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OfflineMtez44
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Why clone?
    #24032871 - 01/23/17 09:56 AM (7 years, 7 days ago)

Sorry for the ambiguous post. But I can't find the answer anywhere.

I took 7 of the largest mushrooms from my tub and put them on agar. Meanwhile I am thinking to myself, why did I do this? I don't know why other than I obviously read it here that this is what people do.

My grow was from MS so wouldn't cloning be the same as collecting spores? Won't I still have the same variation from that tissue as I would the spores?

Isn't it easier to store spores than a master culture?

Isn't it better to stabilize the traits you want via multiple generations of MS grows?

I am trying to learn more about mycology, aside from just cultivating and figured this would be a good starting point. Who knows, I may even buy a microscope :smile: definitely not where I saw this going when I first started.


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InvisibleInocuole
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Re: Why clone? [Re: Mtez44] * 2
    #24032884 - 01/23/17 10:02 AM (7 years, 7 days ago)

Isolating traits through multispore grows would take fucking forever and there'd be a ton of trial and error involved. Too much for me even if I am fairly patient.  Cloning is more like "oh hey look there's the genetics I want right there", now I can grow a tub that are strikingly similar, and take prints from that, which will have a lot more of a chance of producing spores that show the desired traits.

You want to clone BECAUSE multispore is so varied and unreliable.  It's one of the fastest and easiest ways to cut your gene pool down to a reasonable amount of mostly desired traits.  If you have a good clone you can get the same results every time, you'll get to know the culture, and you'll understand over time what makes it special, as you continue to grow other things as well.  Maybe you find something better and clone that, then run them both together in the same tub for fun or whatever reason.  I can think of a ton of reasons to clone.

Granted working with spores is still a good endeavor and having a spore print with highly varying genetics is useful for looking for new traits, so it's really just best to do a bit of everything.

I'd recommend cloning from clusters of reasonable sized mushrooms rather than the biggest in the tub, though.  Unless of course those coincide, in which case, grab it.


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OfflinePeteyboy
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Re: Why clone? [Re: Inocuole]
    #24032895 - 01/23/17 10:08 AM (7 years, 7 days ago)

Innoc hit it on the head...from my own personal experience it's a great method to ensure potency is up to snuff as well. I recently had a tub of GT fruit, it was a dud tub, my buddy bioassay'd 5 grams and didn't feel anything....I was shocked and it was alot of time and effort wasted. This would have been eliminated if I was working with a proven potent clone.


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OfflineMtez44
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Re: Why clone? [Re: Peteyboy]
    #24032908 - 01/23/17 10:15 AM (7 years, 7 days ago)

So I am incorrect in thinking that a clone from MS and the spores from the same mushroom have the same genetic variation?


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InvisibleInocuole
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Re: Why clone? [Re: Mtez44]
    #24033003 - 01/23/17 11:08 AM (7 years, 7 days ago)

Not necessarily. A clone isn't actually an isolated strain, it usually has multiple sets of genetics still.  While the clone print won't have much difference in variation compared to the original mushroom(less if anything), you'll still have dozens or hundreds of that mushroom to take prints from which will increase your chances of finding something good to carry on with.


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InvisibleKauaiOrca
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Re: Why clone? [Re: Inocuole]
    #24033024 - 01/23/17 11:20 AM (7 years, 7 days ago)

Saw a very interesting post a month or so ago (can't remember where it was) on this site about what happens when you keep  using spores from a series of generations and how it starts dramatically limiting the variations that show up on MS grows.  It was really interesting.  After about 6 or 7 generations, mathematically, he showed how you start getting much less variables and it becomes more and more like cloning.  Not exactly the same, of course, but similar.  Sort of like domesticating the genetics, I guess.

I've found with one strain I'm working with that I've done about 7-8 generations MS grows with it and it produces as good and sometimes better than some of the better clones I've done with a lot less effort.  It seemed like he really knew what he was talking about and the formula underneath it about how genetic diversity steadily disappears with each generation was really interesting.


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"The universe is endless, limitless and infinite.  Any effort to define it's boundaries is an attempt to overcome ignorance.  We are physical, mental and spiritual beings ... there is no beginning and there is no end.  There is only memory.  Our repeated loss of memory experiences create the illusion of beginnings and ends.  Immortality is the ability to retain full memory through all consciousness transformations.  Loss of memory is man's greatest curse and, in very real terms, death."

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OfflineAK1000
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Re: Why clone? [Re: KauaiOrca]
    #24033054 - 01/23/17 11:39 AM (7 years, 7 days ago)

Quote:

KauaiOrca said:
Saw a very interesting post a month or so ago (can't remember where it was) on this site about what happens when you keep  using spores from a series of generations and how it starts dramatically limiting the variations that show up on MS grows.  It was really interesting.  After about 6 or 7 generations, mathematically, he showed how you start getting much less variables and it becomes more and more like cloning.  Not exactly the same, of course, but similar.  Sort of like domesticating the genetics, I guess.

I've found with one strain I'm working with that I've done about 7-8 generations MS grows with it and it produces as good and sometimes better than some of the better clones I've done with a lot less effort.  It seemed like he really knew what he was talking about and the formula underneath it about how genetic diversity steadily disappears with each generation was really interesting.




Interesting. I thought if you use spores from a spore print, that it's like starting from scratch because two spores combine to make an entirely new mushroom all over again.


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InvisibleKauaiOrca
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Re: Why clone? [Re: AK1000]
    #24033061 - 01/23/17 11:45 AM (7 years, 7 days ago)

Quote:

AK1000 said:
Quote:

KauaiOrca said:
Saw a very interesting post a month or so ago (can't remember where it was) on this site about what happens when you keep  using spores from a series of generations and how it starts dramatically limiting the variations that show up on MS grows.  It was really interesting.  After about 6 or 7 generations, mathematically, he showed how you start getting much less variables and it becomes more and more like cloning.  Not exactly the same, of course, but similar.  Sort of like domesticating the genetics, I guess.

I've found with one strain I'm working with that I've done about 7-8 generations MS grows with it and it produces as good and sometimes better than some of the better clones I've done with a lot less effort.  It seemed like he really knew what he was talking about and the formula underneath it about how genetic diversity steadily disappears with each generation was really interesting.




Interesting. I thought if you use spores from a spore print, that it's like starting from scratch because two spores combine to make an entirely new mushroom all over again.




I wish I would have bookmarked that post ... It was really eye opening and completely changed my perspective on cloning and MS and genetic diversity.  I think the point he was making is that as you keep doing prints from the same source mushroom, the genetic diversity keeps shrinking rather dramatically ... as that happens, less and less "variety" happens when those two spores combine ... If I run across it, I'll post it.  It was very scientific and it kinda confirmed something I've found myself just intuitively how with successive prints from good grows stemming off the same print for generations, the predictability of the MS grow starts going way up.


--------------------
"The universe is endless, limitless and infinite.  Any effort to define it's boundaries is an attempt to overcome ignorance.  We are physical, mental and spiritual beings ... there is no beginning and there is no end.  There is only memory.  Our repeated loss of memory experiences create the illusion of beginnings and ends.  Immortality is the ability to retain full memory through all consciousness transformations.  Loss of memory is man's greatest curse and, in very real terms, death."

-- Ancient Taoist Master


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OfflineMtez44
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Re: Why clone? [Re: AK1000]
    #24033090 - 01/23/17 12:09 PM (7 years, 7 days ago)

Quote:

AK1000 said:
Quote:

KauaiOrca said:
Saw a very interesting post a month or so ago (can't remember where it was) on this site about what happens when you keep  using spores from a series of generations and how it starts dramatically limiting the variations that show up on MS grows.  It was really interesting.  After about 6 or 7 generations, mathematically, he showed how you start getting much less variables and it becomes more and more like cloning.  Not exactly the same, of course, but similar.  Sort of like domesticating the genetics, I guess.

I've found with one strain I'm working with that I've done about 7-8 generations MS grows with it and it produces as good and sometimes better than some of the better clones I've done with a lot less effort.  It seemed like he really knew what he was talking about and the formula underneath it about how genetic diversity steadily disappears with each generation was really interesting.




Interesting. I thought if you use spores from a spore print, that it's like starting from scratch because two spores combine to make an entirely new mushroom all over again.




I mean think about it, isn't that how you stabilize genetics? 7 generations plus? I'm inly going by what I have read. If MS over generations is the same as cloning as far as genetic diversity is concerned why bother messing with live culture?

In fact can't you much more easily lose a genetic traits by cloning and isolating? I guess you could always save cultures and go back to If the next clone/isolation doesn't show traits you want but still

If cloning doesn't "isolate" a trait more than the spores from the same mushroom, why bother? Unless you just want the head start of not waiting for spore germination

There is clearly something I am not understanding :frown:


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Offlineblackout
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Re: Why clone? [Re: KauaiOrca]
    #24033103 - 01/23/17 12:14 PM (7 years, 7 days ago)

Quote:

KauaiOrca said:
I wish I would have bookmarked that post ...



I would search for the terms F1, F2, F3 which might be mentioned in a thread like that.

Some do not like comparisons to plants, but I think its a reasonable analogy. People will buy a pack of 10 cannabis seeds, and grow them, clone and grow all 10 to maturity, find out 5 are males and discard them then select which they reckon is ideal, which is not simply the most potent, it could be a combination of the best high, potency, good yield, contam resistant, uniformity of maturation, responds well to cloning, ease of harvest etc. Some might want shrooms which produce little or no spores. Some might want massive mushrooms with lower BE than another one as it makes harvesting easier. People can have very different desirable traits, some might value consistent but low potency over more random potency (of course hard to test)

A member called Aero had great success cloning a commercial sclerotia strain, which is presumably carefully selected by commercial growers.


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InvisibleKauaiOrca
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Re: Why clone? [Re: blackout]
    #24033133 - 01/23/17 12:31 PM (7 years, 7 days ago)

Quote:

blackout said:
Quote:

KauaiOrca said:
I wish I would have bookmarked that post ...



I would search for the terms F1, F2, F3 which might be mentioned in a thread like that.





I've not had great success searching on this site.  I need to spend more time with it, obviously.


--------------------
"The universe is endless, limitless and infinite.  Any effort to define it's boundaries is an attempt to overcome ignorance.  We are physical, mental and spiritual beings ... there is no beginning and there is no end.  There is only memory.  Our repeated loss of memory experiences create the illusion of beginnings and ends.  Immortality is the ability to retain full memory through all consciousness transformations.  Loss of memory is man's greatest curse and, in very real terms, death."

-- Ancient Taoist Master


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OfflineMartinsapin
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Re: Why clone? [Re: Inocuole]
    #24033134 - 01/23/17 12:32 PM (7 years, 7 days ago)

Quote:

Inocuole said:

You want to clone BECAUSE multispore is so varied and unreliable.  It's one of the fastest and easiest ways to cut your gene pool down to a reasonable amount of mostly desired traits.




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OfflineRealityCzech
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Re: Why clone? [Re: KauaiOrca] * 1
    #24033234 - 01/23/17 01:22 PM (7 years, 7 days ago)

Quote:

KauaiOrca said:
Saw a very interesting post a month or so ago (can't remember where it was) on this site about what happens when you keep  using spores from a series of generations and how it starts dramatically limiting the variations that show up on MS grows.  It was really interesting.  After about 6 or 7 generations, mathematically, he showed how you start getting much less variables and it becomes more and more like cloning.  Not exactly the same, of course, but similar.  Sort of like domesticating the genetics, I guess.




Very much like domesticating... think about dog breeds.  Pure bred dogs tend to have higher rates of genetic defects due to the amount of inbreeding that takes place to fix targeted traits so that sexual reproduction can reliably produce the targeted traits.

This is what you're doing by selecting a single fruiting body and doing MS with it and then repeating it over and over with multiple generations.  You have a fruiting body with a very limited genetic variability and taking the monokaryons produced by that single parent to create dikaryons with both nuclear genotypes from the same parent.  Keep doing this over and over and you end up with a lineage whose spores are much more reliably stable as to the resultant phenotypes... just like an inbred dog breed.  It is why vendors can sell spore syringes of specific "strains".  The "strain" has been inbred enough to reliably reproduce specific phenotypes from the sexual reproduction taking place in a multispore injection.

When you clone, you're skipping all the inbreeding in order to get a reliably reproducible phenotype... you take a single fruiting body that exhibits the traits you want, asexually reproduce it so as to eliminate (although in practice there is some) genetic variability, and grow fruiting bodies from those cloned dikaryons.

As usual, I'm oversimplifying, but hopefully this helps you get the general idea.


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InvisibleInocuole
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Re: Why clone? [Re: Mtez44] * 1
    #24033245 - 01/23/17 01:26 PM (7 years, 7 days ago)

Quote:

Mtez44 said:
why bother messing with live culture?




Because of the reasons I stated..

Quote:

Mtez44 said:

In fact can't you much more easily lose a genetic traits by cloning and isolating? I guess you could always save cultures and go back to If the next clone/isolation doesn't show traits you want but still




But still what?  You just answered your question, and the answer is that it's super easy to just go back one plate instead of rolling the dice with spores again and seeing what you get.

Quote:

Mtez44 said:
If cloning doesn't "isolate" a trait more than the spores from the same mushroom, why bother?




Because it does...

Quote:

Mtez44 said:
There is clearly something I am not understanding :frown:




Clearly..


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InvisibleKauaiOrca
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Re: Why clone? [Re: RealityCzech]
    #24033259 - 01/23/17 01:32 PM (7 years, 7 days ago)

Quote:

RealityCzech said:
Quote:

KauaiOrca said:
Saw a very interesting post a month or so ago (can't remember where it was) on this site about what happens when you keep  using spores from a series of generations and how it starts dramatically limiting the variations that show up on MS grows.  It was really interesting.  After about 6 or 7 generations, mathematically, he showed how you start getting much less variables and it becomes more and more like cloning.  Not exactly the same, of course, but similar.  Sort of like domesticating the genetics, I guess.




Very much like domesticating... think about dog breeds.  Pure bred dogs tend to have higher rates of genetic defects due to the amount of inbreeding that takes place to fix targeted traits so that sexual reproduction can reliably produce the targeted traits.

This is what you're doing by selecting a single fruiting body and doing MS with it and then repeating it over and over with multiple generations.  You have a fruiting body with a very limited genetic variability and taking the monokaryons produced by that single parent to create dikaryons with both nuclear genotypes from the same parent.  Keep doing this over and over and you end up with a lineage whose spores are much more reliably stable as to the resultant phenotypes... just like an inbred dog breed.  It is why vendors can sell spore syringes of specific "strains".  The "strain" has been inbred enough to reliably reproduce specific phenotypes from the sexual reproduction taking place in a multispore injection.

When you clone, you're skipping all the inbreeding in order to get a reliably reproducible phenotype... you take a single fruiting body that exhibits the traits you want, asexually reproduce it so as to eliminate (although in practice there is some) genetic variability, and grow fruiting bodies from those cloned dikaryons.

As usual, I'm oversimplifying, but hopefully this helps you get the general idea.




THANKS for the helpful and thoughtful reply.  Very helpful!!!


--------------------
"The universe is endless, limitless and infinite.  Any effort to define it's boundaries is an attempt to overcome ignorance.  We are physical, mental and spiritual beings ... there is no beginning and there is no end.  There is only memory.  Our repeated loss of memory experiences create the illusion of beginnings and ends.  Immortality is the ability to retain full memory through all consciousness transformations.  Loss of memory is man's greatest curse and, in very real terms, death."

-- Ancient Taoist Master


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InvisiblebodhisattaMDiscordReddit
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Re: Why clone? [Re: KauaiOrca]
    #24033286 - 01/23/17 01:40 PM (7 years, 7 days ago)

https://www.shroomery.org/forums/showflat.php/Number/21922023

Scroll down to cloning and strain isolation


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OfflineMtez44
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Re: Why clone? [Re: Inocuole]
    #24033442 - 01/23/17 02:51 PM (7 years, 7 days ago)

Quote:

Inocuole said:
Isolating traits through multispore grows would take fucking forever and there'd be a ton of trial and error involved.




Forever as in 7 times?

Blackout out and bod, thanks for the guidance. I will go do some more research in the areas you suggested


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OfflineKenetic
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Re: Why clone? [Re: Mtez44]
    #24033471 - 01/23/17 03:05 PM (7 years, 7 days ago)

Why clone?  Cause you get bada$$ results, thats why. 
Usually.


--------------------
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Re: Why clone? [Re: Kenetic]
    #24033630 - 01/23/17 04:12 PM (7 years, 7 days ago)



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Re: Why clone? [Re: Tookitooki] * 1
    #24033654 - 01/23/17 04:22 PM (7 years, 7 days ago)

This is not the original link, but this was quoted in another post.  I'll find the original link in time.....

https://www.shroomery.org/forums/showflat.php/Number/20115277#20115277

Workman said:
Mushrooom genetics are a little strange since a single mushroom produces spores that can then act as both parents for a new mycelium.  Essentially, you are selfing or inbreeding each time you do a multispore grow.

Now consider a wild collection of Psilocybe cubensis with a high heterozygosity.  This basically means that most or all of each pair of genes in the mushroom are different from each other.  Its the same gene location with the same basic function, but different versions.  For example, if there is a single gene for height, you might have a version that gives short mushrooms and a version that gives tall mushrooms.  If heterozygosity is high, you have one of each which may result in medium mushrooms unless one of the height genes is dominant.

Now, when you do multispore from a single mushroom you randomly get a mix of all the genes.  Sticking to our height gene example, you could get two short copies, two tall copies or one of each.  Obviously the strains with two short copies will be short and the ones with two tall copies will be tall.

Lets say we liked the short mushrooms so we saved that one and took a spore print for later.  In this example the tall version of the height gene is lost to later generations.  There is a net loss of heterozygosity.  Over the entire genome the loss is about 50% per generation.

So mathematically we can figure out how many sequential multispore generations we need until the heterozygosity is reduced to an insignificant level and the strain is stable even from multispore.

Starting with a presumably high (~100%) heterozygosity from a wild collection.  In reality, the heterozygosity is probably lower than 100%, but its an easy number to start with.

100% wild print
50% 1st generation from wild print
25% 2nd generation from 1st generation print
12.5% 3rd generation.....
6.25% 4th generation.....
3.12% 5th generation.....
1.56% 6th generation.....
0.78% 7th generation.....

You can see that the heterozygosity drops off quickly in the first few generations and is less than 1% after the 6th generation.  This highlights the importance of choosing the best traits early on when there are more to choose from.  Attempting to isolate traits in well established strains results in only minimal improvements unless spontaneous mutations increase the heterozygosity in a positive way (rare).

In summary:

Popular classic strains in circulation have all been grown well beyond 6 generations and are relatively stable from multispore with little need for isolation.

New strains, from wild material or cross breeding between different strains of the same species, can be stabilized fairly quickly with 6 or 7 generations of sequential multispore grows.

Selection is most important early in the process and if good genes are bred out, they are gone forever.  Archiving original or early generation prints is recommended for preserving heterozygosity for later selective breeding.  Continuous isolation of a bad strain with hopes of significant improvement is futile.

Does that help?
If I may jump in here on the breeding true from spores aspect.

You lose on average 50% of heterozygosity with each strain generated from multispore (analogous to selfing in plants). What this means is, the more sequential generations you do via multispore, the less variability you will see in the later generations until the variability is nearly undetectable. There will come a point that the only variability will be from new random mutations.

OK, so what is this point, or how many generations until you can be confident you have a true breeding strain? With a wild strain you would assume 100% heterozygosity. Previously domesticated strains will obviously have less heterozygosity but since that is unknown it doesn't hurt to be conservative.

Mathematically you can see the reduction in variability with each generation. It drops off quickly and then around generation 5 or 6 the gains in stability drop off dramatically. After generation 7, with heterozygosity less than 1%, the rate of random mutations will outpace any small amount of remaining variabilty loss in later generations.

Wild 100%
F1 50%
F2 25%
F3 12.5%
F4 6.25%
F5 3.125%
F6 1.5625%
F7 0.78125%

Generate several strains from the spore print and you should expect to see huge differences between these new strains if the heterozygosity is high. Choose the strain(s) with traits you want (in this case, cap color and size) and take prints. Repeat with these new prints. You can probably feel pretty confident that after doing this 5 or 6 times that the strain is stable, especially if
you don't see any new variants in the later generations.

Good luck and I hope this is understandable.
one of my favs
breeding


Yes it is possible and mushrooms do have sex. Of course you have to stick to the same species so technically crossing one strain of cubensis with another strain of cubensis isn't a hybrid. But it still can be used to generate novel strains.

Spores, like eggs and sperm, have only half the chromosomes of somatic cells. A singe spore germinates and grows a thin monokaryotic mycelium until it comes in contact with another strand of mycelium from a different spore. At the point of contact the two myceliums fuse and genes recombine into a dikaryotic mycelium that grows thicker and faster and is "hopefully" capable of fruiting.

The difficult part in breeding is isolating individual spores and growing out the monokaryotic mycelium. "The Mushroom Cultivator" (Stamets and Chilton) covers the spore diltution technique on pages 340-341.

There is another method of crossing strains that isn't as well understood or well known called anastomosis. This is mentioned on page 8 of "The Mushroom Cultivator". Anastomosis is where two dikaryotic myceliums fuse, exchange genetic material and form a new strain. This can sometimes be seen in casings containing two different strains where a few mushrooms seem to be intermediate between the two parent strains. Anastomosis can be done easily on agar where the two different fruiting strains are allowed to grow together in a single petri dish. Typically, a zone of incompatibility forms where the two strains meet. Even though it seems that the two strains are completely rejecting each other, genetic exchange is usually taking place. If a small wedge is taken from the incompatibility zone and culture out to fruiting, new strains often result mixed in with the parent strains. For some reason the crosses appear more abundantly in later flushes. It is suggested that strains very different in appearance are chosen for crossing by this method so that they are easily recognized when they occur.
inbreeding
Inbreeding?  Well, it isn't generally good long term, but it is a useful tool for enhancing and isolating certain traits.

Each multispore culture is selfing (breeding with one's self).  This doesn't seem to be a problem initially, but sequential multispore cultures reduces heterozygosity.  What I mean by sequential multispore is using prints from your current cultivation to start the next cultivation and so on.  A better idea is to save prints from your earliest cultivation and use those to preserve your "strain" as long as possible.  Cubensis spores should last at least 10+ years if kept cool and dry, but viability drops yearly, so you may need to use large amounts of spores to revive a culture from very old spores.

The loss of heterozygosity means that the spores will produce fruits that show less and less variability from multispore, which sounds fine.  You get a nice uniform crop with little variability, so its similar in behavior to a clone.  But this also makes your culture vulnerable to random mutations.  Random mutations that can be recessive and invisible to the grower.  The PE mutation is a good example.

The PE mutation is recessive, so lets say you made a multispore culture from a print, maybe you selected out a single strain on agar or just injected tons of spores into a substrate.  You got a crop of nice looking mushrooms and printed some.  You gave away all the prints you made and just have one print remaining.  Unknown to you, the mushroom you saved the print from was generated by a pair of spores, where one had been hit by a cosmic ray that damaged a key gene necessary for proper cap development.  The mushroom looks fine because each of it's cells also contains the nuclei from the other spore with an undamaged gene.

You only have the one print, you want to grow it out again and expect the same results as before.  But something is wrong, several mushrooms look like PE, with weird malformed caps.  The selfing, or inbreeding has resulted in pairs of the damaged gene appearing in 25% of the generated strains.  Many of the mushrooms in the crop look fine, but most of them will also contain the damaged gene.  And this is just one trait.  More mutations can sneak in over time, almost all of them are bad, some can make the mycelium unable to even fruit.

So save your earliest prints, save slants of your best cultures.  Combining the spores of different varieties can increase heterozygosity which can then be selectively selfed to produce a new custom made variety.

I know it's confusing and I probably didn't explain it very well, but maybe I helped.
more on inbreeding
I should point out that common sense dictates that inbreeding is bad since it can expose recessive genetic defects or undesirable traits.  Ideally you want high heterozygosity to give a particular mushroom strain a wide range of available genes, which in turn makes it better adapted to unpredictable environmental conditions.  This could be achieved by cloning a wild vigorous specimen or by crossing two very different strains.  In my experience, the mushrooms resulting from such a cross are very vigorous and productive (hybrid vigor).

The problem is that to keep this strain going you have to keep the mycelium going.  This isn't a problem with edible cultures but we need stable spores.  The spores from a high heterozygosity strain will be genetically recombined and won't produce the original strain  (although with aggressive isolation you could get something close).  Multispore grows will be terrible, not because the newly generated strains are bad, but because they are so genetically different from each other.  These different strains don't get along nicely in the same tray which reduces productivity.  If we can reduce the differences between the strains generated by multispore to some minimal level, they tend to cooperate better with each other and act more like a single strain.

The point is that we are dealing with spores not clones.  Many (most) Psilocybe growers use syringes to generate multispore grows with no isolation. In this context, stabilized strains are desirable.  If everyone could trade/sell mycelium this wouldn't be an issue.


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