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InvisibleHerbBaker
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Would this Work for Creating Hybrid Strains??
    #8057340 - 02/22/08 04:54 PM (16 years, 29 days ago)

Can i make a hybrid by mixing the spores of two different strains in water?

Edited by HerbBaker (02/22/08 05:44 PM)

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Invisibleim_on_a_boat
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Re: Breeding Spores Question [Re: HerbBaker]
    #8057389 - 02/22/08 05:04 PM (16 years, 29 days ago)


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InvisibleHerbBaker
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Re: Breeding Spores Question [Re: im_on_a_boat]
    #8057483 - 02/22/08 05:24 PM (16 years, 29 days ago)

None of those threads answers my question.

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OfflineRogerRabbitM
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Re: Would this Work for Creating Hybrid Strains?? [Re: HerbBaker]
    #8058527 - 02/22/08 09:42 PM (16 years, 29 days ago)

Quote:

warriorsoul said:
Can i make a hybrid by mixing the spores of two different strains in water?




No. Different strains of the same species will often readily join, but such isn't a hybrid. Now, if you crossed two species, you'd have a hybrid.
RR


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InvisibleHerbBaker
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Re: Would this Work for Creating Hybrid Strains?? [Re: RogerRabbit]
    #8059379 - 02/23/08 06:00 AM (16 years, 28 days ago)

I wasnt talking about mixing species but strains rather.
Wouldnt it still be called a hybrid like pe6 or falbino or is there another word im looking for?

So if i mixed two spore prints from the same species together in water.Would some of the offspring be a cross of the two strains?
I dont see why the monokaryotic hyphae of two different strains wouldnt fuse.
Wouldnt it be the first compatible hyphae that the monokaryon comes in contact with?
Doesnt the system favour outbreeding?


Edited by HerbBaker (02/23/08 07:52 AM)

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Offlinecaricapapaya
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Re: Would this Work for Creating Hybrid Strains?? [Re: HerbBaker]
    #8059800 - 02/23/08 10:34 AM (16 years, 28 days ago)

With plants a hybrid is the offspring of 2 different strains.

usually it is referring to an F1 hybrid, which is the first generation of a cross between 2 pure breeding strains of the same species.

If you were to cross 2 species, you would have an interspecific hybrid, or 2 genera would be an intergeneric hybrid. Interspecific and intergeneric hybrids can be difficult to obtain, but not always.

Is the terminology the same for mushrooms?

That said, I think you would get some crossing, but identifying the crosses might be the tricky part depending on how different the parents are.

you should read workman's post about the 'White Penis' cross.

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InvisibleHerbBaker
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Re: Would this Work for Creating Hybrid Strains?? [Re: caricapapaya]
    #8059822 - 02/23/08 10:42 AM (16 years, 28 days ago)

Thanks.
Ive already read it.
I want to know if there is an easier way to make crosses when breeding for a certain phenotype.
Mixing the spores in water would be alot easier than isolating monokaryotic hyphae.

Edited by HerbBaker (02/23/08 01:05 PM)

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InvisibleSmushroom
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Re: Would this Work for Creating Hybrid Strains?? [Re: HerbBaker]
    #8060452 - 02/23/08 01:27 PM (16 years, 28 days ago)

You need to do a lot more reading if you want to create "hybrids". Mushroom genetics don't work in the same way as normal genetics.

The simple answer to your question like RR said, is no.

The long answer is way more complex than anyone here really wants to go into with you and it would be better if you just read until you figured it out yourself, but the simplified version is this:

Then you start from spores you will have millions of them germinate and grow. If you put spores from 2 strains together and allowed them to germinate you would have some that are Strain A, some that are Strain B, and some that are Strain AB. However you have to understand that the genetics of the Strain A mycelium will vary with every single germination. So if you inoculated a single jar with your "crossed" spores you would get a bunch of different colonies of mycelium growing in the same medium. Some would over power the others, some would grow alongside the others, and others would eventually be overpowered.

If you let that grow out and fruit you would have fruits with various genetics and wouldn't know which is which.

Now lets say you actually found a mushroom with genetics from both Strain A and Strain B. If you took a spore print from that and germinated those spores you would have the same genetics as last time, some A, some B, and some AB. So the cycle continues.

Eventually you would probably lose all AB mushrooms since they would not be as stable as the others.

If you truly want to create a "hybrid" strain you will have to do a lot of agar work and go through several generations to stabilize the genetics of it. You are looking at atleast 6 months worth of culture work to even get a strain that produces fruits 50% of the hybrid strain. You would need over a year of work to get something to yield 80% or more of a strain.

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InvisibleHerbBaker
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Re: Would this Work for Creating Hybrid Strains?? [Re: Smushroom]
    #8061564 - 02/23/08 05:24 PM (16 years, 28 days ago)

System prevents selfing, promotes outcrossing.
As long as the Afactors and B factors are different they mate.
Differences are based on alleles present at each of the A factor loci, and each of the B factor loci.
A(allele set 1)1
A(allele set2)2
B(alllele set1)1
B(allele set2)2

A1B1 mates with A2B2 succesfully. Dikaryon.
A1B1 mating with A2B1 might form clamps but not true clamps, no success.
A2B1 mates with A2B2 no clamps, no success.
Mating between a single strain 1/4 compatability.
Mating between two different strains, that share NO common parents, 100% compatable.
Mating between two strains that share a like A or B factor will result in 3/4 compatability.
If there is no common parent there should be over a 98% of mating. This doesn't apply to mixing Mycelium of different strains, though, of course. Only to different spores mating.

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OfflineRogerRabbitM
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Re: Would this Work for Creating Hybrid Strains?? [Re: HerbBaker]
    #8062153 - 02/23/08 07:52 PM (16 years, 28 days ago)

What constitutes a 'strain'?  Certainly not the name some idiot put on the print.  If someone picks a wild mushroom in one part of florida and gives it the name 'x' and someone else picks a mushroom in the next pasture and names it 'y' are they really two strains? :shrug:

The 'problem' with mixing the spores in water is if they cross or not cross, you won't be able to tell because in most cases there isn't a hill of beans difference between them and you'd have no basis to compare to even know if they crossed or not.

Monokaryons can cross with compatible monokaryons.

Dikaryons can cross with compatible dikaryons or monokaryons. 

The above is how 'strains' avoid degeneration in nature.  Spores from hundreds or even thousands of miles away can germinate and 'cross' with the local mycelium.  Is such hybrids?  No.

If a man from africa and a woman from texas have a baby, is it a hybrid?  Of course not.  If a monkey from africa and a woman from texas crossed, it would be a hybrid. Cross a cubensis with an oyster or shiitake and you have a hybrid.
RR


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OfflineMycoAu
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Re: Would this Work for Creating Hybrid Strains?? [Re: RogerRabbit]
    #8062222 - 02/23/08 08:28 PM (16 years, 28 days ago)

RR, so do you have any examples of fungal hybrids in the gourmet or medicinal mushrooms catagory (even some "commonly known" articles) in the past few years? I am interested in this type of experimentation if it is possible for the home grower to accomplish. How common is it for hybridization in Gourmet/medicinal mushies? In the wild? In the lab setting? (successes, that is)

Just curious if you could point me towards some "dumbed" down basic reading to get into this topic.

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Invisibleim_on_a_boat
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Re: Would this Work for Creating Hybrid Strains?? [Re: RogerRabbit]
    #8062679 - 02/23/08 11:13 PM (16 years, 28 days ago)

Quote:

RogerRabbit said:
If a man from africa and a woman from texas have a baby, is it a hybrid?  Of course not.  If a monkey from africa and a woman from texas crossed, it would be a hybrid. Cross a cubensis with an oyster or shiitake and you have a hybrid.
RR




what you tryin to say RR? :tongue:

:angrykidface:

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InvisibleHerbBaker
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Re: Would this Work for Creating Hybrid Strains?? [Re: im_on_a_boat]
    #8063426 - 02/24/08 06:44 AM (16 years, 27 days ago)

Im talking about breeding for unique genetic identifiers.
like breeding Albino with PE.
Wouldnt that be an intraspecies hybrid?

Doesnt the system favour outcrossing?








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Edited by HerbBaker (02/24/08 10:25 AM)

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Re: Would this Work for Creating Hybrid Strains?? [Re: HerbBaker] * 1
    #8064844 - 02/24/08 03:00 PM (16 years, 27 days ago)

Within species hybridization is well known in animal husbandry. Hybrid vigor is also well known. And yes when "strains" of humans mate, the resulting offspring are often or even usually faster stronger and smarter.

One study conducted with women rating the "attractiveness" of shirts they smelled revealed through blood testing that women find the most dissimilar immune systems in men to be the most attractive. IE promoting genetic diversity is a driving factor of evolution and many times combines the best of both worlds whereas stagnating genetics and inbreeding cause recessive traits to become dominant.

<soapbox>

If you believe all humans are born exactly the same then you must agree to allow a downs or other genetically mentally challenged adult to care for your child to prove it. Though they are far nicer, more selfless, loving and forgiving people than average they also have disadvantages. Just like all of us...

Everyone has strengths and weaknesses, advantages and disadvantages. Fear of inadequacy and hiding from glorious differences is the flip side but the same driving force as racism.

</soapbox>


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The curse of Insanity is the constant perception of scrutiny...

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Re: Would this Work for Creating Hybrid Strains?? [Re: ShivaMeme]
    #8068470 - 02/25/08 12:50 PM (16 years, 26 days ago)

Please lets not get so into human genetics guy's. We should all know by now that in breeding mushrooms, there is no "sex". If man or animal could mate with mushrooms that would be fantastic and I would love to see it.

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Re: Would this Work for Creating Hybrid Strains?? [Re: HerbBaker]
    #8068512 - 02/25/08 01:04 PM (16 years, 26 days ago)

Quote:

warriorsoul said:
Thanks.
Ive already read it.
I want to know if there is an easier way to make crosses when breeding for a certain phenotype.
Mixing the spores in water would be alot easier than isolating monokaryotic hyphae.





What's the difference. You still have to isolate the monokaryotic tissue after they germinate.
I am just now getting into mating tissue and don't know much. so I can't give you much advice on the subject. I think you should just isolate for now and start crossing them and see what happens. Only then will you really understand " with a little help of the experienced" what it is all about. I personally think your jumping the gun on learning here. And I didn't mean that in a bad way.

Edited by lipa (02/25/08 02:43 PM)

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Invisiblefastfred
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Re: Would this Work for Creating Hybrid Strains?? [Re: lipa]
    #8073161 - 02/26/08 03:48 PM (16 years, 25 days ago)

> Mushroom genetics don't work in the same way as normal genetics.

LOL While there are differences in the sexual cycle, the molecular basis is still the same. The same Mendelian genetics apply.

I would have to disagree with RR on the strain aspect. It is simply scientific convention to name each sample collected from the wild as a separate strain. The designations strain or sub-strain just don't really have any firm meaning other than as a naming convention.

If I went to a field and collected several samples they would first be named as samples. If there were any morphological differences they would be named as separate strains. Samples collected from different locations or by different researchers would be named as separate strains. Strains are often later found to be the same and the nomenclature is revised.

As far as "hybrids" you can call things whatever you want for the most part. You could call them intra-species hybrids or inter-strain hybrids. Some might disagree with that nomenclature, but they would know exactly what you mean. Therefore it's a useful term.

Hybrids are generally considered to be between species, but species boundaries are usually defined by mating compatibility, so the terminology is certainly not the clearest.


Anyhow, your best bet for creating strain crosses is to dilute your spore solution to obtain only a couple spores per plate and then use the monokaryons obtained to create dikaryotic cultures. Workman also came up with a useful technique of colonizing some substrate with a monokaryon and then adding spores of the other strain to obtain a good percentage of crosses.

Other techniques would involve genetic markers. Using recessive traits you would need to take the crosses to the F2 generation to see them expressed. i.e. obtain spores from your crosses and then grow them out. Redboy, PE, and albino are all recessive I believe. I'm not really aware of any dominant markers.

Another techniques involves mutation, finding auxotrophs, then using nutritional complementation on minimal media to find verified crosses. Obviously this is beyond most people's capabilities and introduces deleterious mutations into your genetics that will take awhile to breed out, but if you're interested look up "filtration enrichment" + "auxotroph".

Workman's done a good job of coming up with a simple technique. Perhaps we should create a guide on ways to accomplish crosses since there seems to be so much interest in the topic. It might answer most of the basic questions and get some people involved in the more advanced aspects of mycology.

One book that might be useful is "Genetics and Breeding of Edible Mushrooms". It's quite technical and focuses way too much on the parasexual cycle and protoplast fusion, but there is some interesting info and examples of hybrids that have been created.


-FF

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OfflineMycoAu
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Re: Would this Work for Creating Hybrid Strains?? [Re: fastfred]
    #8073201 - 02/26/08 04:03 PM (16 years, 25 days ago)

I've considering purchasing that book, but as mentioned, it seems to be a little too complicated and expensive just to find out that I might not need that level of explanation. I'm interested in the information, as are several other members, I'm sure. It would definitely be appreciated if one or a couple of the "advanced" members could collaborate to create a user friendly approach to "hydrids" (or whatever term you decide to use).

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Re: Would this Work for Creating Hybrid Strains?? [Re: MycoAu]
    #8073329 - 02/26/08 04:39 PM (16 years, 25 days ago)

I think that would be a good idea. I just don't really have the time myself to put much time into it.

I nominate.... Workman and RR!

I'd be happy to help out as much as I can. We might get more people with advanced breeding programs and fewer with "Can I just mix the spores?" questions.


-FF

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Re: Would this Work for Creating Hybrid Strains?? [Re: fastfred]
    #8076090 - 02/27/08 06:08 AM (16 years, 24 days ago)

LOL.
Maybe if someone actually addresses my question..
System prevents selfing, promotes outcrossing.
As long as the Afactors and B factors are different they mate.
Differences are based on alleles present at each of the A factor loci, and each of the B factor loci.
A(allele set 1)1
A(allele set2)2
B(alllele set1)1
B(allele set2)2

A1B1 mates with A2B2 succesfully. Dikaryon.
A1B1 mating with A2B1 might form clamps but not true clamps, no success.
A2B1 mates with A2B2 no clamps, no success.
Mating between a single strain 1/4 compatability.
Mating between two different strains, that share NO common parents, 100% compatable.
Mating between two strains that share a like A or B factor will result in 3/4 compatability.
If there is no common parent there should be over a 98% of mating. This doesn't apply to mixing Mycelium of different strains, though, of course. Only to different spores mating.
why wouldnt some of the offspring be hybrids?

Edited by HerbBaker (02/27/08 06:21 AM)

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Re: Would this Work for Creating Hybrid Strains?? [Re: HerbBaker]
    #8076540 - 02/27/08 09:57 AM (16 years, 24 days ago)

Mushroom breeding!

Until about 1980, button mushrooms had been improved by selection: the discovery and preservation of mutants and of spore cultures that showed improvements relative to their parents.

Between 1970 and 1980 several laboratories learned techniques for cross-breeding two strains of A. bisporus to produce a hybrid strain. The first successful commercial hybrid strains were the Horst 'U1' and 'U3' strains, developed by Dr. Gerda Fritsche at the Proefstation voor de Champignoncultuur in The Netherlands. The U1 hybrid now forms the bloodstock of almost all commercial white button mushroom strains grown outside of Asia.

Dependence upon a single crop genotype is called 'monoculture'. It raises the risk that a large fraction of the global crop could be susceptible to a new or mutated pathogen. Serious pathogen outbreaks have affected the button mushroom crop previously: LaFrance virus disease in the 1960s, and aggressive Trichoderma harzianum strain types in the 1990s.

A basic goal of crop breeding is genetic diversification, to reduce and manage such risks. Further goals of mushroom breeding often include some or all of the following improvements:

Resistance to disease Bacterial blotch, mummy (Pseudomonas spp.)
Verticillium spp.
Trichoderma spp.
Mycogone sp.
LaFrance virus

Better utilization of substrate (compost) nutrients
Flavor
Appearance
Crop reliability
Profitability
Nutritional or health value

Breeding and diversification depend upon the availability of large numbers of genetically different individuals (= strains). Until recently, major culture collections held as few as 12 (or fewer) genetically distinct strains of A. bisporus. The Agaricus Resource Program is the first program designed to increase the amount of Agaricus germ plasm available to mushroom breeders worldwide.
In the early 1970's, California scientists first succeeded at splicing viral and bacterial DNAs in the test tube, heralding the birth of the recombinant DNA (rDNA) era, popularly known as genetic engineering, gene transfer technology, gene splicing, molecular biotechnology, and transgenics. This new biotechnology found immediate application in the production of pharmaceuticals, where synthesis by rDNA microbes provided a quantum leap in efficiency over the laborious extraction of miniscule amounts from other sources. Early on it was stated that "the uses of biotechnology are only limited by the human imagination." Today we are witnessing how this broad-based science is impacting virtually every sector of our society.

It was during the 1980's when the power and potential of the burgeoning discipline of genetic engineering was first brought to bear on the improvement of agricultural productivity. The discovery of techniques to transfer genes to the major agronomic crops, including corn, soybean, and wheat, from unrelated species provided breeders with new vistas for increasing the efficiency of food crop production. Remarkable progress, far exceeding early predictions, has been made during the last two decades in breeding plants with new traits such as insect, viral, and fungal resistance, herbicide, stress, and cold tolerance, delayed senescence, improved nutritional features, and others. The global demand for transgenic crops is projected to be a $25 billion market by the year 2010. The growth of this industry will be propelled, in part, by "Golden" rice, which was engineered using a daffodil gene to be rich in beta carotene and thereby the promising answer to the vitamin A deficiency problem pervading the developing world.

Despite concern for the unforeseeable health and environmental risks posed by genetically-modified (GM) crops, gene transfer technology has irreversibly revolutionized plant breeding. Today, more than 100 plant species have been modified by gene splicing for improved sources of food, fiber, or ornamentation. More than 50 new crop varieties have cleared all federal regulatory requirements and stand approved for commercial retail. Because field testing is an essential step in the commercialization process, the number of permits issued by the U. S. Department of Agriculture, Animal and Plant Health and Inspection Service (APHIS) for GM crops provides a measure of the interest in transgenic breeding. During a 16-year period, more than 8,000 permits and notifications (fast-track permits) were issued, rising from a low of 9 in 1987 to a high of 1,120 in 2001 (Fig. 1). For the first three months of 2002, 536 permits/notifications were recorded by APHIS with 49% involving insect resistance, 33% herbicide tolerance, 7% each for product quality and agronomic properties, and with the balance comprising fungal and viral resistance and other traits. Thus, the "genie out of the bottle" scenario describes the status of agricultural genetic engineering. Despite the anti-GM sentiment expressed by a vocal minority, the potency of the new biotechnology for problem solving has been realized to an extent that is far too compelling for it to be disregarded.



Genetically Engineering the Button Mushroom

For almost as long as scientists have been introducing genes into crop plants using molecular biotechnology, others have attempted with limited success at developing a gene transfer method for Agaricus bisporus. A major breakthrough came in 1995 with the surprising discovery that the bacterial workhorse, Agrobacterium tumefaciens, used to shuttle genes into plants, also operated with yeast fungi. Shortly thereafter, this method was extended to filamentous fungi, including A. bisporus.

Agrobacterium is a common soil bacterium with a worldwide distribution. It causes a disease known as crown gall on hundreds of woody and herbaceous plant species, but most commonly pome and stone fruits, brambles, and grapes. In its normal life cycle, the bacterium transfers a tiny bit of its DNA into the plant DNA resulting in the formation of galls. These galls serve as food factories for the mass production of the bacterium. Over the years, scientists learned how to develop disarmed strains of the bacterium that were incapable of inducing galls, but retained the ability to transfer DNA. In essence, a natural biological process was harnessed to create a bacterial delivery system for moving genes into plants, and now fungi.

Though Agrobacterium was shown to be highly promiscuous in shuttling genes into a spectrum of plant and fungal species, the method was still too inefficient to be applied to the breeding of A. bisporus. More recently, we devised a convenient and effective Agrobacterium-mediated 'fruiting body' gene transfer method holding the promise of a powerful tool for the genetic improvement of the mushroom. In our experiments, a small ring of DNA carrying a gene for resistance to the antibiotic, hygromycin, was transferred to a disarmed strain of the Agrobacterium. The antibiotic resistance gene is referred to as a selectable marker, because mushroom cells receiving this gene from the bacterium become marked by the resistance trait and can be selected based on the ability to grow on a hygromycin-amended medium. The end result is a mushroom strain having the newly acquired characteristic of hygromycin resistance. Such a strain has little commercial value, but rather the resistance trait was a research tool that allowed us to easily determine if the bacterium had transferred the gene to the mushroom, and exactly how efficiently it did so under different experimental conditions. Today, and more so in the future, this gene is being replaced or complemented by genes that will confer commercially relevant traits.

Figure 2 highlights the steps in the 'fruiting body' gene transfer method. In this procedure, gill tissue is taken from mushrooms approaching maturity, but with the veil intact, so as to ensure some degree of sterility. Next, the tissue is cut into small pieces and vacuum-infiltrated with a suspension of Agrobacterium carrying the antibiotic resistance gene. In a process referred to as co-cultivation, the gill tissue and bacterium are grown together in the laboratory for several days, during which time the bacterium transfers the resistance gene to the mushroom DNA. Because not all mushroom cells receive a copy of the gene, those that have can be distinguished from those that have not by the ability to grow on the antibiotic medium. After 7 days on the medium, mycelium of A. bisporus appears growing at the edges of some of the gill tissue pieces. After 28 days, upwards of 95% of the tissue pieces will have regenerated into visible cultures. At this point, the GM cultures can be transferred to a standard growth medium, and used to prepare grain spawn in the ordinary manner.



Figure 3 depicts the first of two cropping trials carried out at the Penn State Mushroom Research Center involving GM mushroom lines. In these trials, all six antibiotic-resistant GM lines mirrored the parental commercial hybrid strain in colonizing the compost and casing layer. Further, the GM lines produced mushrooms having a normal appearance and, in some cases, yielded on a par with the commercial strain (Table 1). Expression of the resistance trait in the mushrooms could be easily demonstrated by placing pieces of the cap or stem tissue on the antibiotic medium and observing for growth (Figure 4). These experiments were crucial, because the results established for the first time that a foreign gene could be introduced into A. bisporus without having a detrimental effect on its vegetative and reproductive characteristics.



Table 1. Productivity of genetically-modified (GM) mushroom lines expressing the antibiotic resistance gene that were derived from a commercial off-white hybrid strain.
Yield (lbs./sq. ft.)
Line Trial I Trial II
Commercial hybrid 3.00 a 3.68 a
GM-1 2.08 d 0.86 d
GM-2 1.73 d 1.45 d
GM-3 2.52 bc 2.70 c
GM-4 2.12 cd 2.99 bc
GM-5 2.90 a 3.63 a
GM-6 2.86 ab 3.59 a


Means within a column having the same letter are not significantly different according to the Waller-Duncan K-ratio t test at P<0.0001


Impact of Transgenic Breeding on Mushroom Cultivation

The overwhelming popularity of the hybrid mushroom strains introduced in the 1980's has created a near global monoculture that is precarious from the standpoint of disease and pest susceptibility, and has limited the choice of production characteristics and the range of tolerance to environmental and cultural stresses. During the last two decades, no notable advances have been made in breeding strains with strikingly improved features. This is due largely to the cumbersome genetics of A. bisporus and a shortage of commercially desirable traits. There is movement afoot in using traditional breeding to explore wild isolates of A. bisporus as a source of new traits. Though this represents an important step towards expanding the genetic base of cultivated A. bisporus, it is not yet clear which traits exist in the wild germplasm collection, and if they can be successfully bred into commercial strains.

The advent of a facile gene transfer technique for A. bisporus enables the exploration of genetic solutions to problems confronting the mushroom industry in a realm never before imagined. The awesome power of transgenics lies in what is known as the universality of the genetic code. The biochemical alphabet consisting of the letters G, A, T, and C that spells the DNA sequences of genes controlling traits is identical for all organisms. A scientist blindly handed a gene would have difficulty determining if its source was a mushroom, mouse, or man. It is this unifying feature of genes from all walks of life that makes transgenics so potentially powerful, while it is the tools of molecular biology that unleashes this power so this potential can be realized. Simply stated, the new biotechnology permits the exchange of genetic information between organisms outside the confines of the natural breeding barrier. No longer is the genetic improvement of the mushroom decided by the question of sexual compatibility or traits found within the species.

At another level, gene transfer technology will vastly accelerate our understanding of the molecular mechanisms underlying commercially relevant characteristics. It also will serve to strengthen the muscle of our industry's scientific arm, growing from a handful of mushroom researchers to the global workforce of molecular biologists. As one hypothetical illustration, the quest to breed robust resistance to dry bubble disease would not be restricted to a few scientists searching within A. bisporus, where it may or not exist. Instead, it would extend to scores of scientists working on unrelated organisms who have discovered resistance genes to other Verticillium species. Importing these genes to the mushroom for an evaluation against dry bubble is now possible. As farfetched as this may seem, it is precisely this trans-species approach that has met with commercial success. Genetic manipulations of this sort have been carried out on crop plants and include, importing cry genes from the Bacillus thuringiensis bacterium for insect resistance, a synthetase gene from Agrobacterium for glyphosate herbicide resistance, the nitrilase gene from the Klebsiella pneumoniae bacterium for bromoxymil herbicide resistance, a hydrolase gene from the Escherichia coli bacterium for modified fruit ripening, the barnase gene from Bacillus spp. for male sterility, and viral genes for virus disease resistance.

It cannot be overstated that gene transfer technology is not a panacea whose arrival marks the departure of traditional breeding. Quite the contrary, it is a new tool at the disposal of the breeder that will complement existing techniques, while offering a far broader range of options for successfully affecting genetic solutions to problems. Gene splicing will expedite the breeding process, transferring much of the time in development from the field to the laboratory. It will enable the introduction of genes with a surgical precision and from exotic sources, which otherwise would be unattainable by more conventional methods. It is important to recognize, however, that in the end, the forces of nature overcoming a trait (e.g., the breakdown of insect resistance) would act with the same intensity on the controlling gene whether introduced by traditional or transgenic breeding.

The melding of gene transfer methods with traditional techniques in a mushroom breeding program may take several forms initially, only to be continually refined, streamlined, and improved for higher efficiency and greater effectiveness. Many transgenic manipulations with A. bisporus will require the transfer of the gene to both parental lines so that their offspring mimic the natural inheritance process by carrying a duplicate copy of the gene. For other applications, introducing a single copy of the gene may achieve the desired effect. In either case, the resulting GM lines may require further selection before emerging as worthy commercial strains.

The Perils of Genetic Engineering

If the decision to exploit genetic engineering for agricultural improvement was left to scientists, the cultivation of GM crops would probably be far more widespread and diverse than it is today. But science does not occur in a vacuum. Political forces reflecting the pendulum of public opinion have a strong bearing on the direction and timetable of scientific progress. The early comment that, "the uses of biotechnology are only limited by the human imagination" was used within the context of its seemingly boundless benefit to humanity. In actuality, human imagination has limited biotechnology. That food crops created by genetic engineering are unnatural to the extreme of threatening human health and the delicate balance of the environment is a perception held by a segment of our society. Whether or not these fears are rationale is irrelevant, because their mere existence has hampered the growth of genetic engineering in agriculture. As with many new technologies, the question of acceptance by society will be answered through a distillation of the benefits to be derived for the risks that must be taken.

Both transgenic and conventional plant breeding strive to increase yield, improve quality, and reduce production cost. However, the two breeding strategies differ enormously in the manner in which the end is achieved. For many, it is the process of genetic engineering and not the final product that is most disconcerting. Removing the element of compatible sexual crosses from breeding and reducing it to the splicing of genes in the laboratory seems highly unnatural, constituting extreme human intervention. True, only transgenic breeding allows genes from exotic sources to be brought together in unique combinations. This has been criticized for the possibility of creating new and unpredictable food-borne allergies and toxicities. But the conclusions drawn by the American Medical Association, Board on Agriculture and Natural Resources and National Research Council, and Institute of Food Technologists, among other organizations, agree that GM food poses no greater threat to human health than conventional food.

Consider the tomato breeder seeking to transfer disease resistance from a wild species to the cultivated species. The traditional approach would be to cross the wild species with the domesticated species producing an offspring having inherited half of its genes from one parent and half from the other. In an effort to filter out the undesirable traits contributed by the wild species, the breeder would repeatedly cross the offspring with the cultivated species. However, this process is imperfect, so the new commercial tomato variety would possess the resistance gene and other contaminating genes from the wild species. Now, in the transgenic approach, the resistance gene alone would be snipped from the DNA of the wild species and transferred to the cultivated variety. Here, the risk of altering the food constituents is greater with the conventionally bred variety than the GM variety. For this reason, a likely backlash of the genetic engineering controversy will be stricter regulation of food crops bred by conventional means. As a matter of fact, GM food is federally regulated and adequate safeguards for quality assurance are in place. There is no logical reason to believe that a GM food product would be any more threatening to human health or any more difficult to evaluate for safety than say, for example, a new drug.

Another safety concern with GM food crops revolves around the unintended consequences associated with introduced genes escaping GM crop plants to other species. Science cannot predict with absolute certainty the non-targeted effects of GM crops on the environment, but it can determine which native species could acquire an escaped gene by cross-pollination. More importantly, science is now beginning to appreciate that genetic exchange among unrelated organisms occurs in nature. Therefore, it can be argued that moving genes between unrelated species by transgenic breeding only accelerates this natural evolutionary process.

In order for GM food to reach mainstream society, a greater emphasis must be placed on trait improvements that will benefit the consumer. Most of the genetic engineering accomplishments with crop plants have involved input traits, such as herbicide tolerance and disease and insect resistance. Farmers have embraced the new biotechnology with open arms, because GM crops have reduced their workload or increased profit. But what incentives exist for the consumer to choose GM over non-GM produce in the marketplace? The Agbiotech giants now realize this and are redirecting research towards output traits offering greater consumer appeal, as for example, improved shelf life, appearance, color, flavor, nutrition, hypoallergenicity, etc.

The Shape of the Future

The pace at which genetic engineering is implemented in the mushroom industry will be determined solely by economic factors related to necessity and the resources committed to R & D. If transgenic breeding offered a solution to a problem threatening the livelihood of the mushroom industry today, then the growing of GM strains would become widespread tomorrow. This 'do or die' scenario played out in Hawaii, where a ringspot virus was literally decimating the papaya industry. Fortunately, the fruits of a transgenic breeding effort underway for many years provided a solution. Virtually all papayas now produced in Hawaii are GM for virus resistance.

Another economic force driving the rate at which transgenic breeding reaches the mushroom industry is the level of emphasis placed on R & D. Mushrooms lag far behind other crops in molecular biotechnology, so it is likely that the path through public opinion to acceptance of GM food will be forged by these other commodity groups. As the climate for GM food improves, so will the research funding for the transgenic breeding of mushrooms. For the time being, scientific meetings will be punctuated by modest advances in mushroom transgenics contributed primarily by laboratories in Europe and Far East.

Early transgenic breeding achievements with mushrooms will likely shadow those on cultivated crop plants. Because of funding constraints and technical ease, traits controlled by single genes will be targeted initially, including viral and fly resistance and possibly resistance to bacterial and fungal pathogens, pesticides, and bruising. With the mapping of the mushroom genome and an increased understanding of genetic mechanisms, complex traits controlled by more than a single gene will be undertaken. Improvements might be expected in the areas of yield, size, color, shelf life, heat and water stress, food constituents, fruiting cycle regulation, sexual compatibility, strain stability, and substrate utilization.

Mushrooms will be explored as bioreactors for the synthesis of valuable pharmaceuticals and other bioproducts. The idea of growing the mushroom as a factory rather than a food offers several possible advantages over existing plant-based schemes (i. e., tobacco and corn). A high biomass of mushrooms can be produced on low-cost waste material in a secure containment facility with a controlled, HEPA-filtered environment, and with the option for mechanical harvesting. Further, it may be learned that proteins manufactured by mushrooms have higher specific biological activities in humans than those produced in plant counterparts.

By virtue of the foreign gene introduced and its location within the mushroom DNA or, alternatively, through the deliberate introduction of small snippets of DNA as molecular signatures, it will be possible for spawn manufacturers to definitively identify their strains. This ability to fingerprint strains with ease will afford greater patent protection, which, in turn, will provide the resources to expand breeding programs. The economic incentives related to patented strains also may attract new, perhaps venture capital funded parties to strain development and spawn manufacturing. The mushroom industry as a whole would benefit from the increased competition through a greater selection and diversity of mushroom strains.

Our industry is on the brink of a new and exciting age of strain improvement of a like never experienced before. Many of the accomplishments being realized for cultivated crop plants through transgenic breeding might now be achieved for mushrooms. The availability of mushroom strains with genuinely novel and obviously improved traits will provide the industry with new options for solving problems, simplifying tasks, increasing the efficiency of production, and usage. Though the timetable for its application to mushroom cultivation remains an uncertainty, to paraphrase, "genetic engineering will be persistent, it will be pervasive, and it will be everlasting."

Relevant Resources

Altman, A. 1999. Plant biotechnology in the 21st century: the challenges ahead. EJB Electronic Journal of Biotechnology 2:51-55. Available at http://www.ejb.org/content/vol2/issue2/full/1/.

American Medical Association. 2001. Genetically-modified crops and foods. Report 10 of the Council of Scientific Affairs (I-00). Available at http:/www.ama.assn.org/ama/pub/print/article/2036-3604.html.

Animal and Plant Health and Inspection Service. APHIS field test permits. Available at http://www.aphis.usda.gov/ppq/biotech/.

Board on Agriculture and Natural Resources and National Research Council. 2002. Environmental effects of transgenic plants: the scope and adequacy of regulation. Committee on Environmental Impacts Associated with Commercialization of Transgenic Plants. National Academy Press. 342 pp.

Bundock, P., A. Den Dulk-Ras, A. Beijersbergen, and P. J. J. Hooykaas. 1995. Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J. 14:3206-3214.

Chen, X., M. Stone, C. Schlagnhaufer, and C. P. Romaine. 2000. A fruiting body tissue method for efficient Agrobacterium-mediated transformation of Agaricus bisporus. Appl. Environ. Microbiol. 66:4510-4513.

Conway, G., and G. Toenniessen. 1999. Feeding the world in the twenty-first century. Nature 402 (Suppl):C55-C58.

Cornell University. 1998. First genetically engineered papaya released to growers in Hawaii. New York Agricultural Experiment Station. Available at http://www.nysaes.cornell.edu/pubs/press/1998/papayarelease.html.

De Groot, M. J. A., P. Bundock, P. J. J. Hooykaas, and A. G. M. Beijersbergen. 1998. Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nature Biotechnology 16:839-842.

Food and Agriculture Organization of the United Nations. Statement of biotechnology. Available at http://www.fao.org/biotech/state.htm.

Food and Drug Administration. Biotechnology main page. Center for Food Safety and Applied Nutrition.

Institute of Food Technologists. 2001. Expert report on biotechnology and foods. Institute of Food Technologies, Chicago, IL. 56 pp.

Pennsylvania State University. Biotechnology: food and agriculture. Available at http://biotech.cas.psu.edu/.

Persidis, A. 1999. Agricultural biotechnology. Nature Biotechnology 17:612-614.

Robinson. J. 1999. Ethics and transgenic crops. EJB Electronic Journal of Biotechnology 2:71-80. Available at http://www.ejb.org/content/vol2/issue2/full/3/.

Snow, A. A., and P. M. Palma. 1997. Commercialization of transgenic plants: potential ecological risks. BioScience 47:86-96.

U. S. Department of State. 2001. Biotechnology creates a green gene revolution. International Information Programs. Available at http://usinfo.state.gov/topical/global/biotech/99072900.html.

World Health Organization. Genetically modified food main web page. Available at http://www.who.int/fsf/gmfood/index.htm.

Ye, X., S. Al-Babili, A. Klöti, J. Zhang, P. Lucca, P. Beyer, and I. Potrykus. 2000. Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287:303-305.

147
Introduction
Basidiomycetes, one of the four branches of the monophyletic
group of Eumycota (true fungi), account for about 35% of
the fungal species currently described, and are of both
ecological and industrial importance. Their ecological impact
varies according to their different life styles: saprotrophs
(feeding on the remains of dead organisms or wastes), which
play a central role in the recycling of organic material because
of their ability to degrade some molecules especially reluctant
to biodegradation (i.e. lignin breakdown by white rot fungi);
symbionts, forming ectomycorrhizae with vascular plants,
which facilitate nutrient absorption; or fungal, plant or animal
pathogens, responsible for crop losses (Ustilago maydis) or
serious human diseases (Criptococcus neoformans). Besides,
some basidiomycetes have been traditionally used for human
consumption because of their organoleptic characteristics
(Boletus edulis, Lactarius spp.), their hallucinogenic properties
(Amanita muscaria) or, even, as poisons (A. phalloides).
There is a growing industry of edible mushroom production
based on a process of solid fermentation of pasteurized or sterilized
substrates inoculated with the appropriate spawn that proceeds
under controlled conditions of temperature and humidity. This
control, however, is far from strict and, in practical terms, the
overall process shares more characteristics with open-air
composting processes, where different populations of
microorganisms compete and establish successions, than with
industrially-controlled axenic fermentations. The many factors
(both biotic and abiotic) involved in this process very often cause
instability in yield or in the quality of the product, as it has been
the case for the production of the oyster mushroom, Pleurotus
ostreatus, over the last few years. Hence, there is a market pressure
to improve the yield and quality of the mushrooms currently
produced, and to increase the number of cultivable fungi that has
fuelled research aimed to develop breeding programs for edible
fungi and to formulate appropriate substrates and culture
conditions for new species. Furthermore, this pressure is based
on three main reasons: (i) the economic value of some highlydemanded
fungal species; (ii) their use to produce enzymes or
chemicals useful in industry or pharmacy; and (iii) their application
in processes aimed to recycle industrial or agricultural wastes.
The development of breeding programs for edible
basidiomycetes, however, has been hampered by the difficulties
in performing directed crosses between fungal strains, due to
incompatibility barriers, by the contradictory data about size
and organization of the genetic material, and by the lack of
linkage maps to localize genes of interest. In our laboratory,
Lucía Ramírez
Luis M. Larraya
Antonio G. Pisabarro
Department of Agricultural Production,
Public University of Navarra, Pamplona, Spain
Received 5 May 2000
Accepted 3 July 2000
Correspondence to:
Antonio G. Pisabarro.
Departamento de Producción Agraria.
Universidad Pública de Navarra.
31006 Pamplona. Spain
Tel.: +34-948169107
Fax: +34-948169732
E-mail: gpisabarro@unavarra.es
REVIEW ARTICLE
INTERNATL MICROBIOL (2000) 3:147–152
© Springer-Verlag Ibérica 2000
Molecular tools for breeding
basidiomycetes
Summary The industrial production of edible basidiomycetes is increasing every
year as a response to the increasing public demand of them because of their nutritional
properties. About a dozen of fungal species can be currently produced for food with
sound industrial and economic bases. Notwithstanding, this production is threatened
by biotic and abiotic factors that make it necessary to improve the fungal strains
currently used in industry. Breeding of edible basidiomycetes, however, has been
mainly empirical and slow since the genetic tools useful in the selection of the
new genetic material to be introduced in the commercial strains have not been
developed for these fungi as it was for other organisms. In this review we will discuss
the main genetic factors that should be considered to develop breeding approaches
and tools for higher basidiomycetes. These factors are (i) the genetic system controlling
fungal mating; (ii) the genomic structure and organisation of these fungi; and (iii)
the identification of genes involved in the control of quantitative traits. We will
discuss the weight of these factors using the oyster mushroom Pleurotus ostreatus
as a model organism for most of the edible fungi cultivated industrially.
Key words Pleurotus ostreatus · Basidiomycetes · Breeding fungi · Mating factors ·
Fungal genome structure
we have used the oyster mushroom Pleurotus ostreatus as a
model system to study these three aspects.
P. ostreatus is an edible basidiomycete that grows wildly
on decaying wood thanks to its lignin-degrading capacity, and
is industrially cultivated on a variety of substrates based on
agricultural wastes (such as straw, cotton wastes, sawdust, etc.).
P. ostreatus is currently the second major mushroom in the
world market led by the button mushroom Agaricus bisporus
[32]. Besides its importance for food production, it is of interest
for industrial applications such as paper pulp bleaching (by the
action of its ligninolytic enzymes) and for cosmetics and
pharmaceutical industries [2, 3, 12, 13, 20, 21]. The life cycle
of P. ostreatus, as well as those of many other higher
basidiomycetes, alternates monokaryotic (haploid) and
dikaryotic (di-haploid) phases [9]. Two monokaryotic
compatible hyphae are able to fuse and give rise to a dikaryotic
mycelium in which the two parental nuclei remain independent
(dikaryon, heterokaryon) throughout the vegetative growth,
and which will fruit under the appropriate environmental
conditions. True diploidy occurs at the basidia where karyogamy
takes place immediately before the onset of the meiosis giving
rise to four uninucleate basidiospores. At this diploid stage,
genetic recombination can occur, although some reports have
also suggested the occurrence of parasexual somatic
recombination in higher basidiomycetes [37]. The basidiospores
can germinate when they find the appropriate environmental
conditions producing monokaryotic mycelia that reinitiate the
fungus life cycle. The monokaryotic or dikaryotic condition of
a mycelium can be distinguished by the presence of clamp
connections (specialized structures which allow nuclei
distribution into daughter cells) in dikaryons and their absence
in monokaryons.
Genetic structure of mating genes in higher
basidiomycetes
Monokaryon compatibility and mating is controlled by two
multiallelic genetically independent loci that ensure the
transmission of the two nuclei of the dikaryotic cell during cell
division [4]: the genes in locus A are responsible for controlling
the pairing of nuclei in the dikaryon, for the formation and
septation of clamp cell, and to coordinate cell division, whereas
genes at the B locus control the migration of the nuclei towards
the hyphal tip, the dissolution septa, and the fusion of clamp cells
to ensure a correct dikaryotic stage after cell division. This system
of mating control is referred to as bifactorial (two loci) or
tetrapolar (as it generates four different incompatibility types in
the monokaryotic offspring of a dikaryon) and is common to
most of the edible basidiomycetes industrially cultivated with
the exception of the unifactorial button mushroom Agaricus
bisporus [1, 8, 31]. Molecular analyses of the A and B genes in
Coprinus cinereus and Schyzophillum commune have revealed
that A genes code for homeodomain proteins that, to be functional,
should form heterodimers (with one subunit coded for by each
one of the two nuclei forming the dikaryon), whereas B genes
code for pheromones and their receptors [4]. The genetic structure
of both factors is complex. The factor A gene complex consists
of a central motif of two genes (coding for the two protein types
present in the heterodimer) transcribed in divergent directions
that appears duplicated one to three times in the different A mating
types and species [4, 6, 14, 19, 24, 25]. The gene complex for
factor B has a central unit formed by a single gene coding for a
membrane pheromone receptor and a variable number of genes
(from two to seven) coding for pheromones [4, 35]. Again, a
variable number of copies of this central motif can be found in
different B factors and species.
In P. ostreatus locus A behaves as a single one [16], whereas
locus B is a complex of two genes (matB aand matB ß) linked
at genetic distances ranging from 17.5 cM to less than 5.0
cM in the different strains, and new B specificities can appear
by recombination between the two loci as it occurs in other
higher basidiomycetes [9, 26]. The bifactorial mating control
system makes it difficult and cumbersome breeding-oriented
crossing of monokaryons. In fact, it is first necessary to
determine the incompatibility factors present in monokaryons
derived from a given strain using testers for the four basic
incompatibility types (Ax Bx, Ax By, Ay Bx and Ay By) appearing
in the offspring of a dikaryon AxAy BxBy. By using this method,
we have studied the mating factors present in P. ostreatus
accessions from a variety of origins and have found nine
different A and 15 different B mating types, some of which are
the result of intra-factorial recombination (Table 1). Moreover,
each different strain analysed carried a different pair of A factors,
with only one exception, and a different pair of B factors.
The determination of the mating type of a given monokaryon
is highly facilitated by the use of molecular markers linked to
the mating factors. These markers can be identified, in a first
step, using a bulked segregant analysis approach [23] to generate
Randomly Amplified Polymorphic DNA (RAPD) markers
genetically linked to the genes controlling A and B mating factors
in P. ostreatus [16] (Table 2). Due to the number of monokaryons
analysed in our study, the minimum linkage distance measurable
between markers and the corresponding genes is 1.25 cM
(centimorgan). RAPD markers behave as dominant and, in
the strain under study, they segregate against a null allele.
Consequently, only alleles matA1, matB a2, and matB ß1 are
directly detectable using these RAPD markers. This limitation,
as well as those derived from the RAPD methodology, can be
avoided by converting RAPD markers in Restriction Fragment
Length Polymorphism (RFLP) markers, which allow a quick
and certain identification of monokaryons because they
distinguish the two alleles present in a dikaryotic individual.
RAPD and RFLP markers, in addition, allow the study of
the genomic areas flanking the mating factors that have been
reported to be highly conserved [4]. The sequences of the RAPD
markers linked to the mating factors in P. ostreatus show no
homology with any other entry in the gene databank, and no
148 INTERNATL MICROBIOL Vol. 3, 2000 Ramírez et al.
obvious open reading frames were found to suggest that they
may correspond to coding sequences rather than to intergenic
regions. Notwithstanding, when those probes were used in
Southern experiments on genomic DNA purified from other P.
ostreatus strains, a strong hybridization signal was obtained,
whereas, in the same conditions, only a weak signal on DNA
from other species of the same genus, and no signal on genomic
DNA purified from other agaricales appeared; this suggests a
high degree of species-specificity [16].
Table 2 RAPD and RFLP alleles genetically linked to the mating alleles
identified in Pleurotus ostreatus var. florida. Alleles placed in the same row
are in coupling phase
Mating allele RAPD marker RFLP marker
matA1 S11900 rS11900 a
S181300 rS181300 a
matA2 – rS11900 ß
– rS181300 ß
matB a1 – rL313001
matB a2 L31300 rL313002
matB ß1 L61800 rL618003
matB ß2 – rL618002
149 Breeding of basidiomycetes INTERNATL MICROBIOL Vol. 3, 2000
Table 1 Mating factors found in different Pleurotus ostreatus strains
Strain Variety Origin B factor Occurrence Sample Recombination
(mating genotype) of B factor size(a) frequency (%)
N001 florida USA B1 63 120 15.8
(A1A2 B1B2) B2 38
B3 11
B4 8
N017 florida UPNA(b) B3 45 102 15.7
(A1A2 B3B4) B4 41
B1 8
B2 8
N002 ostreatus Germany B5 39 98 8.2
(A5A6 B5B6) B6 51
B15 6
B16 2
N018 ostretaus UPNA(b) B15 41 105 4.8
(A5A6 B15B16) B16 59
B5 3
B6 2
N003 ostreatus Spain B7 86 170 0.6
(A7A8 B7B8) B8 83
B17 1
N005 colombinus Italy B11 – – –
(A8A11 B11B12) B12 –
N006 sajor-caju India B13 – – –
(A13A14 B13B14) B14 –
(a)Number of individuals studied.
(b)UPNA: Public University of Navarra, Spain.
150 INTERNATL MICROBIOL Vol. 3, 2000 Ramírez et al.
Fig. 1 Molecular karyotype of Pleurotus ostreatus. A) Clamped Homogeneous
Electric Field (CHEF) separation of the chromosomes present in the dikaryon
(N001) and in each of the two nuclei (PC9 and PC15). B) Idiotype of the two
nuclei (PC9 and PC15) indicating the chromosome length polymorphisms. (Figure
from Larraya et al. [17]. Reproduced with permission.)
Table 3 Characteristics of the molecular karyotype and linkage map of Pleurotus ostreatus
Chromosome Sizea (Mbp) Sizeb (cM) Markers number kbp/cM Average Marker Interval (cM) Cross-over events
I 4.70 103.0 23 45.6 4.5 0.98
II 4.35 173.6 23 25.1 7.5 1.71
III 4.55 178.7 25 25.5 7.1 1.75
IV 3.55 59.2 14 60.0 4.2 0.59
V 3.45 82.0 13 42.1 6.3 0.81
VI 3.10 76.7 20 40.4 3.8 0.76
VII 3.15 74.4 18 42.3 4.1 0.74
VIII 2.95 85.3 14 34.6 6.1 0.84
IX 2.10 74.5 16 28.2 4.7 0.74
X 1.75 33.8 13 51.8 2.6 0.34
XI 1.45 59.5 10 24.4 5.9 0.59
Average 3.19 91 16.7 35.1 5.3 0.89
Total 35.1 1000.7 189
a Average of the two homologous chromosomes [17].
b Sum of the linkage distances between the markers placed on the corresponding chromosome. Sizes in centimorgans (cM) correspond to the sum of all
the distances between adjacent chromosome markers.
151 Breeding of basidiomycetes INTERNATL MICROBIOL Vol. 3, 2000
152 INTERNATL MICROBIOL Vol. 3, 2000 Ramírez et al.



I really didn't want to post all of this, but, I did and I hope it helps some. If it does'nt, I'm sorry for posting it, just trying to help.



Doc


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InvisibleHerbBaker
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Re: Would this Work for Creating Hybrid Strains?? [Re: doc34]
    #8076962 - 02/27/08 11:58 AM (16 years, 24 days ago)

Doesn't really apply to what i'm talking about, but thanks!

What would happen if you made two spore prints on top of each other from different strains?

The laws of genetics should favor the crossing of the two strains.
F1 hybrid vigor should be noticed.
F2 recessive phenotypes should also be noticed.

I guess you could call it passive hybridization for lack of a better term.
But i really think it would work..
Time to make some hybrid prints!

Edited by HerbBaker (02/27/08 12:53 PM)

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OfflineWorkmanV
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Re: Would this Work for Creating Hybrid Strains?? [Re: HerbBaker]
    #8077171 - 02/27/08 01:08 PM (16 years, 24 days ago)

Mixing ungerminated spores together should allow mating between strains of the same species (avoid mixing premade syringes since the spores can germinate within the syringe before use). Since most cubensis look relatively the same and an individual spore print from a single strain can generate a range of phenotypes, you need to use very different looking strains of cubensis to be certain of hybridization (and yes, I would call these hybrids in the broadest sense, just like hybrid corn which is also not an interspecies or intergeneric cross).

The obvious problem is distinguishing hybrids from selfed strains. Randomly mixing spores is a crude method and you are going to end up with a mixture of the original strains and hopefully some hybrids (assuming they are compatible). The hybrids won't necessarily look intermediate between the two original strains. If the parents were relatively true breeding without much variability, all of the hybrids are going to look about the same as each other (uniform) as they will each get 50% of their genes from each parent. Only later generations from spores will give you that mix of traits you are looking for (see below)

My initial hybrid work was between the PF albino and the PE strains, both true breeding from spores and easily distinguished from other strains. The cross looked like a normal unremarkable cubensis lacking the penis shaped caps and albinism of the parents. In this case the normal appearance helped confirm the cross but you can imagine that a normal looking cubensis isn't going to stand out in most other crosses. Controlled crosses are better in this regard since you can be sure the result is a hybrid no matter what it looks like.

Another issue is that you can't perpetuate the hybrid if you use the hybrid's spores to start the next generation. There will be all sorts of new strains revealed in the F2 generation as the mix of genes in the hybrid are recombined. Only after selecting for the traits you want for about 6 generations will the features you are after stabilize into a distinct strain (I use strain in the broadest sense even if its not technically correct).

The fact that a hybrid won't breed true is a method that vegetable seed suppliers count on for repeated sales and to discourage seed saving (the bastards). A package of hybrid corn seed will generate a superior and uniform corn crop. If you save that corn crop to replant the next season (to save money), you will end up with all sorts of mixed traits and not all will be as good as the original hybrid seed. This is bad for farmers but can be fun for the home gardener.

So the short answer is, yes it will work but you may not be able to tell. Hybrids tend to show hybrid vigor so even if you aren't sure or can't prove you were successful with the hybridization you may still end up with a superior strain. A good clue that a hybrid is successful is if the F2 generation from the hybrid's spores produces a wide range of phenotypes.

Go for it and have fun.


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Re: Would this Work for Creating Hybrid Strains?? [Re: Workman]
    #8080493 - 02/28/08 06:59 AM (16 years, 23 days ago)

Thanks!I was hoping you'd chime in!

I plan to take both prints right after one another.
and on top of each other to maximize contact.
I'm considering using an anti-clumping agent.

If only 1 in 4 hyphae with the same parents can mate,
and 4 out of 4 hyphae from different strains mate.
This favors the hybrid.
Hopefully any small differences in germination times will be nullified by the 100% compatibility of unrelated hyphae.

The key may be to use spores that are the same age with the same hydration.
That should help maximize contact between monokaryotic hyphae.
All the spores wont germinate at the exact same time,
there should be enough overlap in germination rates to give the hybrids a chance.

It's my 100th post.. Yay!!


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Edited by HerbBaker (02/28/08 07:05 AM)

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Re: Would this Work for Creating Hybrid Strains?? [Re: HerbBaker]
    #8081475 - 02/28/08 01:21 PM (16 years, 23 days ago)


Edited by HerbBaker (02/28/08 01:33 PM)

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Re: Would this Work for Creating Hybrid Strains?? [Re: HerbBaker]
    #8082273 - 02/28/08 04:49 PM (16 years, 23 days ago)

That is a very good point about using spores of the same freshness to help syncronize germination. That is something I didn't even consider and is potentially very important.

What strains are you thinking of crossing?


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Re: Would this Work for Creating Hybrid Strains?? [Re: Workman]
    #8094245 - 03/02/08 04:30 PM (16 years, 20 days ago)

Your best bet is to take 2 different colonized jars of different strains and spawn them to bulk together. You will most definetly get mushrooms that have crossed genes. A few people have already done it.


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Re: Would this Work for Creating Hybrid Strains?? [Re: bryanbzl]
    #8094273 - 03/02/08 04:41 PM (16 years, 20 days ago)

http://www.shroomery.org/forums/showflat.php/Number/8092635#8092635

that is one example.

NOW EXPERIMENT! and make us proud.


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conclusion:
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Re: Would this Work for Creating Hybrid Strains?? [Re: Workman]
    #8097228 - 03/03/08 11:55 AM (16 years, 19 days ago)

Got a falbino and pe6 coming up..so its looks like i'll try them.
I'd like to try some of the older strains in the future
Also would like to cross two albinos from different strains.
I wanna try to do some back-crossing for certain phenotypes.

I want to start working with albinos to create a true breeding albino strain.

Edited by HerbBaker (03/03/08 02:07 PM)

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Re: Would this Work for Creating Hybrid Strains?? [Re: HerbBaker]
    #8097883 - 03/03/08 02:30 PM (16 years, 19 days ago)

I also would like to cross some Pan cyans.

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Re: Would this Work for Creating Hybrid Strains?? [Re: HerbBaker]
    #8110332 - 03/06/08 10:01 AM (16 years, 16 days ago)

I'm  surprised no one has done this with cubes yet..

I hope it makes things alot easier for breeders.

Time to get back to my studies!:tongue:

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Re: Would this Work for Creating Hybrid Strains?? [Re: HerbBaker]
    #8178955 - 03/22/08 07:53 AM (16 years, 21 hours ago)

I have a good idea for you.

You could stain your spores - hopefully, in a way which will not be detrimental to them. some food colouring MAY work, though I don't know.

anyway, make a very dilute spore solution of type A and of type B, and
inocculate a few drops of each onto agar medium. If your lucky, you will be able to catch germination and growth and see where the monokaryotes meet from A and B. If you get a rhizomorphic sector where they meet you know you have a hybrid. Tbh though, I think what will happen is you will get rhizo straight away from A and from B. so, if your going to try it, make sure you do lots of plates and make sure you dilute the spores quite alot. (you're looking for single spore germination sites, which can be isolated and propagated.)

If you can isolate monokaryons from A and B, theoretically the should mate as soon as they touch under normal growth conditions. That'll be your hybrid.

Don't listen to the naysayers, it shouldn't be TOO hard.Good luck!

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Re: Would this Work for Creating Hybrid Strains?? [Re: shoeareyou]
    #8184625 - 03/23/08 08:15 PM (15 years, 11 months ago)

Im very interested in your findings to date, if any plz post


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Re: Would this Work for Creating Hybrid Strains?? [Re: Duke Adro]
    #8249790 - 04/07/08 08:52 AM (15 years, 11 months ago)



Edited by HerbBaker (04/08/08 01:25 PM)

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Re: Would this Work for Creating Hybrid Strains?? [Re: HerbBaker]
    #8255384 - 04/08/08 01:23 PM (15 years, 11 months ago)

The main reason i overlapped the prints is because i wanted at least some of the spores from the two strains to stick together and hopefully increase the chance of mating.
I got four jars of the hybrid and two jars of the original strains to compare growth.

Have to come up with a name if this works..
So far i've got FATE (f+,albino,texan,envy) or F6 ..
Let me know what you think y'all!



Edited by HerbBaker (04/08/08 04:06 PM)

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Re: Would this Work for Creating Hybrid Strains?? [Re: HerbBaker]
    #8256711 - 04/08/08 06:58 PM (15 years, 11 months ago)

The main thing i'll be looking for in the F1 generation are hybrid vigor and dominant gene expression. Albinism and other unfavorable genes are recessive, so i wont choose from those expressing these recessive genes in the F1, as they likely wont be hybrids.
The F1 hybrids will likely have a normal looking phenotype because of Dominant Gene Expression.
The F2 generation should show a much wider range of phenotype expression than either of the original parent strains.
The change in phenotype should be obvious when compared to the Controls.


The hybrid Mycelium should also consistently outperform the Mycelium of the more homozygous parent strains.

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Re: Would this Work for Creating Hybrid Strains?? [Re: HerbBaker]
    #8264308 - 04/10/08 01:09 PM (15 years, 11 months ago)

FATE sounds good you could have fun with the name for sure FaT4, works for me, not sure if you would manage to cross all four strains into one hybrid simultaneously you may have to break it down into smaller steps. Sounds exciting, will be great to see photos of the mycelium and see if it is thicker/faster etc than the individuals. Enjoy!


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Re: Would this Work for Creating Hybrid Strains?? [Re: solumvita]
    #8266559 - 04/10/08 09:13 PM (15 years, 11 months ago)

48 hours after inoculation, three of the four hybrid jars are showing early growth.:thumbup:


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Re: Would this Work for Creating Hybrid Strains?? [Re: HerbBaker]
    #8269121 - 04/11/08 12:04 PM (15 years, 11 months ago)

12 hours later.... all the jars are doing well.
Both Falbino and PE6 are fast strains but the hybrid jars are clearly ahead.

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Re: Would this Work for Creating Hybrid Strains?? [Re: HerbBaker]
    #8286168 - 04/15/08 10:17 AM (15 years, 11 months ago)

The first pins from the hybrid cakes.



Edited by HerbBaker (05/07/08 08:00 PM)

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Re: Would this Work for Creating Hybrid Strains?? [Re: HerbBaker]
    #8374718 - 05/07/08 08:04 PM (15 years, 10 months ago)

Once I get a good white spored Falbino, i'm gonna try a PF Redspore cross with it.

Edited by HerbBaker (05/07/08 08:18 PM)

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Re: Would this Work for Creating Hybrid Strains?? [Re: fastfred]
    #17913000 - 03/06/13 12:13 PM (11 years, 15 days ago)

I dont know about hybreeding but what I did was take a jar of golden teacher and crumbled it then added it to another jar of a cube that the label fell off but i think it was the orsis india or a cam but I got these results:

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