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Mushroom breeding: a fresh perspective.

The Amycel Spawn Mate Biotechnical Group has been working on developing new hybrid strains for the last 15 years. Our efforts have expanded throughout the years as new techniques and methodologies have become available to the mushroom breeder.

Through the adoption of advance technologies the breeding of new strains has become largely an attainable goal. However there are a number of hurdles to overcome during the development of new strains; some which must be faced in the lab but also some must be faced on the mushroom farm.

We have been working with a number of new hybrid varieties over the last 10 years and recently were granted a patent (US Patent 7,608,760 B2) on a method to develop new brown hybrids. This method uses a wild Agaricus strain, a descendent of U1, and the commercially available OId-Fashioned Brown strain. All of the strains produced using the method described are covered by the patent.

Producing New Hybrids

The actual process of creating a new hybrid is relatively simple. The first and probably most important step is to create and cultivate a library of diverse genetic material with which to work. Our lab has a large collection of wild and commercial strains collected throughout the world; some of these strains were acquired from the Agaricus Recovery (Resource) Program (Kerrigan 1991). Once you have the genetic building blocks in place, the challenge of breeding is choosing the correct cultures to combine.

We spend a lot of time in the lab trying to obtain cells that are called homokaryons. The cells of Agaricus bisporus have two compatible nuclei, often referred to as the "A nucleus" and the "B nucleus". Homokaryons are cells that only contain the same nuclear type (all A or all B). Homokaryons usually lack important cellular information and are not capable of fruiting. To create a new hybrid, homokaryons are combined, or crossed, to form heterokaryons. Heterokaryons are cells that contain multiple genetically different nuclei (Figure 1).

[FIGURE 1 OMITTED]

In a nutshell, mushroom breeding is the combination of compatible homokaryons in new or novel ways.

The Development of U1

In the Sinden Award Lecture of 1985, Dr. Gerda Fritsche described the breeding strategy used for the Horst U1 and U3 hybrid lines (Fritsche 1986). Prior to the development of Ul, white and off-white mushroom strains dominated the industry. Each of these strains had positive and negative traits associated with them. Off-white strains were better for mechanical harvesting but were poor cannery strains due to discoloration. The white strains, although less prolific had smooth white round dense caps, and did not discolor as easily when sliced and canned.

Dr. Fritsche's breeding program combined homokaryons from both the white and off-white strains in an attempt to produce a hybrid exhibiting the best qualities of each strain. Mushroom hybrids were grown; and following six years of spore isolation and testing, the Ul and U3 hybrid strains were released to the market.

When Dr. Fritsche crossed homokaryons together to make the U1 and U3 hybrids, she did so without the knowledge that they had the potential to mate. The crosses were all based on chance and good luck that they would be successful. Today marker assisted selection (MAS) allows for a quick, easy and relatively cheap method of screening many thousands of cultures for their ability to be useful in mushroom breeding.

Critical to mushroom breeding is the ability to obtain and identify homokaryotic cultures from parental strains. Thousands of these homokaryons from commercial and wild sources are used in our breeding program,

The two methods used in the lab are protoplast formation and spore germination. Mushroom scientists have used both techniques successfully for the last 20 years (for a description of the techniques see Horgen et al., 1991 for protoplasts and Kerrigan et al., 1992 for spore germination).

Our lab has had the best luck obtaining homokaryons through spore germination. Spores are plated out on a specific cultivation media and allowed to germinate. Homokaryotic strains grow slower than the hetero-karyotic strains. The slow growing cultures are collected and tested to determine how many nuclear types they contain. Hundreds of homokaryons from individual strains can easily be collected, cataloged, screened and stored for crossing experiments.

Marker Assisted Selection

The primary mode of strain development in our lab is MAS (Rafalski & Tingey 1993, Kerrigan 2000), which uses the polymerase chain reaction (PCR) method to identify specific DNA markers (Khush et. al., 1991) as shown in figure 2.

[FIGURE 2 OMITTED]

These markers can be associated with agronomic traits such as color, cap shape and mushroom quality. MAS allows us to use a DNA-based marker system, to determine which of the homokaryons will provide us with the traits we desire, without having to wait for new crosses to be cropped. MAS makes breeding both easier and more cost effective.

The ability to screen large numbers of crosses easily and quickly has made MAS the key to developing successful new hybrids. We have been lucky enough to find markers associated with desirable traits and focus in on testing only those new hybrids, which carry those markers.

With the large amount of natural genetic variety that is found in the wild populations of A. bisporus, we believe that there is no need to modify strains genetically in a breeding program. All of the traits needed to improve upon the commercial mushroom are available naturally.

A good example of how this method works can be seen with a marker we developed to select for cap color (Loftus et al., 2000). This marker was integral in the development of our patented brown hybrids; where one of the steps was to cross homokaryons from a white commercial strain with homokaryons from an old-fashioned brown strain. This marker allowed us to select for homokaryons that had inherited the cap color from the brown commercial strain. Without MAS we would have had to grow a large number of test crosses to determine which homokaryons were brown and which homokaryons were white.

Many plant breeding programs use DNA-based technology as an integral part in developing new strains (Rafalski & Tingey 1993). This methodology has also been successfully used in previous mushroom breeding efforts (Loftus et al., 1995, Loftus et al., 2000).
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Mapping the Mushroom Genome

The U.S. Department of Energy's Joint Genome Institute has finished sequencing the entire mushroom genome, and it will be in the public domain shortly. What this means for a mushroom breeder using DNA markers is that more detailed and directed breeding of new mushroom strains can be carried out. Having a "roadmap" to the mushroom genome opens up the possibility of identifying regions of DNA that are related to more complicated traits such as disease resistance and cellulose metabolism.

We strongly believe that all the breeding advances that have been made in plants and animals whose genomes have been sequenced could shortly be realized in mushrooms.

Breeding a New Brown Hybrid Using MAS

Almost all the major spawn companies in the world sell a version of what is commonly referred to as the Old-Fashioned Brown strain. This strain is used to produce mushrooms often marketed as the Portabella, Crimini and various trade names.

A number of key agronomic traits were identified as areas of improvement for a new brown hybrid strain; these included the color and size of the mushroom caps. Dark cap color was determined to make the mushrooms more attractive to the consumer, and a breeding program using a darker wild A. bisporus strain began to breed a mushroom with darker caps.

A suitable wild strain was identified (we call it AA-0096) and crosses were made between it, the Old-Fashioned Brown, and a descendent of the U1 hybrid. Hundreds of crosses were made before a number of commercially viable hybrids were created.

The breeding strategy we used was arrived at after a number of unsuccessful experiments trying different combinations of the wild strain, U1, and the Old-Fashioned Brown. All of the crosses we made between AA-0096 and the commercial white strains produced cream or tan colored mushrooms--all of which were too light in color for commercial production. The crosses between AA-0096 with the Old-Fashioned Brown produced hybrids with unacceptable commercial productivity

It was not until we crossed homokaryons from U1 with homokaryons from the Old-Fashioned Brown and created an intermediate hybrid, that we finally started to have success. We crossed these intermediate hybrids with the wild AA-0096 strains and were able to produce darker and more productive hybrids.

So far higher-yielding hybrids have been introduced into mushroom growing regions all over the world. All of these strains produce mushrooms with darker, thicker and denser caps.

Yield

In addition to other improved traits, such as darker colored caps, the new hybrids are more productive than the current strains available. We believe that a large part of this is due to the increased thickness and density of the mushroom cap, as well as the prolific nature of the strains. A comparison of the cap shape can be seen in Figure 3 and Figure 4. Unlike the Old-Fashioned strains which can produce a large number of pins that never fully mature, the new hybrids can be optimized to produce a large number of pins which will fully mature. Table 1 shows data collected at our test facility. As you can see the new strain yielded significantly higher in a series of three trials. All of the trials were optimized for Portabella production using both thinning and cultural manipulation.

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]
Table 1: Shows average yield in lbs/ft2.

           Experimental Strain  Old-Fashioned

Trial#1          6.41 (a)           5.49 (b)

Trial#2          6.39 (a)           5.28 (b)

Trial#3          6.64 (a)           5.65 (b)


Cap Shape

One of the most striking differences between the new hybrids and die Old-Fashioned Brown is the size and shape of the cap. Data comparing cap shape is presented in Table 2. Values are expressed in Cap Shape. Cap Shape is the ratio of cap height and cap diameter. In all experiments, 40 mushrooms were randomly selected from each treatment from small experimental trays, grown according to standard mushroom growing conditions. In all of the experiments, mushrooms were harvested at the same time.
Table 2: Cap shape described as the ratio of cap height and cap
diameter.

                Experimental Strain  Old-Fashioned

Experiment#1       0.29 (a)           0.27 (b)

Experiment#2       0.26 (a)           0.22 (b)

Experiment#3       0.32 (a)           0.30 (b)

Experiment#4       0.30 (a)           0.25 (b)


As can be seen in all four trials the new hybrid strain produced mushrooms with a higher Cap Shape value than the Old-Fashioned Brown. The mushrooms produced by all of the hybrids are noticeably thicker and genuinely unique.

Cap Color

All of the new brown hybrids are darker than the Old-Fashioned Brown strains, with some being quite a bit darker. Figures 4, 5, and 6 show a comparison of color. Mushroom surface color data was evaluated with a chromameter (Konica Minolta BC-10, Osaka, Japan), by measuring the L * and b * parameters. L * is a brightness variable and extends from 0 (black) to 100 (white). The b * value represents yellowness-blueness chromaticity. Mushrooms of cap diameter 8-10 cm were collected from both a new hybrid and an Old-Fashioned Brown at the same crop stage, and measurements were taken on the tops of the caps at random. Thirty L * and b * values for each strain were analyzed using standard t-test analysis (Microsoft EXCEL 2000 Data Analysis Package). Data is analyzed in Table 3.

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]
Table 3: Color and brightness variation between Brown Hybrid and
Old-Fashioned Brown mushrooms.

           Experimental Strain  Old-Fashioned

L * Value       56.66 (b)         60.15 (a)

B * Value       13.64 (b)         11.60 (a)


The new hybrid mushrooms were less bright, or darker, and more yellow (higher b * Value) than the commercial Old-Fashioned Brown.

Additional Brown Hybrids

A number of related brown hybrids are currently in various stages of testing with a goal of commercializing at least one additional hybrid within the next year. A focushas been made on providing the industry with a darker brown mushroom optimized for growing Crimini or medium sized brown mushrooms.

Tailoring Strains for Today's Growing Systems

Even with all of the advancements of the last three decades mushroom breeding has continued to be a very time consuming endeavor, which requires a large investment of capital and patience from both the breeder and the mushroom grower. A number of specific industry standards have been adopted to grow the strains that have been available for the last 30 years. In our experience for a new strain to be successful it requires some modifications in growing parameters for optimal growth.

One of the factors that helped with the introduction of the new brown hybrids is that growers were already used to manipulating growing parameters to get optimal size mixes with the Old-Fashioned Brown strain. Traditionally growers have had to adapt their growing systems to accommodate the cultural needs of the strain. They have had to modify flushing regimes, watering patterns and harvesting practices to optimize strain performance.

We have worked directly with the growers who are successfully growing the new hybrids, to help them pinpoint the growing parameter changes that have been needed for the new strains. Slight modifications of cultural practice, specifically frequency and timing of irrigations, have been needed to optimize performance.

We hope that this hands-on approach, and the development of close relationships with growers, will help ease the culture shock that may be experienced when introducing new and unique strains into the marketplace.

White Hybrid: The Holy Grail

In the 30 years that have passed since the development of U1, no other strain has even come close to usurping its dominance. How do we improve upon this strain? How do we develop new strains that will not just improve upon U1 but will also easily fit into today's specialized growing environments?

We feel that the improvements in the tools available to the mushroom breeder, the exciting potential unlocked by the decoding of the mushroom genome, and the commercial pressures facing the industry will all help propel us into a new era of strain development. Mushroom breeding is not going to become magically less complicated or time consuming, but with the tools we have available now it is achievable. Now is not the time to abandon our efforts or shutter our laboratories. Now is the time to focus our resources and develop new strains that will make the industry profitable for the next 30 years and beyond.

Special thanks to Kim Pitchford for all of her technical assistance in the development of the new brown hybrids.

REFERENCES

Fritsche, G. 1986. Sinden Award Lecture 1985. Breeding Mushrooms. The Mushroom Journal January 157: 4-17.

Horgen, P.A., Jin, T., Anderson, J.B. The use of protoplast production, protoplast regeneration and fragment length polymorphisms in developing a systematic and highly reproducible breeding strategy for Agaricus bisporus. In: Genetics and breeding of Agaricus. Van Griens-ven (Ed.) Pudoc Wageningen ISBN 90 220 1045 7.

Kerrigan, R.W., 1991. What on earth is the Agaricus recovery program? The Mycologist 5: 21.

Kerrigan, R.W., Baller, L.M., Horgen, P.A., Anderson, J.B. 1992. Strategies for the efficient recovery of Agaricus bisporus homokaryons. Mycologia 84:575-579.

Kerrigan, R.W., 2009. Agaricus bisporus mushroom genome sequencing. Mushroom News 57: 4-5.

Kerrigan, R.W. 2000. A brief history of marker assisted selection in Agaricus bisporus. In Science and Cultivation of Edible Fungi, Van Griensven (ed.) Balkema, Rotterdam ISBN 90 5809 143 0.

Khush, R.S., Morgan, L,, Becker, E., Wach, M. 1991 Use of the polymerase chain reaction (PCR) in A. bisporus breeding programs. In: Genetics and breeding of Agaricus. Van Griensven (Ed.) Pudoc Wageningen ISBN 90 220 1045 7.

Loftos, M.C., Lodder, S.C., Legg, E.J., 1995. Molecular Mushroom Breeding. In Science and Cultivation of Edible Fungi, Elliott (ed.) Balkema, Rotterdam ISBN 90 5410 570 4.

Loftus, M.G., Bouchti-King, L., Robles, C. 2000 Use of a SCAR marker for cap color in Agaricus bbporus breeding programs. In Science and Cultivation of Edible Fungi, Van Griensven (ed.) Balkema, Rotterdam ISBN 90 5809 143 0.

Rafalski, J.A. & Tingey, S. 1993. Genetic diagnostics in plant breeding: RAPDs, microsatellites and machines. Trends in Genetics 9: 275-279.

Robles, C.W., Lodder, S.C. Brown mushrooms for commercial production. 2009. US Patent 7,608,760 B2.

Any two means having a common letter are not significantly different at the 5% level of significance, using standard t-test analysis.

Chris Robles Research Scientist

Steve Lodder, Ph.D. Research Scientist

Amycel Spawn Mate Inc. 260 Westgate Drive Watsonville, CA 95076
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Title Annotation:speciality mushrooms
Author:Robles, Chris; Lodder, Steve
Publication:Mushroom News
Article Type:Report
Geographic Code:1USA
Date:Feb 1, 2010
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