I have isolated 10 different fungi from rhizosphere and would like to identify them upto species level.
your fungi are purified? if yes you can firstly begin by DNA EXTRACTION. After that you can amplided some gene such as ribosomal 18s. PCR product should be sequenced
The best way to identify your fungi down to species level is of course to clone & sequence the full length 18S rDNA; for this I normally use the primers given in Medlin et al, 1988.
THE CHARACTERIZATION OF ENZYMATICALLY AMPLIFIED EUKARYOTIC 16S-LIKE RRNA-CODING REGIONS
By: MEDLIN, L; ELWOOD, HJ; STICKEL, S; et al.
GENE Volume: 71 Issue: 2 Pages: 491-499 Published: NOV 30 1988
This will though most likely require cloning and the additional use of internal primers to get a full-length, double-stranded sequence.
If you think that a shorter part of the 18S might be sufficient for the identification, then the discussion is open about which variable region to choose, i.e. V3, V4, V6 or V9. For each region possible primer pairs exist, but which one might give the best results for your particular taxa I don´t know.
Have a look at the paper of Wang et al (2014) which suggest a number of primers and also lists plenty of references with more universal primer pairs for eukaryotes.
Wang Y, Tian RM, Gao ZM, Bougouffa S, Qian P-Y (2014) Optimal Eukaryotic 18S and Universal 16S/18S Ribosomal RNA Primers and Their Application in a Study of Symbiosis. PLoS ONE 9(3): e90053. doi:10.1371/journal.pone.0090053
Good luck!
For fungal identification you should use the ITS region. You can find multiple primer set, but ITS1 and ITS4 will amplify almost all fungi
Analyzing the ITS region is great for identifying fungi. I have used the primers NSI1F and NLB4R from Martin and Rygiewicz 2005 with great success.
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