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jason9086
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Questioning a Roger Rabbit breeding method
#26949915 - 09/22/20 06:42 PM (3 years, 4 months ago) |
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When I breed mushrooms I follow the standard process of isolating monokaryotic mycelium and plating 2 isolates of monokaryotic mycelium together in order to produce dikaryotic mycelium.
However, I have been reading some posts from RR on shroomery in which he claims that you can get successful crosses using the exact same method with dikaryotic mycelium. According to him, the dikaryotic mycelium meet and if they are compatible they fuse and exchange genetic information. He does not explain the genetics of this (the fused cells would have to have 4 nuclei, what happens to the new 'cross', does it revert to dikaryotic somehow? Does it grow as a tetrakaryon?)
It seems he offers no information regarding how this would happen, and I have searched through the literature to no avail to find any other academics doing this (please link something if you know). The way he backs up this method is that a new sector of growth emerges from the zone where the colonies meet, and apparently if you isolate it and plate it together with the other 2 sectors, it forms a zone of inhibition.
I would be very interested in being linked some information on this from an academic source that actually explains it, because I find his explanation lacking. Thank you.
Frankly, I am very suspicious of the method, but he links the book the mushroom cultivator to back up his claim (a Stamets book), as there is a brief mention of plasmogamy and exchanging genetic information. I don't see citations there backing up that claim either though. Usually plasmogomy refers to the life cycle stage of fusion of monokaryotes, i could see cell fusion of somatically compatible dikaryotes occurring, but i cant find info regarding this resulting in genetic exchange.
Edited by jason9086 (09/22/20 07:18 PM)
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trippleblack
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Re: Questioning a Roger Rabbit breeding method [Re: jason9086]
#26950160 - 09/22/20 09:31 PM (3 years, 4 months ago) |
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maybe you have your terminology a little mixed?
from the literature:
""if the nuclei are genetically identical, as in a mycelium derived from a single uninucleate spore, the mycelium is said to be homokaryotic, but where a cell or mycelium contains nuclei of different genotype, e.g. as a result of fusion(anastomosis) of genetically different hypae, it is said to be heterokaryotic. A special condition is found in the mycelium of many basidiomycota in which each cell contains two genetically distinct nuclei. This condition is dikaryotic, to distinguish it from mycelia which are monokaryotic. ""
i'm not sure how many fusions are the maximum, i don't think it can be unlimited. From what i remember(i think), dikaryotic mycelia can fuse with other dikaryotic mycelia, but not in all in all situations like monokaryotic mycelia. I would think monoisolates would be good to collect if introducting specific nuances into your breeding cultures.
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jason9086
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Re: Questioning a Roger Rabbit breeding method [Re: trippleblack]
#26950173 - 09/22/20 09:43 PM (3 years, 4 months ago) |
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What do you think I am confused on? The terms homokaryotic and heterokaryotic refer to the alleles that make up chromosomes within the nuclei. A heterokaryotic cell contains nuclei with different genetic makeup whereas a homokaryotic cell contains nuclei with the same genetic makeup. A monokaryoyic or haploid cell is by definition homokaryoyic since there arent other nuclei and dikaryotic cells are by definition heterokaryotic in basidiomycetes since they do not result from mitotic nuclear division but rather plasmogamy with a different monokaryote.
All the terms i was using refered to the nuclear number within the cell (mono, di, tetra). I am wondering where i misused the terms?
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trippleblack
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Re: Questioning a Roger Rabbit breeding method [Re: trippleblack]
#26950316 - 09/22/20 11:42 PM (3 years, 4 months ago) |
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the information i came across use different wording. at the time it say nobody knows exactly how anastomosis occur; maybe im confused with terms, but was just going over it.
https://aem.asm.org/content/65/12/5571
i'm interesting in more detail too, anything to take my breeding interest to next level.
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bodhisatta 
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Re: Questioning a Roger Rabbit breeding method [Re: trippleblack] 1
#26950478 - 09/23/20 04:36 AM (3 years, 4 months ago) |
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If anyone's actually mated two monokaryons they conveniently never documented it.
I'm sure almost everyone who thinks they had monokaryotic growth actually didn't anyway.
Mixing varieties is easy there's nothing to take to the next level
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trippleblack
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Re: Questioning a Roger Rabbit breeding method [Re: trippleblack]
#26950755 - 09/23/20 09:24 AM (3 years, 4 months ago) |
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do you mean in the space? nobody is mating two monokaryons? - havent seen it either.
my favorite cordycep breeders claim they are good at mating monokaryotic mycelium; i wanted to apply those techniques to this space. I been working with coryceps more than actives but love both.
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jason9086
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Re: Questioning a Roger Rabbit breeding method [Re: bodhisatta]
#26951009 - 09/23/20 12:43 PM (3 years, 4 months ago) |
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Quote:
bodhisatta said: If anyone's actually mated two monokaryons they conveniently never documented it.
I'm sure almost everyone who thinks they had monokaryotic growth actually didn't anyway.
Mixing varieties is easy there's nothing to take to the next level
How so? Are you saying the threads of workman's breeding efforts and various other threads showing the microscopy of isolated monokaryons is bunk?
So what is your method of 'mixing varieties? Are you talking just about multispore from various varieties in the same jar and hoping for some cross variety interaction?
Edited by jason9086 (09/23/20 12:43 PM)
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bodhisatta 
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Re: Questioning a Roger Rabbit breeding method [Re: jason9086]
#26951415 - 09/23/20 04:23 PM (3 years, 4 months ago) |
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Got links to those threads? All of the crosses people grow these days were made by cultivators in their homes. Usually they get crossed on a dish. People don't generally shoot two varieties into a jar but that also could work.
Workman isna vendor. I wouldn't discount the fact that he could make a cross the same way as any of us and then simply post pictures of mycelium and say he did something much more scientific. Like the mysticism around PE spores. You don't need a centrifuge.
I like to go with simple plausible explanations for things especially when people don't show their whole process. This community likes to make teks to document and show methods but the only cross "teks" i see are from people who've actually done it and have crosses that circulate in the mush growing community and are sold by vendors now. Those people made their crosses mixing spores. No microscope necessary.
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Alan Rockefeller
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Re: Questioning a Roger Rabbit breeding method [Re: bodhisatta]
#26951541 - 09/23/20 05:45 PM (3 years, 4 months ago) |
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Quote:
bodhisatta said: If anyone's actually mated two monokaryons they conveniently never documented it.
I'm sure almost everyone who thinks they had monokaryotic growth actually didn't anyway.
Mixing varieties is easy there's nothing to take to the next level
Isn't just about every mushroom culture the result of two monokaryotic cultures mating?
When a spore germinates it makes very wispy mycelium that doesn't have any clamp connections at the septa - until it meets another monokaryotic mycelium, mates, and starts forming thicker dikaryotic mycelium that has clamp connections.
Monokaryotic mycelium is easy to identify, I don't think it's likely that you are right and everyone else who has ever looked into this is wrong.
Proof of this is everywhere, especially visible with Cordyceps militaris. Monokaryotic mycelium is generated via spore germination using serial dilutions, and the monokaryotic mycelium is checked for mating type via PCR. Based on PCR results, it can be known which two monkaryotic cultures to put together to make a fruiting strain. If you skip the PCR and just put them together, you only get a 50% chance of getting a fruiting strain.
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jason9086
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Re: Questioning a Roger Rabbit breeding method [Re: bodhisatta]
#26951551 - 09/23/20 05:49 PM (3 years, 4 months ago) |
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Well i remember one particular thread of someone posting pics of myc without clamp connections and they were claiming it was monokaryotic and i remember you were on that thread calling it bullshit and the guy was very upset about it lol. And i remember a workman tek in which he claims he used entire spawn jars of monokaryotic myc and injected spores in to there.
The thing with just mixing spores is its really hard to confirm unless the strains are very stable and very visually distinct. Like you couldnt cross a b+ and ecuador very easily since it would be next to impossible to visually distinguish them.
I somewhat agree with you that getting monokaryotic myc is very hard with just streaking or dilution and unless it is confirmed as a single spore it is difficult to distinguish. Tbh i am not sure if monokaryotic myc even grows in a visually robust way.
I work in a mycology lab and have experience single sporing ascomycetes to get genetically pure cultures. I do the same process with basidiomycetes and plate them together in close proximity after hyphal tipping so i will catalog my next cross, i am thinking of an albino x albino such as averys x ape. I think confirmation of a cross with mass spore mixing would be difficult as id expect f1s to look mostly like averys.
However, i am wondering if you can give insight to my original question regarding the RR method of basically just subculturing a sector that arises from plating dikaryotes. I cant find any info on any genetic exchange that can result from that.
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jason9086
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Quote:
Alan Rockefeller said:
Quote:
bodhisatta said: If anyone's actually mated two monokaryons they conveniently never documented it.
I'm sure almost everyone who thinks they had monokaryotic growth actually didn't anyway.
Mixing varieties is easy there's nothing to take to the next level
Isn't just about every mushroom culture the result of two monokaryotic cultures mating?
When a spore germinates it makes very wispy mycelium that doesn't have any clamp connections at the septa - until it meets another monokaryotic mycelium, mates, and starts forming thicker dikaryotic mycelium that has clamp connections.
Monokaryotic mycelium is easy to identify, I don't think it's likely that you are right and everyone else who has ever looked into this is wrong.
Proof of this is everywhere, especially visible with Cordyceps militaris. Monokaryotic mycelium is generated via spore germination using serial dilutions, and the monokaryotic mycelium is checked for mating type via PCR. Based on PCR results, it can be known which two monkaryotic cultures to put together to make a fruiting strain. If you skip the PCR and just put them together, you only get a 50% chance of getting a fruiting strain.
Alan, i think his wording was a bit off but i think what he meant was that people on this forum who claim to have monokaryotic myc dont have evidence that it actually is since they mostly only get them from dilution streaking. I think he is working under the paradigm that monokaryotic myc is not visible or robust enough to grow enough biomass to be visible in culture. Therefore, people posting pics of monokaryotic jars are mistaken. However, i have seen some microscopy pics from people here as well showing myc lacking any clamp connections so i kind of doubt it. My experience with breeding is single sporing, hyphal tipping, and plating them right next to each other and i tend to get visible dikaryotic myc and cant really speak to how visible the monokaryotic myc is.
I think i will try to get pics next time i do this.
By the way, do you have any thoughts on the original question i posed in the thread?
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bodhisatta 
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Quote:
Alan Rockefeller said:
Quote:
bodhisatta said: If anyone's actually mated two monokaryons they conveniently never documented it.
I'm sure almost everyone who thinks they had monokaryotic growth actually didn't anyway.
Mixing varieties is easy there's nothing to take to the next level
Isn't just about every mushroom culture the result of two monokaryotic cultures mating?
When a spore germinates it makes very wispy mycelium that doesn't have any clamp connections at the septa - until it meets another monokaryotic mycelium, mates, and starts forming thicker dikaryotic mycelium that has clamp connections.
Monokaryotic mycelium is easy to identify, I don't think it's likely that you are right and everyone else who has ever looked into this is wrong.
Proof of this is everywhere, especially visible with Cordyceps militaris. Monokaryotic mycelium is generated via spore germination using serial dilutions, and the monokaryotic mycelium is checked for mating type via PCR. Based on PCR results, it can be known which two monkaryotic cultures to put together to make a fruiting strain. If you skip the PCR and just put them together, you only get a 50% chance of getting a fruiting strain.
Yes and this happens very quickly. By the time you see the culture with your naked eye hundreds to thousands or more monokaryotic filaments have met each other and been compatible making dikaryotic mycelium. So you have many dikaryotic strains in a single grow. They manage to grow as a single organism despite being many strains of the same species. I suspect anastomosis plays a role in these varietal hybrids. Like mixing a rust spore variety of cubes with a leuistic variety to yield hopefully a leuistic and rust spored variety. Which is what pasty did. I'm not convinced all the crosses people have managed are because of two monokaryons of each variety meeting but rather some and most are from the dikaryotic mycelium of each mixing
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Alan Rockefeller
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Re: Questioning a Roger Rabbit breeding method [Re: bodhisatta]
#26951881 - 09/23/20 09:20 PM (3 years, 4 months ago) |
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That could be, more experiments with careful documentation and controls are definitely needed! This is some publishable research that anyone at home with agar and a microscope could do.
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jason9086
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Re: Questioning a Roger Rabbit breeding method [Re: bodhisatta]
#26951975 - 09/23/20 10:29 PM (3 years, 4 months ago) |
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Quote:
bodhisatta said:
Quote:
Alan Rockefeller said:
Quote:
bodhisatta said: If anyone's actually mated two monokaryons they conveniently never documented it.
I'm sure almost everyone who thinks they had monokaryotic growth actually didn't anyway.
Mixing varieties is easy there's nothing to take to the next level
Isn't just about every mushroom culture the result of two monokaryotic cultures mating?
When a spore germinates it makes very wispy mycelium that doesn't have any clamp connections at the septa - until it meets another monokaryotic mycelium, mates, and starts forming thicker dikaryotic mycelium that has clamp connections.
Monokaryotic mycelium is easy to identify, I don't think it's likely that you are right and everyone else who has ever looked into this is wrong.
Proof of this is everywhere, especially visible with Cordyceps militaris. Monokaryotic mycelium is generated via spore germination using serial dilutions, and the monokaryotic mycelium is checked for mating type via PCR. Based on PCR results, it can be known which two monkaryotic cultures to put together to make a fruiting strain. If you skip the PCR and just put them together, you only get a 50% chance of getting a fruiting strain.
Yes and this happens very quickly. By the time you see the culture with your naked eye hundreds to thousands or more monokaryotic filaments have met each other and been compatible making dikaryotic mycelium. So you have many dikaryotic strains in a single grow. They manage to grow as a single organism despite being many strains of the same species. I suspect anastomosis plays a role in these varietal hybrids. Like mixing a rust spore variety of cubes with a leuistic variety to yield hopefully a leuistic and rust spored variety. Which is what pasty did. I'm not convinced all the crosses people have managed are because of two monokaryons of each variety meeting but rather some and most are from the dikaryotic mycelium of each mixing
This is the crux of my question. It is known that each fruiting body is a single dikaryon (if i remember correctly) not mixes of dikaryons so what ypu are talking about is genetic exchange between nuclei of different dikaryons with somatic compatability during vegetative growth. In other words the genetic exchange would have to occur before fruiting.
The only way i could see this happening is if dikaryotic cells fuse and exist for some period of time as a tetrakaryon and somehow either the genetics would have to be exchanged OR subsequent cells resulting from mitosis revert to a dikaryotic state in which the nuclei pair randomly resulting in daughter cells with a different pair of nuclei than either parent cell, which i havent seen literature on.
Alan, how would you suggest someone show this using simply a microscope? I am thinking nucleic staining and sequencing work at least would have to be done to show what is essentially breeding of dikaryons through somatic compatability.
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Alan Rockefeller
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Re: Questioning a Roger Rabbit breeding method [Re: jason9086]
#26951977 - 09/23/20 10:31 PM (3 years, 4 months ago) |
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I haven't heard of anyone using a microscope to see the nucleus. Perhaps they are too small to see, or maybe a stain would work.
Most sequencing just shows the species, perhaps full genome sequencing could be more informative, if you could figure out how to make sense of the data.
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bodhisatta 
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Re: Questioning a Roger Rabbit breeding method [Re: jason9086]
#26952158 - 09/24/20 04:29 AM (3 years, 4 months ago) |
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As far as i and most people know a single mushroom can be made of many strains of dikaryotic mycelium. As cloning isn't some easy way to get a monoculture
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