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Good morningThis Scutellinia from July 9 grew at 1
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Villalonga PacoSmall Scutellinia growing in garden soil (calcareo
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Andgelo Mombert
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Question about sequencing
Juuso Äikäs,
30-03-2022 16:08
If I've understood correctly, generally for tiny ascomycetes it would be preferable to make an agar culture of the fungi in order to avoid contaminants that you would get by just gathering a bunch of fruitbodies.
1:
In what form is it customary to send a cultured mycelium to a lab to be sequenced?
Would it be ok to for example scrape the mycelium off the agar plate, dry it in e.g. a closed glass jar with silica gel, and put the dried mycelium into a small plastic test tube?
Just made my first test ascomycete culture from a small piece of (a fairly fresh) dry Nectria cf. dematiosa fruitbody, and the mycelium has started to grow nicely on the sterilized agar plate :).
2:
At least with gilled fungi the ITS region seems to be the area to sequence. How does this apply to ascomycetes? Is it generally useful to get started by just sequencing that region or are there some other areas that are also usually needed (LSU for example)? Or does it depend entirely on the case of the genus/family etc.?
Andrew N. Miller,
30-03-2022 16:16
Re : Question about sequencing
1. We routinely scrape fresh mycelia from a plate into a tube, but there is no need to dry it before DNA extraction.
2. All fungi should be sequenced for the ITS and LSU. After that, the genes you chose are highly dependent on the group you are working on.
Cheers,
Andy
2. All fungi should be sequenced for the ITS and LSU. After that, the genes you chose are highly dependent on the group you are working on.
Cheers,
Andy
Juuso Äikäs,
30-03-2022 16:24
Re : Question about sequencing
Thanks for the answer!
1. I was thinking about the time for the sample to be posted, and maybe lie in a queue to be sequenced. Or also generally with longer term storage there'd be a risk of it starting to grow some extra stuff? I would assume drying it wouldn't be harmful for the sequence?
1. I was thinking about the time for the sample to be posted, and maybe lie in a queue to be sequenced. Or also generally with longer term storage there'd be a risk of it starting to grow some extra stuff? I would assume drying it wouldn't be harmful for the sequence?
Andrew N. Miller,
30-03-2022 16:28
Re : Question about sequencing
You are welcome.
1. No, not harmful... just uneccessary.
1. No, not harmful... just uneccessary.
Kosonen Timo,
31-03-2022 08:14
Re : Question about sequencing
Hi Juuso,
Answering your questions (a bit indirectly), and hopefully adding something useful:
a) an ITS-LSU sequence using eg primers ITS1-LR3 is c. 1100 bp long piece. THis usually copies/is sequenced very well by just using those two primer (cheap, easy, fast). LR5 (instead of LR3) streches a bit further, but sometimes you have an "intron" in between and things go wrong (with Helotiales). This is a good piece of data and makes a fine barcoding seq. There are thousand of species lacking all seq data, it's valuable, but, still:
b) Seeing the trouble of producing cultures, you'd be able to produce other sequences as well, sequences that copy less well from tiny dried material (with possible contaminants). FOr some groups this would be essential. If you aim for this it helps if the extraction is made from very fresh culture (rich with DNA). Preferably straight from the plate. If the delivery is fast, scraped mycelia in an eppendorf is ok for some days I suppose. ...but you should get the ITS-LSU always as that part is all around the DNA and copies very easy. You can long-term storage the mycelia, but you need some room.
c) not sure how you produce your cultures, but cultures made directly from apos (touching the growth medium) have a tendency to produce contaminations. Sometimes its hard to say which is a contaminant and which is the target. THey could even be sitting on the same branch - so to speak =8-). ...Aim for spores, try diverse set of mediums.
I'll gladly help if you need/want more advice,
cheers
Timo
Juuso Äikäs,
31-03-2022 12:57
Re : Question about sequencing
Thanks! This is useful info.
This was just my first test attempt to make a culture. I cut a pink (anamorph) fruitbody open and scraped a bit of the inside flesh with the tip of tweezers and quickly put it on the agar. In a few days the mycelium has grown to a diameter of around 5 cm.
But I reckoned spores would be better. Last year I tried a few times to catch a spore print to a cover glass raised a little above apos, with success. Should just replace the cover glass with an agar plate. But there isn't much material to try it at the moment. I'll try it later when spring begins.