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Breeding Dikaryons Through Anastomosis ?
    #6203531 -

i have started a few agar plates and have selected some nice isolates from 2 strains of the same species. i've read it was possible to place these isolates on the same agar plate and in time (and luck) a third or maybe fourth sector will pop up, thus being a hybrid of the 2 strains.

does anyone know more about this and how it can be achieved with less than 100 plates etc?


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Re: Breeding Dikaryons Through Anastomosis ? [Re: BlimeyGrimey]
    #6204356 -

You might get lucky with less. Put two small pieces no larger than a grain of rice from each strain 1" apart on a dish and let them grow together. A line of isolation will develop between them as you can see from the picture below on the left. If a third growth emerges from the junction of the first two, it's a cross between them as can be seen at the 12 o'clock position from the picture on the right. Bear in mind, you must start with pure strains. In other words, single sector isolates, so you'll have to do a few transfers to get to that point. It also helps your final outcome if both strains are proved awesome fruiting strains.

Toxins such as rattlesnake venom can be mixed into the agar after it cools that will injure the cell walls of the mycelium enough to help the nuclei to come together. You can read about the process by searching for the thread "Return of the Redboy" here in advanced and/or read John Holidays paper on using the technique with cordyceps here: http://www.nwbotanicals.org/nwb/lexicon/hybridcordyceps.htm
RR



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Re: Breeding Dikaryons Through Anastomosis ? [Re: RogerRabbit]
    #6204882 -

thanks
now i know i'm on the right track. so in theory this technique should work using isolates from the Redboy and Pf Albino strains?

also, what would be the best way to isolate monokaryons using a syringe?
Dilution or perhaps putting a drop of spore solution on a sterile swab and streaking the plate. assuming that near the end of the zigzag streaking should be a few monokaryon colonies?


edit:
know of any other substances to use instead of snake-venom (i'm sure its not easy to get your hands on rattlesnake venom).
i was thinking about the old science lab we did in class once. the "extract dna from peas using detergent and meat tenderizer" thing.
maybe use a detergent or enzyme to break down the cell walls, in a weak enough mixture as to not kill the mycelium.

Edited by BlimeyGrimey (10/24/06 02:31 AM)

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Re: Breeding Dikaryons Through Anastomosis ? [Re: BlimeyGrimey]
    #6205214 -

Try anything you can think of to degrade the cell walls. Read the article by John Holliday. He lists some other aides to hybridization besides snake venom. Snake venom is available commercially, but there's quite a 'clearing process' before they can ship to you. It's also several hundred dollars per gram.

Massive spore dilution or streaking in a zig zag pattern on agar will help you isolate single spores to generate monokaryons, but that isn't what you're looking for here. You were wanting to breed dikaryons. You want to make numerous transfers until there is no more sectoring on the petri dish.

I've been wanting to cross redboy or pf's redspore with an albino, but have not been able to find the time, so I hope you're successful.
RR


--------------------
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Re: Breeding Dikaryons Through Anastomosis ? [Re: RogerRabbit]
    #19503271 -

I'm having trouble understand this:
RogerRabbit said:
Toxins such as rattlesnake venom can be mixed into the agar after it cools that will injure the cell walls of the mycelium enough to help the nuclei to come together. 


because as far as I am aware the two nuclei of dikaryotic mycelium do not fuse except during sporogenesis in the basidium.

If cellular fusion occurs between two dikaryotes as described, would the result not be a tetrakaryote? I fail to see how this method would result in nuclear fusion in addition to cellular fusion.

The only way for the described method to yield a hybridized haploid dikaryote would be for a tetrakaryote yielded from this method to undergo cellular division, resulting in two haploid dikaryotes (which would only result in hybridization if an exchange between one nuclei from either or the original dikaryote cultures occurred). Furthermore, this would form two new hybridized strains, not one!

If nuclear fusion were also to occur during this process, would the result not then be a diploid dikaryote?

Assuming they are tetrakaryotes, I wonder how nuclear fusion/gametogenesis would unfold in the basidium? Perhaps all four nuclei would fuse resulting in a 4n zygote, or perhaps they would fuse in pairs to form two 2n zygotes.

Edited by Delay (01/31/14 09:15 PM)

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Re: Breeding Dikaryons Through Anastomosis ? [Re: Delay]
    #19503452 -

This may help Blimey.  But it may also just be another useless patent.  It lists some mixtures they were using to make sporeless strains of Oysters and other edibles by pairing a sporeless dikaryon with a monokaryon of the mushroom they desired to make sporeless,  resulting in a sporeless or semi sporeless mushroom.  In theory this is basically the same thing you want except instead of sporeLESS you want a albino with red spores.  :smile:

I can't wait to see some albino redspores.

http://www.google.com/patents/us4242832


CH


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Re: Breeding Dikaryons Through Anastomosis ? [Re: CH HELL]
    #19503521 -

CH HELL said:
This may help Blimey.  But it may also just be another useless patent.  It lists some mixtures they were using to make sporeless strains of Oysters and other edibles by pairing a sporeless dikaryon with a monokaryon of the mushroom they desired to make sporeless,  resulting in a sporeless or semi sporeless mushroom.  In theory this is basically the same thing you want except instead of sporeLESS you want a albino with red spores.  :smile:

I can't wait to see some albino redspores.

http://www.google.com/patents/us4242832


CH


This is different than what BlimeyGrimey was suggesting, however, in that the methodology you mention involves the chemically induced division of a sporeless dikaryote into daughter monokaryotic cells. One of the resulting sporeless monokaryotes could then be forced to undergo anastomosis with a monokaryote of sporulating variety, resulting in a sporeless hybrid. The method you mention makes much more sense.

BlimeyGrimey and RR were suggesting anastomosis between two dikaryotes, which can yield only: a tetrakaryote (via cellular fusion), a diploid dikaryote (through cellular fusion and nuclear fusion in pairs), a tetraploid monokaryote (through cellular fusion and nuclear fusion of all four nuclei), two hybridized haploid dikaryotes (through cellular fusion, an exchange of one nuclei per parental dikaryote, followed by a cellular division).

As far as I am aware, RR's method does not facilitate nuclear fusion, nor does it cause cell division in the absence of mitosis (monokaryotes to be formed from dikaryotic cell lines). Hence why I am inclined to say RR's technique would yield a tetrakaryote.

I think the biggest confusion here is that, as far as I understand, there is NOT an exchange of information between nuclei during anastomosis; crossing-over occurs only during meiosis, and meiosis occurs only during sporogenesis, which occurs only in the basidium, which are found exclusively on the gills of mushrooms!

Edited by Delay (01/31/14 09:39 PM)

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Re: Breeding Dikaryons Through Anastomosis ? [Re: Delay]
    #19503673 -

You are over thinking this Delay.  This is the same species of mushroom he is trying to mate and will occur naturally with a little luck.  Blimey is just looking for something to help with the luck part so there isn't as much work.
If you look at RR's picture you can see three distinct sectors from two isolates.  This can happen without any help from us but it may take 100-1,000 tries before it happens.  If they don't mate with each other there while be a clear line between the two and no third sector making it easy to tell without microscopy.  Only by growing the third sector to a fruit body will you truly know what you have,  it will contain the genetics of both but one maybe dominant.

CH


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Re: Breeding Dikaryons Through Anastomosis ? [Re: Delay]
    #19503734 -

Furthermore, I don't believe that RR's method would guarantee that the new sector is in fact a hybrid with a nuclei from either original dikaryote (Assuming that his method somehow yield a 2n dikaryote, which is not explained via his methodology, which would actually yield a 4n tetrakaryote).

For example:
Dikaryote 1:
-(1)nuclei genotype A
-(1)nuclei genotype B

Dikaryote 2:
-(1) nuclei genotype C
-(1) nuclei genotype D

Whether RR's method involves 2n->2x(1n)->2n or 2n->4n->2n, there is no guarantee that the new sector would include a genotype from Dikaryote 1 and Dikaryote 2; genotypes AA, BB, CC, or DD could be yielded just as easily as genotypes AC, AD, BC or BD; two cells from the same dikaryotic cell line could fuse, resulting in an inbreeding. In fact, it would seem more likely for inbreeding to occur given the respective dikaryotic cultures keep to their own.

Unless I am missing something, RR's technique cannot guarantee hybridization.

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Re: Breeding Dikaryons Through Anastomosis ? [Re: CH HELL]
    #19503737 -

CH HELL said:
You are over thinking this Delay.  This is the same species of mushroom he is trying to mate and will occur naturally with a little luck.  Blimey is just looking for something to help with the luck part so there isn't as much work.
If you look at RR's picture you can see three distinct sectors from two isolates.  This can happen without any help from us but it may take 100-1,000 tries before it happens.  If they don't mate with each other there while be a clear line between the two and no third sector making it easy to tell without microscopy.  Only by growing the third sector to a fruit body will you truly know what you have,  it will contain the genetics of both but one maybe dominant.

CH


false. Third sector could be inbreeding. Also from a genetics perspective, his method does not make sense in terms of what it claims to be achieving. You cannot fuse two 2n cells to produce a 2n cell. If two 2n cells are fused, you get a 4n cell, meaning you've either increased the number of nuclei or you've increased the ploidy level. In order for the organism to remain dikaryotic, it would either have to become a diploid through nuclear fusion, or it would have to divide into TWO haploid dikaryotes, which would be either the same as the original parents or TWO new hybrid strains. It doesn't make sense for a new haploid dikaryotic hybrid strain to be produced via his 3-sector-yielding-method, because you would see FOUR sectors as a result of this, not three.

The lack of a fourth sector suggests his third sector is a 4n hybrid (most probably tetrkaryote, or less likely diploid dikaryote).

To the contrary, I would say that RR is underthinking this. I respect his mycological expertise, but I feel that his logic is wrong/incomplete on this matter, which relates much more to genetics and microbiology than it does to mycology.

Edited by Delay (01/31/14 11:42 PM)

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Re: Breeding Dikaryons Through Anastomosis ? [Re: Delay]
    #19503893 -

Inbreeding?

With true isolate you should never have more than one sector, Mycelium can recognize its own mycelium and grow nicely together.  Placing two piece of the same isolated culture on agar they will grow into one sector.  Placing one piece of two different isolated cultures on agar it will most likely grow into two sectors with a distinct line of aversion.  If they do cross there will be a third sector.  Chemicals can be used to help aid the connection by breaking down the cellular barriers.

Isolate is the key word here. 
it would be more like..
For example:
Dikaryote 1:
-(1)nuclei genotype A
-(1)nuclei genotype A

Dikaryote 2:
-(1) nuclei genotype B
-(1) nuclei genotype B


Fungi are different from plants,  fungi only have one set of chromosomes and fungal cells have pores that nuclei can pass through.

May I ask what your back ground is Delay?

CH


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Re: Breeding Dikaryons Through Anastomosis ? [Re: CH HELL]
    #19503995 -

I have experience as a researcher in genetics/biochemistry and a background in agricultural sciences.

From spore to monokaryotic mycelium, there is one set of chromosomes per cell in fungi. From dikaryotic mycelium to zygote, there are two sets of chromosomes per cell. Dikaryote = two nuclei. Ploidy level = number of sets of chromosomes per nucleus. Dikaryotic mycelium is produced when two monokaryotic cells fuse. Ploidy remains haploid throughout the entire fungal life cycle, but the number of nuclei per cell varies. Dikaryotic mycelium has two sets of chromosomes, and two nuclei (one set of chromosomes per nucleus). One sector consists of a single genotype of dikaryotic mycelium (which has two genetically different nuclei per cell). There is only one genotype of cells in a sector, but because each cell has two nuclei that are different from one another, there are two genotypes of nuclei in a sector.


A dikaryotic isolate implies that each cell has two nuclei, which originated from two spores. During sporogenesis, crossing over occurs, implying that each spore from a given mushroom is unique; no two spores are the same. The probability that two spores be the same after crossing over is exceedingly low. Even if that happened, the probability that those two spores should combine to form dikaryotic mycelium is also exceedingly low. It would also be exceedingly low for that dikaryotic mycelium to be selected by whoever is doing this exeriment. Exceedingly low probability x exceedingly low probability x exceedingly low probabilty = not going to happen. Therefore I can guarantee you that you will never get two nuclei of the same genotype in dikaryotic mycelium, unless an inbreeding event such as what I described were to occur. The sets of chromosomes in either nucleus of dikaryotic mycelium are genetically different, which is largely what gives rise to genetic variation. Crossing over will occur between those two sets of chromosomes during meiosis, which occurs when the spores are being produced in the basidium.

An example of such an inbreeding event:

Dikaryote 1 fuses with Dikaryote 1, which would then consist of:
-nuclei genotype A
-nuclei genotype A
-nuclei genotype B
-nuclei genotype B

As is, this would simply be a chromosomal doubling event resulting in a tetrakaryote. If that tetrakaryote were to then divide, it would become either:
Dikaryote AB + Dikaryote AB(wouldn't form new sector, same as Dikaryote 1)
or
Dikaryote AA + Dikaryote BB (inbreeding event)


Notice that with inbreeding you would get TWO new types of dikaryotic mycelium = 4 sectors.


From an intercellular-exchange-of-genetics standpoint, it would only be possible to yield 3 sectors if the new sector were a tetrakaryote or if a hybridization/inbreeding event were to occur in which only one of the two newly formed dikaryotic cell lines be able to undergo mitosis. If this method consistently yields 3 sectors, I'm inclined to say that tetrakaryotes are being formed.

As an alternative explanation for yielding 3 sectors, snake venom might simply induce mutations that cause the mutated cell line to be "rejected" by it's former non-mutated cell line without any intercellular exchange of genetic information occuring?

Perhaps I am missing something, but I'm inclined to say that it's merely a hypothesis that the method RR cited results in hybridization. It would be remarkable if it is a result of hybridization, because the effects of increasing ploidy level/number of chromosomes per cell in an organism can have significant effects on the organism's biochemistry, which can translate into differences in traits of interest such as yields, concentrations of given compounds, etc...

Edited by Delay (02/01/14 12:25 AM)

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Re: Breeding Dikaryons Through Anastomosis ? [Re: Delay]
    #19504213 -

I call bullshit on that method unless I see the genetic sequencing of these so-called hybrids. Assuming it actually promotes anastomosis, which I'm not sure of as being proven or not, it would be an excellent method using two monokaryotic cultures, however  :thatsayes:

Edited by Delay (02/01/14 12:29 AM)

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Re: Breeding Dikaryons Through Anastomosis ? [Re: Delay]
    #19507039 -

Delay said:
I have experience as a researcher in genetics/biochemistry and a background in agricultural sciences.


Okay that makes sense.
  I really feel you are getting some botany in your thinking here and putting too much emphasis of the Genotypes.  Fungi can have multiple nuclei with the same genetics.  Try not to over think it and just think in terms of how the mushroom reproduces/life cycle.

It was late last night but most of what I said stands and I am not going to edit it.

The fact remains that fungi cross breeding is not genetic engineering and there are many ways a fungi can cross breed.  Some ways will cause mutations or none fruiting mycelium.  The parasexuality of fungi has been used for over twenty years to produce mushroom strains with desirable traits.
The easiest way would be to place one spore of one mushroom and another spore from a different mushroom on agar and have them grow together.  The problem with this is that it would require equipment to place two microscopic spores next to each other to grow.
  The second easiest way and most realistic is to create monokaryotic mycelium and then place spores from another mushroom on the mycelium to create dikaryotic mycelium.  This should create a hybrid of the two mushrooms only problem here is going through the work and you could end up with a non fruiting strain but chances are rare of that happening,  or it could end up only growing mycelium from the spores which would not give you a hybrid of any type.  You will also have to isolate after growing this out because you could have several different variation of the hybrid
  Using chemicals isn't always necessary if you are crossing same species, it can make things much easier though.  As I stated last night fungi are much different from plants because they can cross DNA through cellular pores.  With that in mind the chemicals can break down the chitan to allow the dna to flow much easier from one cell to another, this dna migration can be spore to spore,  spore to mycelium or mycelium to mycelium.
There are many other ways to cross breed a mushroom due to the cell pores and someone else can go through all of them but you get the idea, I hope.
  The easier plant reference I can think of would be taking pollen from a Habanero pepper plant to pollinate a Jalapeno pepper plant to create a hotter Jalapeno.  You are not creating a new species just a altered version of the Jalapeno.  Even that isn't a very good example. :/

There are plenty of examples on this site alone.  RR's work on Redboy and Workman has a write up on APE,  I am sure there are more but those are the two to look at.  I have done it with a albino cubensis that was sporeless crossed with another cube to get a white spore bearing cubensis but I don't think I have a thread on this site of it.

If that isn't enough for you delay then you are just arguing to argue.

I apologize for the thread jack Blimey I am done. (for now);)

CH


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Edited by CH HELL (02/01/14 05:00 PM)

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Re: Breeding Dikaryons Through Anastomosis ? [Re: CH HELL]
    #19507529 -

I understand what you are saying, but it is a very incomplete explanation in my mind which does not negate any of the points I raised. I remain unconvinced.

I will look into these pores that you speak of though.

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Re: Breeding Dikaryons Through Anastomosis ? [Re: Delay]
    #19514512 -

Delay said:
Furthermore, I don't believe that RR's method would guarantee that the new sector is in fact a hybrid with a nuclei from either original dikaryote


It doesn't.  This is why the new sector must be plated against the original two to see if it truly is unique.  It also isn't a hybrid when it's mycelium from the same species.
RR


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Re: Breeding Dikaryons Through Anastomosis ? [Re: RogerRabbit]
    #19515276 -

RogerRabbit said:
Delay said:
Furthermore, I don't believe that RR's method would guarantee that the new sector is in fact a hybrid with a nuclei from either original dikaryote


It doesn't.  This is why the new sector must be plated against the original two to see if it truly is unique.


My logic goes to follow that two sister chromosomes wouldn't be found in the same cell of dikaryotic mycelium naturally, because those genetics orgiginated from the same spore, which would have had to connect to a different spore of different genetics to form dikaryotic mycelium. However, if you are including snake venom to damage cell walls and promote nuclear exchange intercellularly, would it not then be possible for a dikaryotic mycelium to arise consisting of sister chromosomes?

If that is possible, why are you convinced that a homozygous dikaryotic mycelium (nucleus A + nucleus A) would not form a sector relative to a heterozygous dikaryotic mycelium (nucleus A + nucleus B) (with one of it's chromosomes being a sister chromosome to that in the homozygous dikaryote)?

Quote:
It also isn't a hybrid when it's mycelium from the same species.
RR


I assume you are referring to variety/cultivar/strain, and not species? Hybridization is simply the mating of two germplasms with significant genetic variance between the two, probably due to populations evolving separately. Hybrids happen all the time in nature. Inter-special mating doesn't produce offspring or viable offpspring, which is what defines speciation. If an African human mates with an Asian human, then their offspring would be a hybrid, but the Asian and the African are of the same species.

Edited by Delay (02/03/14 10:49 AM)

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Re: Breeding Dikaryons Through Anastomosis ? [Re: Delay]
    #19515299 -

Quote:
If an African human mates with an Asian human, then their offspring would be a hybrid, but the Asian and the African are of the same species.


By that definition, nearly the entire human race is composed of hybrids.  At any rate, none of that applies to fungi.  Mycelium in the ground, fruiting and obviously polynucleate is constantly getting an influx of fresh genetics via spores from its own and other colonies.  This mononucleate mycelium can combine with the polynucleate mycelium already in place.  We also know mycelium can crawl over or 'eat' spores (for lack of a better term) and get the genetics that way without the spores ever germinating.
RR


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Re: Breeding Dikaryons Through Anastomosis ? [Re: RogerRabbit]
    #19515466 -

RogerRabbit said:
Quote:
If an African human mates with an Asian human, then their offspring would be a hybrid, but the Asian and the African are of the same species.


By that definition, nearly the entire human race is composed of hybrids.


It's not a question of being a hybrid or not, it's about degrees of hybridization. The more distantly related you are from your mate, the more hybridized your offspring will be. In other words, the greater the genetic variance in terms of alleles between the parents, the greater the degree of hybridization between the offspring. In practical terms, if two Labradors mated we wouldn't call their offspring hybrids due to a high degree of homozygosity, whereas if a Labrador mated with a Pitbull we would refer to the offspring as hybrids due to relatively high degree of heterozygosity. Nonetheless, you could have two populations of Labradors  with significant allelic variance between the two, which would therefore result in hybridization in the offspring if you crossed individuals from those two populations.

Hybridization occurs within species. When it happens between species, the offspring are usually not viable. For a horse can mate with a donkey to produce hybridized offspring (a mule), but the mule will always be infertile. This is usually because of a different number of chromosomes between species, which results in inability to reproduce of infertility in the offspring. Speciation is defined as inability to reproduce due to genetic, geographic or behavioral differences, so there are instances where two species have the potential to yield fertile offspring, but that is not the norm. You're statement that hybridization only occurs between species is false and generally not possible. It is therefore plausible to breed to different psilocybe species, for example, and yield a fertile hybrid, but such matings are exceptions to the norm...not the norm.

Quote:
At any rate, none of that applies to fungi.  Mycelium in the ground, fruiting and obviously polynucleate is constantly getting an influx of fresh genetics via spores from its own and other colonies.  This mononucleate mycelium can combine with the polynucleate mycelium already in place.  We also know mycelium can crawl over or 'eat' spores (for lack of a better term) and get the genetics that way without the spores ever germinating.
RR

Interesting, but that still doesn't dismiss my hypothesis that this method could produce homozygous dikaryotic mycelium, because if that is the case then the question is whether homozygous polynucleate mycelium could infact combine with heterozygous polynucleate mycelium, under circumstances where the duplicate chromosomes in the homozygote would be sister chromosomes with one of the heterozygote's chromosome as well. Or would they in fact sector?

If they would in fact sector, then your sectoring method could also yield inbreeding instead of hybrids, as it would in your verification method. I guess if you grew the sector out and it happened to exhibit traits of both mushrooms, you could be fairly certain, but to simply plate it against the original two sectors would not dismiss my theory.

Edited by Delay (02/03/14 12:08 PM)

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Re: Breeding Dikaryons Through Anastomosis ? [Re: Delay]
    #19516535 -

Mushrooms have many many sexes. Why do you keep referencing humans and animals?

Lipa

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