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InvisiblePsychedel.EXE
AKA Old Uncle Nutty
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Registered: 07/04/16
Posts: 211
genetic diversity after multiple transfers
    #23539138 - 08/13/16 09:58 PM (7 years, 6 months ago)

This may sound like a stupid question.
After my initial plate was inoculated from MS syringe, I am now 5 transfers deep, narrowing down the genetics. I don't want to narrow it down so much that there is not much variation, right(?), because I have not fruited anything from this yet so there is no way to know where to start isolating.
So I guess my question is, how many transfers would it take, roughly, starting from ms to finally get down to an isolate?
Like I said, i'm 5 transfers deep so could I still expect a lot of variation? (i hope)
I am going to use these plates that I currently made on my different grain jars, fruit them, and try to isolate my best fruits of course, my hope is that there is a bit of diversity still at this point.


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InvisibleSupalemonhaze
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Registered: 10/02/15
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Re: genetic diversity after multiple transfers [Re: Psychedel.EXE]
    #23539174 - 08/13/16 10:14 PM (7 years, 6 months ago)

5 transfers is nothing, there will be plenty of strains in there still. When you are getting an isolate, you are basically taking a shot in the dark. You will not be testing mid-way, that's why you have to end up with a lot of isolates and not just one. Say you are doing isolating, you want to end up with ~20 isolates so you will have a much better chance of getting something worthwhile.

If I were you, I wouldn't bother with this current method. If you want less diverse genetics, get to cloning. Cloning allows you to cherry pick fruiting strains so whatever you pick will always fruit. Now wheter or not that clone is a great one will be purely based on luck and how well you choose your clones. To make clones even more stable, isolate them. It will take away some variety but not all of it. You will still end up with a different result than the original clone but at least you start off with something that is guaranteed to fruit.

If you are not wanting to do a proper isolation where you end up with isolates, cloning is the way to go. Doing a semi-isolation gives you less chance of ending up with something worthwhile.

And to answer your other question regarding how many transfers, it depends on how many spores you used and how small your transfers are. It takes a lot of transfers though, much more than 5. People usually don't even start isolating until they do ~10 transfers. IME, I needed much more than that. I ended up thrashing my isolation project, ended up neglecting my cultures too much as I was too busy to work them. I do know that I spent at least 2-3 months isolating and it didn't seem like I was even close.

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InvisiblePsychedel.EXE
AKA Old Uncle Nutty
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Registered: 07/04/16
Posts: 211
Re: genetic diversity after multiple transfers [Re: Supalemonhaze]
    #23539193 - 08/13/16 10:24 PM (7 years, 6 months ago)

Thanks for the info.
In my last sentence I meant to say clone my best fruits, not isolate my best fruits. That is my intention, to go down that route. I just wanted to be sure that there will still be plenty of diversity in what I have after 5 transfers to be able to even find some fine fruits to clone.

Thanks bro!


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InvisibleSupalemonhaze
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Registered: 10/02/15
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Re: genetic diversity after multiple transfers [Re: Psychedel.EXE]
    #23539210 - 08/13/16 10:33 PM (7 years, 6 months ago)

If diversity is what you are looking for, don't bother isolating your inoculation. Just do a couple of transfers for cleanliness' sake and grow it out. Grow it out and pick your best shrooms for cloning. Isolate them and test them out side by side. Keep the best one and trash the others. Always good to repeat this in search of a better clone. Unless you are getting wall to wall flushes that is :lol:

An isolate obtained from a clone will still have multiple strains in it, possibly hundreds. Strains will go through anastomosis (fusing) when they meet and are compatabile. This is why an isolate obtained from a clone is not a true isolate. It's also the reason why when you're getting a true isolate, you are always transferring as early as you can, so you can avoid anastomosis as much as you can.

Basically what happens with anastomosis is that 2 strains become one. The result is a single strain that has the traits of both the parent strains. This is one of the ways you can go about making a hybrid. It's also the easiest way to get a hybrid. Now given that there are thousands of strains in a single spore inoculation, a clone will have a lot of these fused strains. The reason why a clone will still sector on agar is because the remaining strains are not compatabile with each other but each of them probably have hundreds of strains fused together already. Plenty of diversity but you can still get a pretty stable culture from them.

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InvisiblePsychedel.EXE
AKA Old Uncle Nutty
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Registered: 07/04/16
Posts: 211
Re: genetic diversity after multiple transfers [Re: Supalemonhaze]
    #23539279 - 08/13/16 11:03 PM (7 years, 6 months ago)

Quote:

If diversity is what you are looking for, don't bother isolating your inoculation. Just do a couple of transfers for cleanliness' sake and grow it out. Grow it out and pick your best shrooms for cloning. Isolate them and test them out side by side. Keep the best one and trash the others. Always good to repeat this in search of a better clone. Unless you are getting wall to wall flushes that is :lol:



Yea, I just got caught up in a number of transfers just testing out my sterile procedure and ensuring cleanliness. Got caught up falling in love with the agar lol.



Quote:

An isolate obtained from a clone will still have multiple strains in it, possibly hundreds. Strains will go through anastomosis (fusing) when they meet and are compatabile. This is why an isolate obtained from a clone is not a true isolate. It's also the reason why when you're getting a true isolate, you are always transferring as early as you can, so you can avoid anastomosis as much as you can.

Basically what happens with anastomosis is that 2 strains become one. The result is a single strain that has the traits of both the parent strains. This is one of the ways you can go about making a hybrid. It's also the easiest way to get a hybrid. Now given that there are thousands of strains in a single spore inoculation, a clone will have a lot of these fused strains. The reason why a clone will still sector on agar is because the remaining strains are not compatabile with each other but each of them probably have hundreds of strains fused together already. Plenty of diversity but you can still get a pretty stable culture from them.



:takingnotes:


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InvisiblebodhisattaMDiscordReddit
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Re: genetic diversity after multiple transfers [Re: Psychedel.EXE]
    #23539323 - 08/13/16 11:26 PM (7 years, 6 months ago)

A clone can have multiple strains but if you isolate from a clone its still an isolate...

An isolate is one strain. A clone is a culture or perhaps an isolate. If you isolate from a clone it is one strain. I don't suggest isolating from a clone.

This is why it can easily take ten transfers to get a isolate from MS or a clone because you have to get the strains to come apart after anastomosis. Hence sectoring

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InvisibleSupalemonhaze
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Re: genetic diversity after multiple transfers [Re: bodhisatta]
    #23539575 - 08/14/16 01:29 AM (7 years, 6 months ago)

An isolate from a clone is not a true isolate though. A true isolate will be more stable and grow more evenly than an isolated clone. Actually, I like to isolate my clones before testing so they won't change their performance from accidental isolation in case I get a keeper.

I don't know why you are saying that strains will come apart after anastomosis, if that was true, making a hybrid would be more difficult since the strains would sector out of each other again. Once anastomosis happens, the two strains that have fused will be one and the same. Same as what happens when monokaryons mate to make a dikaryon strain, before anastomosis the strains are useless but once they fuse they become a single strain capable of fruiting. That's how I always understood it at least, and it's definitely what RR seemed to think.

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InvisiblebodhisattaMDiscordReddit
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Re: genetic diversity after multiple transfers [Re: Supalemonhaze]
    #23540176 - 08/14/16 09:00 AM (7 years, 6 months ago)

It would be better for you just to read what RR has said

Quote:

RogerRabbit said:

There can be dozens of strains conspiring to form a single fruiting body, without these strains combining into a single organism.  This is well known and established in mycology.  Paul Stamets reports isolating up to 200 organisms from a single fruit.  It's not science fiction, it's science.  Picture thousands of individual soldiers in an army going to battle.  Each is separate, yet conspire to form the coherent fighting force.

An isolate can either come from a pairing of two compatible hyphae from separate spores, or from mycelium that has later combined via anastomosis into a single organism.  There can be monokaryotic pairings with other mononucleate mycelium, dikaryotic pairings with other multinucleate mycelium, or mono-dikaryotic pairings.  You know you have an isolate when there is no more sectoring on agar.

To the OP, rhizomorphic mycelium is not the only characteristic we isolate for.  The important thing is to get to single sectors and then fruit out each strain.  This is the only proved way to find the best performing strains.  Many rhizomorphic strains will be poor performers, but some will blow your socks off.  The latter are the ones you keep, discarding the former.  The only way to know is to fruit each one.  It's a long process, but it's fun and how we come up with the best commercial strains.
RR




Sectors come out of already anastomosis'd strains. Kind of the whole point of isolation.

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InvisibleSupalemonhaze
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Re: genetic diversity after multiple transfers [Re: bodhisatta]
    #23542813 - 08/15/16 02:35 AM (7 years, 6 months ago)

That's not really what he's saying though.

Here's one that sheds a better light on the subject.

Quote:

*One swipe of spores on agar will yield hundreds of strains. By selecting a dozen or so of the best rhizomorphic strains and fruiting each one separately, you can find the super performer that will cover every spot of your casing layer with healthy pins. It might take hundreds of multispore grows to find that strain, if ever, because multispore inoculated substrates usually end up with only one or two strains by the time they fruit because they've all combined. (anastomosis). This means the good fruiting and potent strains combine with the poor fruiting and bunk strains.




The ones that he was talking about in your quote are the ones that aren't compatable but each of those non-compatabile strains have already fused with other compatable ones. This is why a great isolate has the ability to outperform a clone, because you don't have any bunk or poor performing strains mixed in with your great ones. God knows how many isolations it would take to find that strain but this is why RR was more pro-isolation rather than pro-cloning, he had plenty of time and resources for such a thing so it was worth it to him in the long run.

Now granted, he was exaggerating when he said that one or two strains remain but one thing is for sure, isolation takes much less time on a clone than it does on a spore inoculation. I always attributed this to anastomosis.

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InvisiblebodhisattaMDiscordReddit
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Re: genetic diversity after multiple transfers [Re: Supalemonhaze]
    #23543422 - 08/15/16 10:00 AM (7 years, 6 months ago)

By the time they fruit its all one organism working as a unified creature then tell me how fruits have multiple strains. They're anastomosis together but they come apart..

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InvisiblePsychedel.EXE
AKA Old Uncle Nutty
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Registered: 07/04/16
Posts: 211
Re: genetic diversity after multiple transfers [Re: Supalemonhaze]
    #23544173 - 08/15/16 01:53 PM (7 years, 6 months ago)

This hobby just became even more interesting. I guess I need to pick up a few books and geek out.

Thanks for all the superb information TC and Supa!


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InvisibleSupalemonhaze
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Registered: 10/02/15
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Re: genetic diversity after multiple transfers [Re: bodhisatta]
    #23544245 - 08/15/16 02:16 PM (7 years, 6 months ago)

Quote:

Trusted Cultivator said:
By the time they fruit its all one organism working as a unified creature then tell me how fruits have multiple strains. They're anastomosis together but they come apart..




Because they are not all compatable. That's how I always understood it, at least.

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InvisibleSupalemonhaze
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Re: genetic diversity after multiple transfers [Re: Supalemonhaze]
    #23544292 - 08/15/16 02:35 PM (7 years, 6 months ago)

Some more info on the subject

Quote:



Of course there's thousands of parents if thousands of spores germinate from multispore inoculation. Eventually, most of the substrains combine into a single organism through the process of anastomosis. If one wishes to separate out the individual strains, or substrains as they're sometimes called in order to preserve the diversity, you want to place the multispore culture on agar where you can observe the growth in the flat, two dimensional plane of the Petri dish. This process needs to begin within a day or two of germination so you can catch the individual strains before they combine with other dikaryons via anastomosis. Some strains are not compatible, so this is the reason on many substrates that were inoculated by multispore you'll see drastically different looking(and performing) mushrooms on the same flush. It's because those that are not compatible usually lay claim to their niche of real estate and fruit from there. By the time it gets to that point, my liquid slides are so thick with growth, the microscope light can't shine through them. According to stamets, and it seems to match my observation, most of the time all the substrains combine into a coherent whole. In other words, if you took a sample of mycelium from each corner of a fruiting tray and placed them on the same petri dish, they'd grow together seamlessly without forming a zone of inhibition. Still, there are times when it's fairly obvious by observation that more than one substrain is at work in the same fruiting tray. SPORE GERMINATION




Quote:

An alternative is to isolate a couple of strains down to single sector dikaryons, and then place those on a petri and allow them to run together. If a third sector opens up and starts growing, isolate it. You can then test for the cross by placing all three on a new dish. A line of isolation should develop between all three.



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