Auto-SummaryThis post discusses mushroom genetics and the process of inbreeding through multispore cultures. When a single mushroom produces spores, these spores act as parents for a new mycelium, essentially resulting in selfing or inbreeding. High heterozygosity in wild Psilocybe cubensis collections means that most gene pairs are different, leading to variable mushroom sizes. Sequential multispore generations result in a loss of heterozygosity, reducing variability and making strains more stable but also more vulnerable to random mutations. The post emphasizes the importance of selecting good traits early in the process, as later generations show minimal improvements. It also touches on the concept of anastomosis, a crossbreeding method that can introduce new genetic variability.
Just dropping this quote from Workman that I like to put out there whenever this topic comes up. Increased my understanding significantly.
Quote:
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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That's the way she goes, boys. Sometimes she goes, sometimes she doesn't, cause that's the fuckin way she goes.
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