The hygromycin B phosphotransferase gene from Escherichia coli and the pyrithiamine resistance gene from Aspergillus oryzae are two dominant selectable marker genes widely used to genetically manipulate several fungal species. Despite the recent development of CRISPR/Cas9 and marker-free systems, in vitro molecular tools to study Aspergillus fumigatus, a saprophytic fungus causing life threatening diseases in immunocompromised hosts, still rely extensively on the use of dominant selectable markers. The limited number of drug selectable markers is already a critical aspect, but the possibility that their introduction into a microorganism could induce enhanced virulence or undesired effects on metabolic behavior, constitutes another problem. In this context, here we demonstrate that the use of ptrA in A. fumigatus leads to secretion of a compound that allows recovery of thiamine auxotrophy. In this study we developed a simple modification of the two commonly used dominant markers in which the development of resistance can be controlled by the xylose-inducible promoter PxylP from Penicillium chrysogenum. This strategy provides an easy solution to avoid undesired side effects since the marker expression can be readily silenced when not required.

Biotechnological application of the green microalga Chlamydomonas reinhardtii hinges on the availability of selectable markers for effective expression of multiple transgenes. However, biological safety concerns limit the establishment of new antibiotic resistance genes and until today, only few auxotrophic markers exist for C. reinhardtii. The recent improvements in gene editing via CRISPR/Cas9 allows directed exploration of new endogenous selectable markers. Since editing frequencies with CRISPR/Cas9 techniques are often low, the Cas9-sgRNA ribonucleoprotein (RNP) delivery protocol was strategically optimized by applying nitrogen starvation to the pre-culture, increasing editing frequencies from 10% to 66% after pre-selection. Probing the essential polyamine biosynthesis pathway, the spermidine synthase gene (SPD1) is shown to be a potent selectable marker with versatile biotechnological applicability. Very low levels of spermidine (0.75 mg/L) were required to maintain normal mixotrophic and phototrophic growth in newly designed spermidine auxotrophic strains. Complementation of these strains with a synthetic SPD1 gene was achieved when the mature protein was targeted to either the cytosol or the chloroplast. This work highlights the potential of new selectable markers for biotechnology as well as basic research and proposes an effective pipeline for the identification of new auxotrophies in C. reinhardtii.

The genetic engineering of plants often relies on the use of antibiotic or herbicide resistance genes for initial selection of primary transgenic events. Nevertheless, the commercial release of genetically modified crops containing any marker gene encounters several challenges stemming from the lack of consumer acceptance. Development of strategies enabled the generation of marker-free transgenic plants is a possible alternative to deal with public concerns over the safety of biotech crops. Leveraging the high in vitro regeneration and transformation capabilities of potato, this study examined the factors associated with the development of transgenic plants without presence of selective substances in medium. To compare the antibiotic-assisted with the antibiotic-free transformation, nodal explants from five highly regenerative potato cultivars were inoculated with the Agrobacterium strain AGL0 carrying the pBIN-mGFP5-ER (35SGFP/nosNPTII) plasmid. The efficiency of transient expression and the formation of chimeric and completely transgenic shoots were assessed through vital GFP observations. Under non selective conditions, the regeneration efficiency of stable non-chimeric transgenic shoots varied from 0.9 (‘Chicago’) to 2.7% (#12-36-42). In order to streamline transformation protocol, a specific timeline for collecting transgenic plantlets was established for each analyzed cultivar. This timeline strategy was implemented to generate transgenic potato plants, which demonstrated the suppression of homologous SteIF4Es family genes as a result of the successful introduction of the marker-free hpRNAi construct.