Thanks anna. But it is also used to identify microalgae. Is there any options to left non-target groups in the BLAST analysis?

hi,

i have faced the same problem in a different type. when you are getting Blast results like this

there are many reasons

first you should check about your culture age

by any cross contaminants of fungi in spore form could be embedded in your microalgal cell wall which has abundant EPS, that can serve as a reservoir for spore.

At particular generation there will be chance for fungal spore growth in midst of your microalgae population

even in a trace level contaminant DNA , using 18 srRNA primer there was a chance for getting amplicon of fungal DNA source

Hence I am suggesting you a solution with my known experience, try to purify your culture once more through antibiotic and physical mode of treatment. after confirming unialgal strains through plating, then you can repeat the process

Thank you

I would also suggest you to purify your culture. Then screen it properly before extracting DNA to check for any contamination.

There are a couple of primer pairs routinely used for DNA barcoding of microalgae. Frequently, the internal transcribed spacer 2 (ITS2) region of nuclear ribosomal DNA is used as a target region. There are also some other suitable target regions for barcoding. From my point of view, the 18S rRNA gene is not the best choice for barcoding of Chlorophyta.

For example, see this recent paper:

Darienko T, Gustavs L, Eggert A, Wolf W2, Pröschold T. (2015) Evaluating the Species Boundaries of Green Microalgae (Coccomyxa, Trebouxiophyceae, Chlorophyta) Using Integrative Taxonomy and DNA Barcoding with Further Implications for the Species Identification in Environmental Samples. PLoS One 10(6):e0127838.

If you also have a contamination problem with your cultures, I would suggest that you try to obtain fresh axenic cultures, if possible. Purification of green algae frequently is a tricky business. In most cases, simple addition of antibiotics does not work. The laborious mechanical purification methods also depend on the respective algae species.

Ya....Thank you very much for your information Prof. Armin Hallmann. But ITS2  region mainly used for bar-coding of fungi?

There is any taxonomic relationship between fungi and microalgae? because most of the earlier studies on microalgae mentioned there are some symbiotic relationship between these two.....they also observed while the culture reach log phase at that time fungal contamination. It means the microalgae can able to adjust their environmental pH while they are doubling the number.

I am not familiar with axenic culture........is there any specific method to determine the purity of culture or only microscopic observation?

ITS2 is also used for green microalgae. See for example the mentioned paper by Darienko et al. (2015). Of course, even if the target region is the ITS2 region, the primers for fungi or microalgae applications are not identical.

18S rRNA genes are highly conserved and, in addition, there is no need for a perfect match to get a PCR product. 18S rRNA genes are used for phylogenetic purposes both in algae and in fungi.

Regarding purity of the culture: There are different methods to determine the purity of a culture. One strategy is to plate the cultures on highly nutrient-rich media (e.g., with glucose) that are preferred by bacteria and fungi, incubate it and see if you get bacteria colonies or fungi. Moreover, there are also PCR-based methods to identify contaminations by bacteria or fungi. You might see (heavy) contaminations by microscopy at high resolution. However, if you don’t see anything, you cannot be sure that there is really no contamination.

Regarding ITS2 analyses you should also look at the ITS2 Database, which is described in this paper:

Merget, B., Koetschan, C., Hackl, T., Förster, F., Dandekar, T., Müller, T., et al. The ITS2 Database. J. Vis. Exp.(61), e3806, doi:10.3791/3806 (2012).

And here is the link to the ITS2 Database:

http://its2.bioapps.biozentrum.uni-wuerzburg.de/