Hello Muhammad

If you really want to make DNA-barcoding you have to preferably use the standardized primers (DNA-barcoding is a standardized method). For fungi it is ITS (proposed by Schoch et al. 2012). If ITS does not work in your group you still can propose an alternative primer (see Guidelines for non-CO1 Selection http://www.barcodeoflife.org/content/resources/standards-and-guidelines (ok in your case Substitute ITS for  CO-1 in the title of the document)).

Why do you want to use new markers? For phylogeny or for species delimitation. Or do you think that Resolution power of ITS is not sufficient in your group?

Depending of what you wat the answer might be different: It can range from .) try with RPB1 or 16S, to try to make ESTs or whatsoever.

Greetings

Nikola

Hi Muhammad,

I think the preferred ITS sequence is the best for overall fungal barcoding. There are several advantages, (1) The ribosomal genes are duplicated perhaps a hundred times in each cell so the signal from the environment is relatively strong. This would of course be the case for other ribosomal genes too, and also for mitochondrial sequences. (2) the variation is optimal in most (but of course not all) fungal groups so that variation within species is limited while there are large differences (at least detectable with sequencing) among species. (3) the primers that are commonly used will discriminate against other groups of organisms so you can get a fungal community read of your environmental sample. (4) although the reference data bases for ITS are not complete and perfect, they are far better than for any other target sequence.

Other genes that have been tested do not have all these advantages but might work well for specific groups of fungi. If you embark on a new barcoding effort, you do have to go through a bioinformatics step to ensure specificity and coverage of the taxonomic variation. Then you should perform tests of artificial controlled mixes of species and also natural communities and ecosystem samples.

A reference that did part of this is: Ihrmark, K, Bödeker, I, Cruz-Martinez, K, Friberg, H, Kubartova, A, Schenck, J, Strid, Y, Stenlid, J, Brandström-Durling, M, Clemmensen K & Lindahl, BD. 2012. New primers to amplify the fungal ITS2 region – evaluation by 454-sequencing of artificial and natural communities. FEMS Microbiology Ecology 82:666-677.

Cheers,

Jan

I support all the comments above - ITS is the region for barcoding fungi.  I'd recommend you check the BOLD (barcode of life database) too.

ITS seems to be the best region for modern samples and for degraded samples see BELLEMAIN, E. et al. Fungal palaeodiversity revealed using high-throughput metabarcoding of ancient DNA from arctic permafrost. Environmental Microbiology 15, 1176–1189 (2012).

ITS will normally work, so I would not develop new primers. Please see the BOLD database for possible primer options and be sure to try different primer combinations and tweak experimen conditions if a species so close to yours has not been reported before. 

Good luck