemental analysis data, and further confirmed by NMR peak areas in the case of AP. These new derivatives showed the capability to condense with each other, forming nanoparticles with the ability to associate a model protein (BSA) and displaying adequate size for drug delivery applications, therefore making them good candidates for the production of pullulan-based nanocarriers by polyelectrolyte complexation.