I would like to identify fungi (mostly Penicillium and Aspergillus) by molecular methods. I used two pair of primers ITS1 and ITS4, and beta tubulin 2a and beta tubulin 2b. After sequencing I obtain for same sample (DNA extraction) two different species (e.g. by beta tubulin P. terrigenum, and by ITS primers P. solitum).
Hi: Did you increased the fungal colony by using single spore technique before DNA extraction?
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You repeat the single spore then you do ITS only , and see the result If it is ok, continue with other primers
Aside of ensuring a single spore culture...
What identity scores did you obtain?
Did you check the quality of your chromatogram?
What database did you search against?...
Thank you for answers.
Xabier Vázquez Campos,
The scores were same, 99 to 100%
I didn't perform chromatogram because I do not have equipment
I searched BLAST and CBS database. Can you suggest some other?
NCBI won't solve much if you don't check the sequences from type material only as it is not very reliable regarding names... CBS is more reliable.
99-100% for both genes or both organisms?
In any case, you shouldn't just pick the first name you get as often you have plenty of other organisms with the same or similar scores and identities, and based on the ITS, it seems that many Penicillium are pretty much the same.
You should check the literature on molecular taxonomy of the taxa you are working on.
Don't you have the original ab1 files of the sequencing? That's what I mean by chromatogram, suposing you did Sanger sequencing.
Dear all!
I have made a DNA isolation from apparently clear fungi cultures, however I haven't done the single spore technique. Nevertheless, the both fragments and corresponding chromatograms (sorry for missunderstanding) turned out really good, i.e. clear sequences without any background. Regarding the literature, a lot of authors are using the same principle or they are performing only ITS sequencing...but as you Xabier have said, it is difficult to pick the species as similarities between them are in 98-100% range. So even if I perform a single spore technique prior to DNA isolation, the problem in defining a species on each fragment remains. I should probably sequence an additional fragment...what is your opinion on that...mitohondrial fragment or some other? Thank you!
Given that the sequence is correct, there are two possibilities for your results:
1) For P. terrigenum only B-tubulin is known and/or for P. solitum only the ITS; therefore the name does not appear in the hitlist of the other sequence
2) as Xabier said; don't just pick the first Name. If you have a long list with sequences above 98% it's going to get difficult or even impossible to differentiate. Maybe some signature sequences are known which help.
The paper linked below contains accession numbers for the ITS and other phylogenetically relevant markers, mainly beta-tubulin, and should be useful for what you try to do.
According to what the authors say in the paper, the annotated sequences of Penicillium are a mess, so I would personally try to use those provided in the paper as reference and recheck the ITS
Article Identification and nomenclature of the genus Penicillium
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