Method for the production of high proportions of homokaryons in breeding stock of the mushroom Agaricus bisporus

A method for the production of high proportions of homokaryons among spores from breeding stock of the mushroom Agaricus bisporus includes the steps of providing a first strain of Agaricus bisporus having tetrasporic ancestry and which carries a gene or genes that determines the trait for production of basidia which predominantly bear at least three spores rather than two, and crossing the first strain of Agaricus bisporus with a second, different strain of Agaricus bisporus to form at least one hybrid heterokaryotic culture. The culture is capable of producing mushrooms having basidia which predominately bear at least three spores, many of which are homokaryotic. The homokaryons taken from the homokaryotic spores can be used in crosses to produce new hybrid strains of Agaricus bisporus mushrooms which may exhibit improvements with respect to various traits including productivity, rate of development, disease resistance, aesthetic qualities and the like. Also, the homokaryons can be used to map genes that control phenotypic traits of interest.

TECHNICAL FIELD 
This invention relates to the production and improvement of mushrooms, the 
sporocarps of edible agaric fungi. More particularly, this invention 
relates to the method of production of, and the utilization of, high 
proportions of homokaryons in hybrid breeding stock of the mushroom 
species Agaricus bisporus (Lange) Imbach, strains of which are 
commercially cultivated. Specifically, this invention relates to the 
introduction of a heritable elevated basidial spore number trait into 
stocks of Agaricus bisporus mushrooms, by producing hybrid Agaricus 
bisporus mushrooms which carry and express the trait and which produce 
relatively large percentages or fractions of homokaryons that may be 
easily recovered from among the offspring of the hybrid strains. These 
homokaryons can be used in crosses to produce new hybrid strains of 
Agaricus bisporus mushrooms which may exhibit improvements with respect to 
various traits including productivity, rate of development, disease 
resistance, aesthetic qualities and the like. Also, these homokaryons can 
be used to map genes that control phenotypic traits of economic 
importance. 
BACKGROUND OF THE INVENTION 
The mushroom species Agaricus bisporus (Lange) Imbach, also known as 
Agaricus brunnescens Peck, is a well known and widely cultivated 
commercial mushroom. At least one distinct horticultural variety, i.e., 
cultivated strain, of this species of mushroom has been the subject of a 
U.S. Plant Patent, No. Plant 7,636, incorporated herein by reference. 
Notably, a distinctive characteristic of Agaricus bisporus, that 
historically has defined the species, is that virtually all previously 
known strains have predominantly produced only two spores on each 
basidium. Generally, only small percentages (less than 10 percent) of 
basidia having more than two spores have been shown to occur in various 
Agaricus bisporus strains. In very rare instances, laboratory strains 
derived from predominantly bisporic strains have been reported to exhibit 
basidia in which basidia bearing more than two spores may predominate. 
These reports appear to involve traits which are either (1) unstable or 
inconsistently expressed, (2) possible artifacts of the sampling method 
(mushrooms too immature to provide representative data), or (3) associated 
with aberrant gross sporocarp morphologies which are unsuitable for the 
commercial market. In any event, although a long-felt need exists for 
increasing the production of homokaryons in breeding stock of Agaricus 
bisporus, none of the foregoing reports have led to a useful method for 
addressing this need. 
The characteristic of producing only two spores on each basidium is 
disadvantageous to the mushroom breeder. Following meiosis in a typical 
two-spored basidium of Agaricus bisporus, each spore receives two nuclei 
which are jointly necessary for fertility. As a result, most spores of 
this species of mushroom, as historically known, produce fertile, 
heterokaryotic progeny. Such a trait of self-fertility poses a problem for 
the mushroom breeder because heterokaryons apparently undergo little, if 
any, hybridization. The bisporic trait characterizes all commercially 
cultivated strains as well as the great majority of naturally occurring or 
"wild" strains of Agaricus bisporus thus far discovered. 
In contrast, all other known species of Agaricus produce predominantly 
fourspored basidia. In most agaric fungi, tetrasporic basidia are usually 
associated with the production of mononucleate homokaryotic spores which 
germinate to produce infertile homokaryons. In mushroom breeding, 
homokaryons, haploid strains which function in a manner similar to the 
gameres of plants and animals, are generally required for the practical 
crossbreeding of stocks to produce new hybrid strains. Homokaryons mate 
easily with other compatible homokaryons. However, less conventionally, it 
is sometimes possible to cross a homokaryon and a heterokaryon or, in some 
instances, to cross two heterokaryons. 
To produce hybrids in the conventional manner, homokaryons such as those 
obtained from the homokaryotic spores of the parent varieties of mushrooms 
must fuse and establish a common heterokaryotic cytoplasm. However, 
homokaryons are presently very difficult to obtain by conventional spore 
isolation from the two-spored Agaricus bisporus strains because typically 
less than three percent of the spores produced by such strains are 
homokaryotic. The great majority of spores of these strains produce 
fertile, heterokaryotic progeny as noted hereinabove. 
Moreover, heterokaryotic and homokaryotic offspring are generally 
indistinguishable from one another except by genetic screening, such as by 
the use of allozyme or DNA markers, which is time consuming and costly. 
Homokaryons are also difficult to obtain by other presently available 
methods. For a more complete description of some conventional 
methodologies for the recovery of Agaricus bisporus homokaryons, and some 
difficulties and drawbacks thereof, see "Strategies For The Efficient 
Recovery of Agaricus bisporus Homokaryons" by Kerrigan et al. in 
Mycologia, 84(4), 575-579 (1992), hereby incorporated by reference. 
In order to overcome the difficulties associated with obtaining homokaryons 
from the two-spored Agaricus bisporus strains, attempts have been made to 
interbreed a four-spored strain from another known species of Agaricus 
with a two-spored strain of Agaricus bisporus. For example, in Raper, 
"Sexuality and Life Cycle of the Edible, Wild Agaricus bitorquis," Journal 
of General Microbiology, 95, 54-66 (1976) a homokaryon from the species 
Agaricus bitorquis was crossed with a homokaryon from the species Agaricus 
bisporus. However, Raper was unable to establish stable, fertile 
heterokaryons, and thus, was precluded from cross-breeding. In fact, no 
attempt to interbreed a four-spored species of Agaricus with a two-spored 
Agaricus bisporus has ever been successful. Accordingly, heretofore, any 
very highly four-spored strain of Agaricus was believed not to belong to 
the species Agaricus bisporus and was believed not capable of 
interbreeding with the species Agaricus bisporus. 
Nevertheless, the need has remained for a process which will permit the 
mushroom breeder to obtain relatively large percentages or fractions of 
homokaryons relatively quickly, efficiently and inexpensively from the 
breeding stock of the Agaricus bisporus mushroom. Moreover, the mushroom 
breeder has always strived to increase the mushroom productivity yield and 
to shorten the duration of the mushroom crop cycle. A crop of mushrooms 
which are higher yielding and earlier fruiting than most commercial 
mushrooms would be economically desirable. 
SUMMARY OF INVENTION 
It is, therefore, an object of the present invention to provide a method 
for producing relatively high proportions of homokaryons from the breeding 
stock of the cultivated mushroom species Agaricus bisporus. 
It is another object of the present invention to provide a method, as 
above, which is less costly and more efficient than the present methods 
for producing and screening for homokaryons from breeding stocks of this 
particular species of mushroom. 
It is a further object of the present invention to provide a method for 
introducing an elevated basidial spore number trait into the bisporic 
stocks of Agaricus bisporus. 
It is still another object of the present invention to provide a method, as 
above, which will produce homokaryons of Agaricus bisporus which are 
useful for mapping genes that control phenotypic traits of economic 
importance. 
It is yet another object of the present invention to provide a method, as 
above, which would produce a higher yielding and earlier fruiting crop of 
Agaricus bisporus mushrooms. 
These and other objects of the present invention, together with the 
advantages thereof over known methods, which shall become apparent from 
the description which follows, are accomplished by the invention as 
hereinafter described and claimed. 
In general, the present invention provides a method for the production of 
homokaryons from breeding stock of the mushroom Agaricus bisporus which 
includes the steps of providing a first strain of Agaricus bisporus having 
tetrasporic ancestry and which carries at least one gene that determines a 
trait for production of basidia which predominantly bear at least three 
spores, and crossing the first strain with at least a second strain of 
Agaricus bisporus to form at least one hybrid heterokaryotic culture. At 
least one hybrid heterokaryotic culture is capable of producing mushrooms 
having basidia which predominantly bear at least three spores, a fraction 
of the spores being homokaryotic. 
The present invention also provides a method for introducing a trait for 
the production of basidia which predominantly bear at least three spores 
into stocks of the mushroom Agaricus bisporus which includes the steps of 
providing a first strain, from a first stock of Agaricus bisporus, having 
tetrasporic ancestry and which carries at least one gene which determines 
the trait for production of basidia which predominantly bear at least 
three spores, and incorporating the gene or genes into the genetic 
background of at least a second strain, from a second stock of Agaricus 
bisporus, such that the new resultant stock combines characteristics of 
the second stock with the trait. 
The present invention also provides for homokaryons produced from spores on 
hybrid mushrooms and their descendants, the hybrid mushrooms being formed 
by providing a first strain of Agaricus bisporus having tetrasporic 
ancestry and which carries at least one gene that determines a trait for 
production of basidia which predominantly bear at least three spores, and 
crossing the first strain with at least a second strain of Agaricus 
bisporus to form at least one hybrid heterokaryotic culture which is 
capable of producing the hybrid mushrooms and their descendants. 
The present invention further provides for heterokaryons, and their inbred 
and outcrossed descendants, produced from a cross between at least two 
strains of Agaricus bisporus, at least one of the strains having 
tetrasporic ancestry and carrying at least one gene which determines a 
trait for production of basidia which predominantly bear at least three 
spores. 
Still further, the present invention provides a method for selectively 
introducing an elevated basidial spore number trait into stocks of 
Agaricus bisporus including the steps of mapping a gene locus which 
determines the elevated basidial spore number traits; cloning an allele of 
the gene locus, which causes expression of the elevated basidial spore 
number trait, from a strain of Agaricus bisporus having tetrasporic 
ancestry; incorporating a DNA sequence encoding the allele into a nucleic 
acid vector construct; and introducing the vector construct into the 
cytoplasm of a recipient strain of Agaricus bisporus. 
The present invention also provides a method for mapping gene loci in the 
nuclear genome of a hybrid strain of Agaricus bisporus which is 
heterozygous for at least one trait-determining locus, and which expresses 
an elevated basidial spore number trait, including the steps of isolating 
a sufficient number of homokaryon cultures from spores obtained from 
mushrooms produced by the hybrid strain; characterizing the homokaryons 
with respect to the presence of a trait of interest; further 
characterizing the genotypes of the homokaryons using genetic markers; and 
analyzing the results of the characterizations for joint versus 
independent segregation. 
Finally, the present invention provides a method for predicting the 
inheritance in homokaryotic offspring of alleles at a first locus, the 
locus selected from the group consisting of MAT and SNT, including the 
step of determining the genotype of the homokaryon at least one other 
different locus, the different locus selected from the group consisting of 
MAT, SNT, PEP1, PEP2, R4-1, R4-3, P1N17, P1N31, P1, N148, P1N150, 
P33N25-11, P33N25-4 and R18-6.

PREFERRED EMBODIMENT FOR CARRYING OUT THE INVENTION 
The production of homokaryotic offspring from both wild and cultivated, 
two-spored breeding stock of the commercial mushroom Agaricus bisporus has 
always been time consuming and costly. This species of mushroom, as 
historically known, characteristically predominantly produces two-spored 
basidia. Thus, the resultant offspring from the spores are mostly, and 
sometimes always, heterokaryotic. Consequently, much effort and expense is 
associated with testing, either genetically or otherwise, to determine 
which of the spores of each mushroom are homokaryotic and which are 
heterokaryotic. For example, research experience indicates that, on 
average, it generally takes a researcher one to two days of effort to 
obtain a single homokaryon using conventional random spore isolation 
methods followed by genetic screening. Even greater effort and expense may 
be associated with obtaining homokaryons by alternative means such as 
microsurgery or protoplast production. 
The present invention provides a novel method for obtaining homokaryons of 
Agaricus bisporus by producing new hybrid strains, each of which results 
from the cross of a first strain of Agaricus bisporus having tetrasporic 
ancestry with at least a second strain of Agaricus bisporus. By the term 
"tetrasporic ancestry", it is meant that the strain is genealogically 
descended from a strain belonging to a taxonomic variety of Agaricus 
bisporus which produces mushrooms characteristically having predominantly 
tetrasporic basidia. One such tetrasporic taxonomic variety is the rare, 
recently discovered Agaricus bisporus var. burnettii. In contrast, 
Agaricus bisporus var. bisporus, which encompasses all historically known 
strains of Agaricus bisporus, is characterized by mushrooms which 
typically produce predominantly two-spored basidia. The term 
"genealogically descended" is specifically intended to distinguish this 
relationship from evolutionary descent, i.e., a naturally occurring 
process of genetic divergence typically involving at least hundreds of 
generations or thousands of years. 
These first-generation hybrid strains have basidia which, without any known 
exception, predominantly bear at least three spores, a relatively large 
fraction of these spores being homokaryotic. As such, these strains 
produce homokaryons in higher proportions than what exists in today's 
two-spored commercial strains. It is meant by the terms "large fraction" 
and "higher proportions" as well as "high proportions" as noted in the 
title of the invention, a fraction or percentage of spores and/or 
homokaryons greater than the 0 to about 10 percent that is typically 
observed in the two-spored variety, Agaricus bisporus var. bisporus. 
Generally, in hybrids containing the elevated basidial spore number trait, 
this percentage is above about 10 percent and has been observed to exceed 
about 50 percent as shown hereinbelow. Moreover, the theoretical limit 
approaches 100 percent. 
With respect to the present invention, it has been found that such hybrids 
can be produced because the homokaryons of the four-spored var. burnettii 
of Agaricus bisporus will interbreed freely with homokaryons of both wild 
and cultivated strains of the two-spored var. bisporus of Agaricus 
bisporus when conventional crossbreeding techniques are used. Thus, 
hybrids combining the desirable genetic background of the traditional 
breeding stocks of Agaricus bisporus var. bisporus with the gene or genes 
that confer the elevated basidial spore number trait found in strains of 
or descended from strains of Agaricus bisporus var. burnettii may be 
formed. By the term "elevated basidial spore number", it is meant that a 
relatively larger number of basidia, relative to the bisporic variety, or 
a bisporic progenitor, have more than two spores. Even more significantly, 
when these hybrids are induced to produce mushrooms, for example, 
following transfers to grain medium and compost medium, the hybrid 
mushrooms have basidia which predominantly bear three or more spores, most 
of which are homokaryotic. 
The reason the elevated basidial spore number trait results in a high 
proportion of homokaryotic offspring is thought to be that the four 
post-meiotic nuclei tend to migrate singly into the four spores of a 
tetrasporic basidium. Therefore, each new spore is typically mononucleate 
and homokaryotic. A three-spored basidium may produce two homokaryotic 
spores and one binucleate, heterokaryotic spore, or three homokaryotic 
spores. Homokaryotic strains germinate to produce homokaryotic isolates 
(homokaryons). 
Because first-generation intervarietal hybrids always express the elevated 
basidial spore number trait, the trait must therefore exhibit genetic 
dominance. Thus, a hybrid strain of Agaricus bisporus which carries the 
trait for the production of basidia which predominantly bear at least 
three spores, i.e, the elevated basidia spore number trait, will itself 
produce relatively large fractions of homokaryotic offspring which can be 
used to produce further hybrid descendants. The dominance of this trait is 
of great practical value, since in the case of a recessive trait, most 
carriers, including all first generation intervarietal hybrids, would not 
express the trait. 
This trait can be passed down to all generations of descendants. The 
percentage of hybrid homokaryotic offspring that will carry the gene or 
genes for the elevated basidial spore number trait will depend upon the 
number of genetic loci involved (present evidence indicates that only one 
locus has a major effect in determining basidial spore number) and upon 
whether the hybrid receives copies of any allele of such a gene 
responsible for the elevated basidial spore number trait from both, one or 
neither parents. By selecting homokaryotic offspring of hybrids on the 
basis of whether they carry any allele responsible for the elevated 
basidial spore number trait or not, it is then possible to design and 
produce at will subsequent generations of hybrids which exhibit one of the 
three following behaviors: (i) elevated basidial spore number (typically, 
trisporic or tetrasporic) hybrids whose direct descendants will all carry 
and express the elevated basidial spore number trait, (ii) elevated 
basidial spore number hybrids whose direct descendants will show 
segregation for two-spored and elevated basidia spore number traits, and 
(iii) two-spored hybrids whose offspring carry only the two-spored trait. 
Furthermore, through repeated back-crossing, using appropriate hybrid 
homokaryotic offspring, a gene determining the elevated basidial spore 
number trait can be incorporated independently within the genetic 
background of any traditional two-spored breeding stock. 
A preferred technique to produce hybrid heterokaryons of Agaricus bisporus 
is to transfer inoculum of each of two substantially pure cultures of 
homokaryons to a suitable medium, for example, agar media such as potato 
dextrose agar (PDA) or complete yeast medium agar (CYMA). The inocula are 
placed about 1 to 2 centimeters apart. The colonies that grow from the 
inocula are allowed to grow until a junction zone of extensive contact 
along the opposing margins of the two colonies has occurred. Mating 
between sexually compatible homokaryons occurs via cell-fusion and the 
formation of common or shared cytoplasm. Fluffy or strandy mycelium is 
sometimes seen at this point in compatible crosses. Inoculum from the 
junction zone of successful crosses, which will contain newly 
heterokaryotic hybrid mycelium, is then transferred to a fresh medium to 
establish a heterokaryotic culture. On this latter culture, tests to 
confirm heterokaryosis, which indicates a successful cross, may 
subsequently be performed. Heterokaryosis can be determined by genetic 
evaluation, demonstration of fertility or comparable methodologies. 
Once the elevated spore number trait has been incorporated into breeding 
stock, it becomes easier to manipulate germ plasm for the production of 
hybrids improved with respect to various traits of economic importance, 
such as mushroom color, size, yield, growth rate, temperature optima, 
disease resistance, and the like. Furthermore, because the homokaryotic 
offspring can greatly predominate, the necessity for genetic screening may 
be eliminated. The heterokaryons present among spore offspring may not 
need to be screened out prior to using the homokaryotie offspring for 
breeding purposes. 
An additional use of homokaryons of Agaricus bisporus is to observe whether 
genetic markers segregate jointly or independently at meiosis. Segregation 
usually cannot be observed in heterokaryotic offspring of this species. 
Analysis of segregation at multiple loci permits economically important 
trait loci to be located on genetic maps, associated with linked markers, 
followed through crosses and identified among offspring, and, if desired, 
cloned and sequenced. Because the present invention facilitates the 
production of homokaryons, it is an important adjunct to genetic mapping 
and genetic engineering in this species. 
At this point, it should be understood that the initial material employed 
in the invention was not known to be a tetrasporic variety of Agaricus 
bisporus. In fact, no such variety was known to exist. Furthermore, there 
were strong indications, based upon morphological data, that the initial 
material did not belong to the species Agaricus bisporus. Moreover, as 
noted hereinabove, the literature available prior to the demonstration of 
the present invention indicates that trying to cross breed material of a 
four-spored Agaricus species with two-spored Agaricus bisporus was very 
likely to be unsuccessful. Specimens of the initial material have been 
collected from nature in Riverside County, Calif., USA, since November, 
1989. These specimens are identified along with their dates of collection 
in Table I. 
TABLE I 
______________________________________ 
Specimens of Agaricus bisporus var. burnettii 
Date of Date of 
Specimen ID 
Collection Specimen ID Collection 
______________________________________ 
JB 2 10 Nov 1989 JB 3 Nov 1990 
JB 10 Feb 1992 JB 11 Feb 1992 
JB 12 Feb 1992 JB 13 Feb 1992 
JB 14 Feb 1992 JB 15 Feb 1992 
JB 16 Feb 1992 JB 17 Feb 1992 
JB 18 Feb 1992 JB 19 Feb 1992 
JB 20 Feb 1992 JB 21 Feb 1992 
JB 22 Feb 1992 JB 23 Feb 1992 
JB 24 Feb 1992 JB 25 Feb 1992 
JB 26 Feb 1992 JB 27 Feb 1992 
JB 28 Feb 1992 JB 29 Feb 1992 
JB 30 Feb 1992 JB 31 Feb 1992 
JB 32 Feb 1992 JB 33 Feb 1992 
JB 34 Feb 1992 JB 35 Feb 1992 
JB 36 Feb 1992 JB 37 Feb 1992 
JB 38 Feb 1992 JB 39 Feb 1992 
JB 40 Feb 1992 JB 41 Feb 1992 
JB 101 07 Mar 1992 JB 102 07 Mar 1992 
JB 103 07 Mar 1992 JB 104 07 Mar 1992 
JB 105 07 Mar 1992 JB 106 07 Mar 1992 
JB 107 07 Mar 1992 JB 108 07 Mar 1992 
JB 109 07 Mar 1992 JB 110 07 Mar 1992 
JB 111 07 Mar 1992 JB 112 07 Mar 1992 
JB 113 07 Mar 1992 JB 114 07 Mar 1992 
JB 115 07 Mar 1992 JB 116 07 Mar 1992 
JB 117 07 Mar 1992 JB 118 08 Mar 1992 
JB 119 08 Mar 1992 JB 120 08 Mar 1992 
JB 121 08 Mar 1992 JB 122 08 Mar 1992 
JB 123 08 Mar 1992 JB 124 08 Mar 1992 
JB 125 08 Mar 1992 JB 126 08 Mar 1992 
JB 127 08 Mar 1992 JB 128 08 Mar 1992 
JB 129 08 Mar 1992 JB 130 08 Mar 1992 
JB 131 08 Mar 1992 JB 132 09 Mar 1992 
JB 133 09 Mar 1992 JB 134 09 Mar 1992 
JB 135 09 Mar 1992 JB 136 09 Mar 1992 
JB 137 09 Mar 1992 JB 138 09 Mar 1992 
JB 139 09 Mar 1992 JB 140 09 Mar 1992 
JB 141 09 Mar 1992 JB 142 09 Mar 1992 
JB 143 09 Mar 1992 JB 144 09 Mar 1992 
JB 145 09 Mar 1992 JB 146 09 Mar 1992 
JB 147 09 Mar 1992 JB 148 09 Mar 1992 
JB 149 09 Mar 1992 JB 161 13 Mar 1992 
JB 162 13 Mar 1992 JB 163 13 Mar 1992 
JB 164 13 Mar 1992 JB 165 13 Mar 1992 
JB 166 13 Mar 1992 JB 155 Apr 1992 
JB 157 Apr 1992 JB 167 Apr 1992 
JB 168 Apr 1992 JB 169 Apr 1992 
JB 171 Apr 1992 JB 172 Apr 1992 
JB 173 Apr 1992 JB 174 Apr 1992 
JB 175 Apr 1992 JB 176 Apr 1992 
JB 177 Apr 1992 JB 178 Apr 1992 
JB 179 Apr 1992 JB X Apr 1992 
RWK 1840 07 Mar 1992 RWK 1841 07 Mar 1992 
RWK 1842 07 Mar 1992 RWK 1843 07 Mar 1992 
RWK 1844 07 Mar 1992 RWK 1845 07 Mar 1992 
RWK 1846 07 Mar 1992 RWK 1847 07 Mar 1992 
RWK 1848 07 Mar 1992 RWK 1849 07 Mar 1992 
RWK 1850 07 Mar 1992 RWK 1851 07 Mar 1992 
RWK 1852 07 Mar 1992 RWK 1853 07 Mar 1992 
RWK 1854 07 Mar 1992 RWK 1855 07 Mar 1992 
RWK 1856 07 Mar 1992 RWK 1857 07 Mar 1992 
RWK 1858 07 Mar 1992 RWK 1859 07 Mar 1992 
RWK 1860 07 Mar 1992 RWK 1861 07 Mar 1992 
RWK 1862 07 Mar 1992 RWK 1863 07 Mar 1992 
RWK 1864 07 Mar 1992 RWK 1865 07 Mar 1992 
RWK 1866 07 Mar 1992 RWK 1867 07 Mar 1992 
RWK 1868 07 Mar 1992 RWK 1869 07 Mar 1992 
RWK 1870 07 Mar 1992 RWK 1871 07 Mar 1992 
RWK 1872 07 Mar 1992 RWK 1873 19 Mar 1992 
RWK 1874 19 Mar 1992 RWK 1875 19 Mar 1992 
JB 191 10 Apr 93 JB 192 10 APR 93 
JB 193 10 Apr 93 JB 194 10 APR 93 
JB 195 10 Apr 93 JB 196 10 APR 93 
JB 197 10 Apr 93 JB 198 10 APR 93 
JB 199 10 Apr 93 JB 200 10 APR 93 
JB 201 10 Apr 93 JB 202 10 APR 93 
JB 203 10 Apr 93 JB 204 10 APR 93 
JB 205 10 Apr 93 JB 206 10 APR 93 
JB 207 10 Apr 93 JB 208 10 APR 93 
JB 209 10 Apr 93 JB 210 10 APR 93 
______________________________________ 
For several of these field specimens, tissue cultures have been prepared. 
For the remainder, samples of viable spores have been preserved, and 
single- and multiple-spore cultures have been prepared from some of these 
samples. However, for many of these field specimens, no genetic, 
inteffertility, micromorphological, or cultural confirmation of identity 
has been made. Consequently, it is possible that some of the collections 
listed might actually represent taxa other than var. burnettii, although 
this number is unlikely to exceed a very small percentage. 
The initial steps of the invention utilized germ plasm from these wild 
strains of Agaricus, now placed in the newly proposed, characteristically 
four-spored taxon Agaricus bisporus var. burnettii. Two of these 
heterokaryotic stocks (JB2 and JB3) were each preserved as multiple-spore 
samples which were prepared from the specimens collected from nature in 
Riverside County, Calif., between November, 1989 and November 1990. One 
heterokaryotic multi-spore culture, prepared from the JB2 sample, having 
the proposed taxonomic description of Agaricus bisporus var. burnettii and 
designated as JB2-ms (Accession No. 76072), has been deposited with the 
American Type Culture Collection under the terms of the Budapest Treaty on 
the International Recognition of the Deposit of Microorganisms for the 
Purposes of Patent Procedure and has been available to the scientific 
public upon request since the 2nd of May, 1990. Genotype data was obtained 
for the heterokaryotic cultures JB2-ms and JB3-ms. These strains have 
unique combinations of genetic markers as reported in Table II set forth 
hereinbelow. 
TABLE II 
______________________________________ 
Genotype Data on Stocks JB2 and JB3 of 
Agaricus bisporus from Riverside County, 
California, Based upon Multi-Spore Cultures 
Nuclear marker loci 
JB2-ms Alleles 
JB3-ms Alleles 
______________________________________ 
P33N5 2/2 13/13* 
P33N6 1/1 1/1 
P33N7 2/2 .sub.-- /.sub.-- 
P33N10/1 3/3 3/5 
P33N10/2 6/6 2/6 
P33N13 5/5 5/5 
P33N14 8/8 5/7 
P33N18 1/1 1/1 
P33N25 8/8* 3/4 
P4N6 4/4 4/4 
P4N27 1/1 1/2 
GPT 1/1 .sub.-- /.sub.-- 
ADH 3/3 4/4 
PEP1 3/3 4/4 
PEP2 .sup. 5/5.sup.1 
3/3 
BGLU 3/5 5/5 
AAT 3/3* 1/1 
PGM .sub.-- /.sub.-- 
2/2 
EST 2/2 3/3 
MPI S/F S/S 
______________________________________ 
.sup.1 Heterozygosity was lost at PEP2 in JB2ms; based on genotypic 
analysis of singlespore cultures of the JB2 stock, the parental genotype 
was 3/5. 
The uniqueness of these genotypes may be seen by comparison to the table of 
cultivar and wild Agaricus bisporus genotypes presented in Appendix 2 of 
Kerrigan et at., "The California Population of Agaricus bisporus Comprises 
At Least Two Ancestral Elements," Systematic Botany, 18:123-136, (1993), 
hereby incorporated by reference. An asterisk (*) denotes a novel allele 
not previously observed in an extensive global sample of Agaricus bisporus 
stocks. 
A similar pattern was observed when mitochondrial DNAs of JB2 and JB3 were 
subjected to restriction fragment analysis. Three mitochondrial fragments, 
B 1, B4b, and B13 were hybridized to EcoRI cut DNAs from JB2-ms and 
JB3-ms. The resulting patterns of fragments were compared to a world-side 
sample of over 200 strains of Agaricus bisporus var. bisporus. The JB2-ms 
and JB3-ms patterns observed with B13, B1 and B4b were observed 
respectively in 46 and 4, 2 and 1, and 0 and 0 var. bisporus isolates. As 
with the nuclear genotype data presented hereinabove, there were 
indications of similarity to Agaricus bisporus as well as indications of 
uniqueness, evolutionary isolation, and genetic divergence. 
Other relevant characteristics of the JB2 stock include the fact that, 
unlike Agaricus bisporus var. bisporus mushrooms of the culture JB2-ms had 
a fertile lamellar margin (i.e., basidia were present there) and 
cheilcystidia were notably absent. Moreover, the size of the spores from 
the mushrooms of the JB2-ms culture, and from other samples of var. 
burnettii mushrooms, were significantly smaller than those of both wild 
and cultivated bisporic strains, now placed in the taxon known as Agaricus 
bisporus var. bisporus. A statistical analysis of the spore sizes was 
performed after obtaining length and width measurements on 10 to 60 spores 
from each of a total of 37 individual specimens from five different taxa 
(species or varieties). The mean of the length and width is provided for 
each specimen in Table III hereinbelow. Table IV sets forth a comparison 
of the overall means, and their standard deviations, for the five 
different taxa from which specimens were taken. 
TABLE III 
______________________________________ 
Spore Size Measurements of Five Agaricus Taxa 
Taxa Mean Length (.mu.) 
Mean Width (.mu.) 
______________________________________ 
BISP 6.45 4.97 
BISP 6.95 5.49 
BISP 6.73 5.24 
BISP 6.38 5.13 
BISP 6.64 5.31 
BISP 7.24 5.55 
BISP 7.08 5.84 
BISP 6.79 4.99 
BISP 6.83 5.18 
BISP 7.20 5.33 
BISP 6.94 5.55 
BISP 7.02 5.52 
BISP 6.75 5.48 
BISP 7.09 5.52 
BISP 6.45 5.10 
BISP 7.58 5.74 
BURN 5.56 4.49 
BURN 5.37 4.27 
BURN 5.47 4.44 
BURN 5.74 4.47 
BURN 5.51 4.56 
BRUN 6.33 4.86 
BRUN 6.34 4.88 
SBPR 6.30 5.00 
SBPR 5.83 4.55 
SBPR 6.16 4.55 
SBPR 6.03 4.65 
SBPR 6.35 4.59 
SBPR 5.99 4.59 
SBPR 5.78 4.39 
SBPR 6.34 4.68 
SBPR 6.00 4.48 
SBPR 6.08 4.50 
SBPR 5.89 4.62 
SBPR 6.08 4.50 
SBPR 6.04 4.47 
SFLC 6.33 4.86 
______________________________________ 
Legend for Table III 
BISP = Agaricus bisporus var. bisporus 
BURN = Agaricus bisporus var. burnettii 
BRUN = Agaricus brunnescens 
SBPR = Agaricus subperonatus 
SFLC = Agaricus subfloccosus 
TABLE IV 
______________________________________ 
Statistical Comparison Spore Sizes of Five Agaricus Taxa 
Mean Length Mean Width 
Taxa (.mu.) (SD) (.mu.) 
(SD) 
______________________________________ 
BISP 6.88 0.312 5.37 0.248 
BURN 5.53 0.122 4.45 0.096 
BRUN 6.34 NA 4.87 NA 
SBPR 6.07 0.175 4.58 0.143 
SFLC 6.33 NA 4.86 NA 
______________________________________ 
Legend for Table IV 
BISP = Agaricus bisporus var. bisporus 
BURN = Agaricus bisporus var. burnettii 
BRUN = Agaricus brunnescens 
SBPR = Agaricus subperonatus 
SFLC = Agaricus subfloccosus 
Upon viewing the Tables III and IV, it should be clear that the spores 
taken from the var. burnettii strains were much smaller than the spores 
taken from specimens known to be Agaricus bisporus var. bisporus. The 
greatest mean spore length observed for var. burnettii fell 3.65 standard 
deviations (SD) below the grand mean for what is now known as Agaricus 
bisporus var. bisporus. The greatest mean spore width observed for var. 
burnettii fell 3.27 SD below the grand mean for what is now known as 
Agaricus bisporus var. bisporus. The least mean spore length observed for 
what is now known as Agaricus bisporus var. bisporus fell 6.97 SD above 
the grand mean for var. burnettii. The least mean spore width observed for 
what is now known as Agaricus bisporus var. bisporus fell 5.42 SD above 
the grand mean for var. burnettii. The difference in mean spore lengths 
for the two groups was equivalent to 4.33 SD, based upon the traditional 
Agaricus bisporus var. bisporus distribution, and equivalent to 11.07 SD, 
based on the var. burnettii distribution. The difference in mean spore 
widths for the two groups was equivalent to 3.71 SD, based upon the 
traditional Agaricus bisporus var. bisporus distribution, and equivalent 
to 9.58 SD, based on the var. burnettii distribution. Furthermore, without 
expressly detailing the difference, it is clear the hiatus in both spore 
length and spore width between the smallest Agaricus bisporus var. 
bisporus values and the largest var. burnettii values are substantially 
different when based upon either distribution. Thus, the new material did 
not fall within the existing circumscription of Agaricus bisporus. 
Nevertheless, in view of certain similarities between the genotypic data 
for Agaricus bisporus and the genotypic data presented in Table II, it was 
deemed worthwhile to take the unusual step of performing interfertility 
testing between these new strains and strains of Agaricus bisporus. The 
tests were constructed (1) to determine whether hybrid heterokaryons could 
be formed in confrontation between homokaryons of Agaricus bisporus and 
those of the new tetrasporic stocks, and (2) to ascertain the heritability 
of the spore number trait in any hybrids that might be produced. With 
respect to the latter issue, some background data on the spore number 
traits exhibited by the stocks utilized in the experiment is in order. 
Accordingly, in Table V, the number of spores per basidium for several 
commercially cultivated bisporic strains were determined by conventional 
light microscopy techniques as discussed in detail hereinbelow. Each of 
these strains was cultured under locally standard industry conditions. 
Once the mushrooms produced began to sporulate abduntantly, the spore 
number data were obtained. 
TABLE V 
______________________________________ 
Spore Number Data on Certain Agaricus bisporus var. bisporus 
Percentage of N-spored Basidia 
Trial # 
Stock 4-spored 3-spored 
2-spored 
1-spored 
______________________________________ 
1 FS 40 0.0 0.7 98.3 1.0 
2 FS 25 0.0 9.0 89.7 1.3 
3 TV 2 1.0 4.3 95.7 0.0 
4 RWK 1547 0.0 1.0 98.7 0.3 
5 RWK 1634 0.0 3.0 97.0 0.0 
6 RWK 1646 0.0 5.7 94.3 0.0 
7 303 0.0 0.0 100.0 0.0 
8 S381 0.0 5.3 94.0 0.7 
9 56B 0.3 9.7 87.0 3.0 
10 S600 0.0 0.0 92.7 7.3 
______________________________________ 
Similarly, spore number data were obtained for a number of cultures of the 
new tetrasporic strains and are reported in Table VI. For the JB2 and JB3 
stocks, spores were taken from the mushrooms found in nature and 
multi-spore cultures JB2-ms and JB3-ms were formed as noted hereinabove. 
For the other stocks noted in Table VI, cell tissue was obtained from each 
original mushroom and was regrown so as to form heterokaryotic tissue 
cultures. 
TABLE VI 
______________________________________ 
Spore Number Data on Certain Agaricus bisporus var. burnettii 
Percentage of N-spored Basidia 
Trial # 
Stock 5-spored 4-spored 
3-spored 
2-spored 
______________________________________ 
1 JB 2 0.0 99.0 1.0 0.0 
2 JB 3 1.7 92.3 6.0 0.0 
3 JB 102 2.0 97.7 0.3 0.0 
4 JB 104 0.0 95.0 14.3 0.7 
5 JB 105 0.0 92.7 7.0 0.3 
6 JB 106 0.0 100.00 0.0 0.0 
7 JB 107 0.0 92.0 7.3 0.7 
8 JB 108 0.0 95.0 5.0 0.0 
9 JB 109 0.0 53.0 41.3 5.7 
10 JB 110 0.0 96.7 3.3 0.0 
11 JB 111 0.0 72.0 27.0 1.0 
12 JB 117 0.0 54.0 38.0 8.0 
13 JB 118 0.0 93.0 6.0 1.0 
14 JB 121 0.3 92.0 7.3 0.3 
15 JB 123 0.0 96.0 4.0 0.0 
16 JB 125 0.3 88.0 11.7 0.0 
______________________________________ 
Crosses were attempted as described hereinabove, between homokaryons from 
JB2 and known homokaryons from certain of the wild and cultivated, 
two-spored heterokaryotic breeding stocks of Agaricus bisporus as shown in 
Table V. 
In Experiment 1, individual homokaryons from JB2 were each crossed with a 
homokaryon from a commercial, nonhybrid, white, two-spored Agaricus 
bisporus var. bisporus stock belonging to the genotypic class No. 2 as set 
forth in Royse and May, "Use of Isozyme Variation to Identify Genotypic 
Classes of Agaricus brunnescens" Mycologia 74:93-102 (1982), which is 
hereby incorporated by reference and maintained in the Sylvan Spawn 
Laboratory Incorporated ("Sylvan Spawn") culture collection under the 
Stock 303 designation as shown in Trial 7 of Table V. 
In Experiment 2, individual homokaryons from JB2 were each crossed with a 
homokaryon from a wild, heterokaryotic, two-spored, white Agaricus 
bisporus var. bisporus stock, belonging to the genotypic class No. 36, as 
identified in Kerrigan and Ross, "Allozymes for a Wild Agaricus bisporus 
Population: New Alleles, New Genotypes," Mycologia 81:433-443 (1989), 
hereby incorporated by reference. This strain of Agaricus bisporus is 
maintained in the Sylvan Spawn culture collection and is designated as 
Stock R (also known as Stock RWK 1420). 
In Experiment 3, individual homokaryons from JB2 and a different homokaryon 
taken from the same Stock R as employed in Experiment 2 were crossed. 
In Experiment 4, individual homokaryons from JB2 were each crossed with a 
homokaryon from a pre-hybrid, heterokaryotic, two-spored, brown Agaricus 
bisporus var. bisporus stock. This strain of Agaricus bisporus is 
maintained in the Sylvan Spawn culture collection and is designated as 
Stock 56B as shown in Trial 9 of Table V. 
In Experiment 5, an individual homokaryon (-s11) from JB2 was crossed with 
homokaryons (-h25, -h33, and -h12) from a hybrid, heterokaryotic, 
two-spored, brown Agaricus bisporus var. bisporus stock. This strain of 
Agaricus bisporus is maintained in the Sylvan Spawn culture collection and 
is designated as Stock S600 as shown in Trial 10 of Table V. Notably, 
stock S600 is the commercial horticultural variety of Agaricus bisporus 
claimed in U.S. Plant Pat. No. Plant 7,636. 
For the convenience of the reader, a pedigree chart of three hybrids which 
are treated in subsequent experiments is presented in Table VII 
hereinbelow. 
TABLE VII 
__________________________________________________________________________ 
Pedigrees of J81, J99, J102 and J154 
##STR1## 
__________________________________________________________________________ 
In accordance with commercial practice, the novel hybrid heterokaryons 
formed from each cross were transferred to grain culture, and then to 
compost culture, to permit the production of hybrid mushrooms. Mushrooms 
were produced in all crosses in all cases. These mushrooms have basidia 
which predominantly bear at least three spores as reported in Table VIII 
hereinbelow. The proportions of four-spored and other basidia were 
determined by light microscopy as discussed in Kerrigan and Ross, "Dynamic 
Aspects of Basidiospore Number in Agaricus,"Mycologia, 79(2), 204-215 
(1987). Specifically, light microscopy was performed with a Nikon compound 
microscope with a 20x objective and 10x ocular. Lamellae were excised from 
the freshly harvested subject mushrooms and placed on a glass slide to 
form a dry mount. Observations were made immediately by transmitted light. 
Basidia were sampled by selecting an area of the lamella at random, and 
most or all basidia at the appropriate developmental stage within that 
visual field were scored. Successive fields were scored until one hundred 
(100) basidia had been scored. This was typically repeated on two 
additional lamellae to furnish a total sample of three hundred (300) 
basidia from each hybrid mushroom. Basidia which had aborted spores or 
asynchronous spore development were excluded from scoring, as were basidia 
so mature that one or more spores might have been discharged prior to 
observation. 
TABLE VIII 
______________________________________ 
Results of Individual Crosses Between 
the Four-Spored Stock JB2 and Various 
Two-Spored Stocks of Agaricus Bisporus 
Percentage of N-spored Basidia: 
Homokaryon 5- 4- 3- 2- Hybrid 
Trial # 
No. Spored Spored 
Spored 
Spored 
ID 
______________________________________ 
Experiment 1 -- Stock JB2 Crossed with 
Homokaryon -3105 of Stock 303 
1 s1 0.7 68.0 30.0 1.3 J51 
2 s2 0.0 67.7 31.7 0.7 J54 
3 s3 0.3 65.7 32.3 1.7 J57 
4 s4 0.3 82.7 14.7 2.3 J60 
5 s5 0.0 90.0 9.7 0.3 J63 
6 s6 0.0 64.0 35.3 0.7 J66 
7 s7 0.0 72.7 26.3 1.0 J69 
8 s8 0.0 78.7 21.3 0.0 J72 
9 s9 0.3 62.0 37.7 0.0 J75 
10.sup.a 
s10 0.0 65.7 32.0 2.3 J78 
10.sup.b 
s10 0.0 66.3 33.7 0.0 J78 
11 s11 0.0 51.0 43.0 6.0 J81 
Experiment 2 -- Stock JB2 Crossed with 
Homokaryon -9559 of Stock R 
12 s1 0.0 36.0 60.0 4.0 J52 
13 s2 0.0 62.3 37.0 0.7 J55 
14 s3 0.3 59.4 38.3 2.0 J58 
15 s4 0.0 43.3 52.3 4.3 J61 
16 s5 0.0 51.3 46.7 2.0 J64 
17 s6 0.0 55.0 44.0 1.0 J67 
18 s7 0.0 71.7 28.3 0.0 J70 
19 s8 0.0 72.7 27.3 0.0 J73 
20 s9 0.0 68.0 29.0 3.0 J76 
21 s10 0.0 60.3 38.0 1.7 J79 
22 s11 0.0 36.0 58.3 5.7 J82 
Experiment 3 -- Stock JB2 Crossed with 
Homokaryon -s5 of Stock R 
23 s1 0.0 94.0 5.7 0.3 J53 
24 s2 0.0 85.0 14.33 0.7 J56 
25 s3 0.0 96.3 3.7 0.0 J59 
26 s4 0.0 93.7 6.3 0.0 J62 
27 s5 0.0 86.0 13.7 0.3 J65 
28 s6 0.0 64.7 33.7 1.7 J68 
29 s7 0.0 97.7 2.3 0.0 J71 
30 s8 0.3 91.3 8.0 0.3 J74 
31 s9 0.0 93.7 6.3 0.0 J77 
32 s10 0.0 85.7 14.3 0.0 J80 
33 s11 0.0 83.3 16.3 0.3 J83 
Experiment 4 -- Stock JB2 Crossed with 
Homokaryon -4186 of Stock 56B 
34 s1 0.0 91.3 8.7 0.0 J87 
35 s9 0.3 88.0 11.7 0.0 J89 
Experiment 5 -- Stock S600 Crossed with 
Homokaryon -s11 of Stock JB2 
36 h25 0.0 25.0 65.0 10.0 J99 
37 h33 0.0 40.0 54.0 6.0 J102 
38 h12 0.0 37.0 60.0 3.0 J108 
______________________________________ 
Thus, based upon data collected and present evidence, it should be clear 
that at least 90 percent of the basidia produced in mushrooms of each 
hybrid strain are elevated to a higher spore number with respect to the 
two-spored condition prevailing in the bisporic parent. While the 
two-spored condition is not explicitly shown for Stock R in Table V, which 
is employed in Experiments 2 and 3 in Table VIII, it will be appreciated 
that tests have been performed on this stock which do show that it is, in 
fact, predominantly bisporic. Notably, the percentage of two-spored 
basidia has been reduced in the hybrid strains to less than 10 percent. 
Inasmuch as every first generation, intervarietal hybrid always expresses 
this elevated basidial spore number trait, the trait exhibits genetic 
dominance. 
Furthermore, the percentage of homokaryons and heterokaryons produced by 
the spores of three of these first-generation intervarietal hybrids are 
shown in Table IX. Based upon the results presented in this Table, it is 
noted that the 54 to 57 percent production rate of homokaryons from the 
spores of hybrids J99 and J102 is more than five times the percentage 
production rate observed in any other normal bisporic strain, and is 
closer to 10 to 50 times the rate ordinarily obtained. Clearly, high 
proportions of homokaryons are produced by the spores of these hybrid 
mushrooms. 
TABLE IX 
______________________________________ 
Percentage of Homokaryons Found in Hybrids 
No. of Spores 
Hybrid ID 
% Homokaryons 
% Heterokaryons 
Examined 
______________________________________ 
J81 12.0 88.0 25 
J99 54.2 45.8 24 
J102 56.7 43.3 30 
______________________________________ 
With respect to the rather low proportion of homokaryons shown for J81 in 
Table IX this result can be explained best by the existence of a greater 
mortality rate among homokaryotic spores. While spore mortality is high 
for both heterokaryotic and homokaryotic spores (80 to 99% is common), 
mortality may be higher among homokaryotic spores produced by certain 
heterokaryons. With particular reference to FIG. 2, empirical evidence for 
this fact is found in the article "Strategies for the Efficient Recovery 
of Agaricus bisporus Homokaryons" by Kerrigan et al. in Mycologia, 84(4), 
575-579 (1992). Specifically, the growth rate frequency distribution 
curves for homokaryotic single spore isolates may be sharply truncated at 
the zero growth (inviability) point in contrast to those for 
heterokaryotic single spore isolates, suggesting that a disproportionately 
greater number of homokaryotic spores are inviable. The theoretical basis 
of this hypothesis is that secondary homothallism may favor the 
accumulation of recessive deleterious alleles, including those producing 
lethality in the haploid or homoallelic state. 
To demonstrate that the present invention can utilize virtually any strain 
of var. burnettii as a tetraspofic parent in a cross to produce a hybrid 
heterokaryon having the elevated spore number trait, a series of 
intervarietal hybrid crosses were constructed using a very diverse 
selection of wild var. burnettii isolates. This sample, drawn from the 
list in Table I hereinabove, were collections that represented all of the 
known field sites and two different habitats from which isolates of var. 
burnettii were collected in March of 1992. The field sites are all within 
about 5 kilometers from each other. The geographical range of var. 
burnettii is not known. 
The collections chosen for this experimental demonstration, the two var. 
bisporus to which they were crossed, the hybrid strain identifiers 
assigned to the new, resultant intervarietal hybrids, and the spore number 
data on the resultant hybrids are disclosed in Table X hereinbelow. 
TABLE X 
__________________________________________________________________________ 
Results of Individual Crosses Between Various Four-Spored Stocks 
of Agaticus bisporus and Two Two-Spored Stocks of Agaricus Bisporus 
Percentage of N-spored Basidia 
Sample 
Homokaryon 
Crossed with 
Hybrid ID 
2-Spored 
3-Spored 
4-Spored 
__________________________________________________________________________ 
JB 105 
s3 303-3105 
J264 0 7 93 
JB 106 
s3 56B-4186 
J265 6 41 53 
JB 106 
s3 303-3105 
J266 no data available 
JB 106 
s9 56B-4186 
J267 no data available 
JB 108 
s4 56B-4186 
J268 1 17 82 
JB 108 
s4 303-3105 
J269 1 25 74 
JB 108 
s9 56B-4186 
J270 0 6 94 
JB 108 
s9 303-3105 
J271 0 12 88 
JB 109 
s4 56B-4186 
J272 0* 9 90 
JB 109 
s4 303-3105 
J273 3.5 
39 57.5 
RWK 1841 
s1 56B-4186 
J274 4 45 51 
RWK 1841 
s1 303-3105 
J275 0 13 87 
RWK 1842 
s3 56B-4186 
J276 1 6 93 
RWK 1842 
s3 303-3105 
J277 3 44 53 
RWK 1842 
s4 56B-4186 
J278 0 6 94 
RWK 1842 
s4 303-3105 
J279 8 50 42 
RWK 1845 
s2 56B-4186 
J280 0 18 82 
RWK 1845 
s2 303-3105 
J281 3 49 48 
RWK 1855 
s1 56B-4186 
J282 1 59 40 
RWK 1855 
s1 303-3105 
J283 no data available 
RWK 1855 
s4 56B-4186 
J284 0 34 66 
RWK 1855 
s4 303-3105 
J285 4 44 52 
RWK 1856 
s8 56B-4186 
J286 no data available 
RWK 1856 
s8 303-3105 
J287 no data available 
RWK 1856 
s9 56B-4186 
J288 no data available 
RWK 1856 
s9 303-3105 
J289 2 38 60 
RWK 1857 
s5 56B-4186 
J290 5 74 21 
RWK 1857 
s5 303-3105 
J291 2 56 42 
RWK 1857 
s6 56B-4186 
J292 0 12 88 
RWK 1857 
s6 303-3105 
J293 4 46 50 
__________________________________________________________________________ 
*had 1 onespored basidium 
Spore number data on the parents of 303-3105 and 56B-4186 are provided 
hereinabove in Table V. Both are highly bisporic isolates of var. 
bisporus. Therefore, the dominant expression of the elevated spore number 
trait in the above intervarietal hybrids, seen most clearly as a reduction 
of the percentage of bisporic basidia to about 8 percent or less, confirms 
that most and perhaps all stocks of var. burnettii may be employed in the 
method of the present invention. A small number of crosses in the above 
test were unsuccessful. For example, JB 105-s1 mated only with 303-3105, 
and JB 106-s9 mated only with 56B-4186. This is consistent with mating 
type incompatibility, resulting from the sharing of a common allele at the 
mating type locus (MAT) by both homokaryons. This is a common occurrence 
in var. bisporus matings and does not reflect negatively on the method of 
the present invention. Six other homokaryons grew poorly on grain and were 
not useful in matings; again, this is also rather common among var. 
bisporus homokaryons. One of the tested stocks, RWK 1854, produced 2 such 
homokaryons, therefore no successful matings were obtained from this 
stock. This is believed to be a chance result, but no confirmation of this 
is available at the present time. However, the discovery that some stocks 
of Agaricus bisporus having tetrasporic ancestry are less desirable for 
the purposes of the present invention than others, or axe even unsuitable, 
would not materially affect the practice of the method of the present 
invention. 
To demonstrate that virtually any strain of Agaricus bisporus var. bisporus 
can be used to form heterokaryons in crosses with var. burnettii 
homokaryons, the JB2 homokaryons -s8 and -s 1 were each crossed 
individually with three homokaryons of the off-white, pre-hybrid 
commercial Stock 8132, with two homokaryons of the golden-white, 
pre-hybrid commercial Stock 671, and with two homokaryons of Stock V1, a 
proprietary experimental hybrid between homokaryons of wild Stock RWK 
1420. In all cases fertile heterokaryons were formed, and in about half of 
these trials, the yield equaled or exceeded that of the control, the 
high-yielding commercial strain S301. Three crosses between JB2 and JB3 
homokaryons were also successful, resulting in fertile hybrid var. 
burnettii heterokaryons. In these experiments, the basidial spore numbers 
of the hybrid mushrooms were not determined. 
It has also been determined that the elevated spore number trait which is 
found in the tetrasporic var. burnettii of Agaricus bisporus is a 
genetically-determined trait. Based upon results of the initial 
experiments provided in Table VIII, this trait is always inherited and 
expressed in the first hybrid generation. This is a hallmark of genetic 
dominance, and no other explanation satisfactorily explains the 
inheritance of this stable trait in all first-generation hybrids. 
Formal genetic analysis was undertaken by preparing a series of 60 
second-generation intervarietal hybrids. In these hybrids (J203 - J262), 
the first homokaryon was selected from among 12 prepared from spores of 
first-generation hybrid J102, and the second (non-intervarietal hybrid) 
homokaryon was from one of five strains of the bisporic var. bisporus. 
These five strains included: 
______________________________________ 
Homokaryon Strain type 
______________________________________ 
S381-907 Hybrid White Cultivar 
8132-8010 Pre-hybrid Off-white Cultivar 
303-3105 Pre-hybrid White Cultivar 
WQ-9525 Pre-hybrid White Cultivar 
RWK 1420-s5 
Wild Non-hybrid Coastal-Californian isolate 
______________________________________ 
The results of each cross are set forth in Table XI hereinbelow. Each of 
the five bisporic strains has been grouped as a separate experiment for 
the convenience of the reader. 
TABLE XI 
______________________________________ 
Results of Individual Crosses Between the Four-Spored 
Hybrid Stock J102 and Various Two-Spored Stocks of 
Agaricus Bisporus 
Percentage of N-spored Basidia 
Trial 
Homokaryon Hybrid ID 2-Spored 
3-Spored 
4-Spored 
______________________________________ 
Experiment A - Homokaryons from Hybrid Strain J102 
Crossed with Homokaryon -907 of Stock S381 
1 s11 J203 -- NS -- 
2 s12 J208 -- NF -- 
3 s25 J213 9 59 32 
4 s30 J218 -- NF -- 
5 s15 J223 -- NS -- 
6 s23 J228 99 1 0 
7 s31 J233 -- NS -- 
8 s40 J238 -- NF -- 
9 s10 J243 7 49 44 
10 s19 J248 -- NF -- 
11 s22 J253 -- NS -- 
12 s27 J258 -- NS -- 
Experiment B - Homokaryons from Hybrid Strain J102 
Crossed with Homokaryon -8010 of Stock 8132 
13 s11 J204 30 63 7 
14 s12 J209 -- NF -- 
15 s25 J214 2 52 46 
16 s30 J219 53 41 6 
17 s15 J224 36 63 1 
18 s23 J229 29 63 8 
19 s31 J234 20 69 11 
20 s40 J239 23 57 20 
21 s10 J244 10 60 30 
22 s19 J249 18 67 15 
23 s22 J254 14 68 18 
24 s27 J259 -- NF -- 
Experiment C - Homokaryons from Hybrid Strain J102 
Crossed with Homokaryon -9525 of Stock WQ 
25 s11 J205 96 4 0 
26 s12 J210 90 10 0 
27 s25 J215 26 63 11 
28 s30 J220 23 61 6 
29 s15 J225 84 16 0 
30 s23 J230 54 33 3 
31 s31 J235 87 13 0 
32 s40 J240 79 21 0 
33 s10 J245 2 53 45 
34 s19 J250 91 9 0 
35 s22 J255 60 40 0 
36 s27 J260 98 2 0 
Experiment D - Homokaryons from Hybrid Strain J102 
Crossed with Homokaryon -s5 of Stock RWK 1420 
37 s11 J206 43 56 1 
38 s12 J211 77 23 0 
39 s25 J216 16 62 22 
40 s30 J221 -- NF -- 
41 s15 J226 22 75 3 
42 s23 J231 13 65 22 
43 s31 J236 48 49 3 
44 s40 J241 81 18 1 
45 s10 J246 11 54 35 
46 s19 J251 73 27 0 
47 s22 J256 24 64 12 
48 s27 J261 -- NF -- 
Experiment E - Homokaryons from Hybrid Strain J102 
Crossed with Homokaryon -3105 of Stock 303 
49 s11 J207 96 4 0 
50 s12 J212 90 10 0 
51 s25 J217 12 59 29 
52 s30 J222 27 65 8 
53 s15 J227 99 1 0 
54 s23 J232 87 13 0 
55 s31 J237 95 5 0 
56 s40 J242 70 26 4 
57 s10 J247 6 59 35 
58 s19 J252 93 7 0 
59 s22 J257 84 16 0 
60 s27 J262 100 0 0 
______________________________________ 
NF = nonfruiting 
NS = nonsporulating 
Crosses to 8132-8010 and RWK 1420-s5 exhibited a range of elevated basidial 
spore numbers, but Mendelian segregation could not be clearly discerned. 
However, in crosses to 303-3105 and to WQ-9525, the spore-number trait of 
these second-generation intervarietal hybrids resolved clearly into two 
groups. Nine of the 12 crosses to 303-3105 had at least about 70% 2-spored 
basidia, while the remaining three crosses had about 27% or fewer 2-spored 
basidia. An inverse relationship was observed for 4-spored basidia in 
these 12 crosses. In the 12 crosses to WQ-9525, the cutoffs for percentage 
of 2-spored basidia that separated the two groups were greater than about 
54% versus less than about 26%. The spore number values within each pair 
of crosses to WQ-9525 and 303-3105 were similar, indicating that the 
genetic makeup of the particular J102 homokaryon determined the expression 
of the trait in hybrid offspring. Thus, the data from the 24 crosses to 
WQ-9525 and 303-3105, in conjunction with the pedigree and spore number 
data presented above, clearly indicate that the elevated basidial spore 
number trait (in this experiment seen best as the replacement of 2-spored 
basidia with 3- and 4-spored ones) is controlled primarily by a single 
segregating genetic determinant. This proves that elevated spore number is 
a genetically determined trait. The gene locus that determines this trait 
has been provisionally named SNT. It further shows that the trait is 
expressed in the second, as well as the first, hybrid generation. 
Additional data below, presented in an example of the application of this 
technology to gene mapping, further support this assertion. Other data 
presented herein demonstrate that the elevated spore number trait exhibits 
genetic dominance. 
Homokaryon S381-907 is a direct progenitor (essentially a gamete of the 
grandparent S381) of J102, and few of the back-crosses between J102 
homokaryons and S381-907 developed normally, probably due to the effects 
of inbreeding. However, of those which did fruit and sporulate normally, 
two (J213 and J243) had about 32% and about 44% 4-spored basidia, 
respectively, and about 91% and about 93% at least 3-spored basidia, 
respectively. This ratio is not significantly different from the range of 
percentages given above for J102. Therefore, it is clear that the elevated 
spore number trait was retained in two back-crosses to the commercial var. 
bisporus cultivar S381. In contrast, a third back-cross (J228) had no 
four-spored basidia and 99 percent two-spored basidia. The influence of 
each individual J102 homokaryon on spore number in these three crosses was 
in complete agreement with observations on the crosses to Stocks 303 and 
WQ described hereinabove. 
J213 and J243 are expected to have, on average, about 5/8 (62.5%) ancestry 
from S381, as opposed to the 50% that a first-generation hybrid, such as 
J154 (see Pedigree Chart hereinabove), will have. Ancestry, used 
quantitatively, is equivalent to the fraction of DNA inherited from a 
progenitor. However, because chromosomes assort independently and because 
of the infrequency of crossing over which occurs in Agaricus bisporus 
during meiosis, this percentage could actually be much higher (or lower) 
in any individual case. By using various breeding strategies and making 
additional generations of back-crosses, and by using genetic markers to 
select for homokaryons which have inherited more than 50% of their genome 
from the back-cross parent (e.g., S381), it is ultimately possible to 
construct one or a series of heterokaryons or homokaryons in which all of 
the genome, excluding a small chromosomal segment encompassing the spore 
number trait determining locus, comes from the back-cross parent. In other 
words, one or more strains having about 99% of their DNA from a var. 
bisporus strain like S381, and only about 1% from a var. burnettii strain 
like JB2, but expressing the elevated spore number trait originating in a 
var. burnettii strain, can be constructed using this method. The problem 
of inbreeding depression, which may be responsible for the high frequency 
of developmental problems in these back-crosses discussed hereinabove, can 
be ameliorated by back-crossing to sibling homokaryons of those from which 
the hybrid intermediates are descended. In other words, a different, 
sibling homokaryon of S381-907, when used in a cross between S381 and 
I102, will exhibit enough genetic differences from S381-907 to produce 
hybrids with less homozygosity than what is predicted to exist in the 12 
back-crosses to S381-907. Homozygosity is an underlying cause of 
inbreeding depression. 
At this point, it is noted that although the crossing of homokaryons is the 
preferred method of obtaining hybrids, it is also sometimes possible to 
cross a homokaryon with a heterokaryon or, in rare instances, to cross two 
heterokaryons. For example, some single-spore isolates from wild stocks of 
the tetrasporic var. burnettii of Agaricus bisporus are both self-fertile 
(and therefore heterokaryotic) and produce only white mushrooms (therefore 
lack a functional gene necessary for brown pigment production). When one 
such single-spore isolate (RWK 1845-sl) was paired with the homokaryon 
56B-4186 of var. bisporus (which is not self-fertile, but which when mated 
always produces brown mushrooms because it carries a functional, dominant 
allele for brown pigment production), brown mushrooms were produced 
abundantly and exclusively as shown in Table XII. RWK 1845-sl is a 
single-spore isolate from field collection RWK 1845 of the tetrasporic 
vat. burnettii of Agaricus bisporus, collected within 3 kilometers of JB2 
and JB3. Yield is the arithmetic mean of all replicates (usually three) of 
about 0.3 square feet each. The data shows that a mating between the var. 
burnettii heterokaryon and the var. bisporus homokaryon occurred and 
produced a high yielding brown hybrid. 
TABLE XII 
______________________________________ 
Demonstration of Heterokaryon-Homokaryon Mating 
Color of 
Isolate(s) Test Yield (grams) 
Mushroom 
______________________________________ 
RWK 1845-s1 1 63 White 
2 235 White 
56B-4186 x* 0 
RWK 1845-s1 .times. 56B-4186 
1 280 Brown 
2 230 Brown 
Control (S600) 1 214 Brown 
2 192 Brown 
______________________________________ 
*will not colonize compost; never fruits unless mated 
Continuing, the present invention may also include genetically engineering 
the tetrasporic trait into bisporic strains or stocks. That is, it is now 
possible to transform a diverse array of eukaryotic cells with exogenous 
DNA, thereby selectively incorporating individual genes determining 
economically important traits into the genetic background of an individual 
line or stock of the mushroom in question. It is now reasonable to believe 
that Agaricus bisporus may be transformed with respect to the spore number 
trait using recombinant DNA techniques. 
The first step in this process involves locating (mapping) and isolating 
the DNA segment that encodes the gene locus responsible for expression of 
the "tetrasporic" trait in Agaricus bisporus var. burnettii. As noted 
hereinbelow, it has already been determined that a single genetic 
determinant, hereinafter called the Spore Number Trait locus (SNT), or 
possibly a cluster of linked loci within a single DNA segment, controls 
most or all of the expression of this trait in Agaricus bisporus, and that 
this locus lies very close to the PEPI locus, as more specifically 
detailed hereinbelow. In Kerrigan et ai., "Meiotic Behavior and Linkage 
Relationships in the Secondarily Homothailic Fungus Agaricus bisporus," 
Genetics, 133:225-236 (1993), incorporated herein by reference, several 
allozyme and DNA RFLP and RAPD markers, including PEP2, R4-1, R4-3, PINt7, 
P1N31, HNI48, HNI50, P33N25, P33N25 and R18-6, have been mapped to within 
about 58.3, and to within as little as about 5.1 centiMorgans of PEP1, on 
Chromosome I of the nuclear genome. By quantifying the joint segregation 
of markers with SNT, the location of SNT can be pinpointed. 
With this information, a chromosome walk is performed from one or more of 
these mapped DNA RFLP markers to the SNT locus. First, one may construct 
an ordered cosmid library of genomic DNA from Agaricus bisporus, using 
lambda phage as vector and maintaining the recombinant clones in E. coli. 
Ideally this library would contain only DNA from Chromosome I, which can 
be isolated by an alternating field electrophoresis technique such as 
contour-clamped homogeneous electrical field (CHEF) electrophoresis of 
whole-chromosome DNAs, following a preferred procedure discussed in Royer 
et al., "Electrophoretic Karyotype Analysis of the Button Mushroom 
Agaricus bisporus," Genome, 35:694-698 (1992), herein incorporated by 
reference, or a modification thereof. Overlapping homologous segments of 
DNA inserts in cosmids would permit the sequential ordering of the library 
via repeated hybridization of blots of clones in the library to probes 
incorporating ever more distant contiguous segments. 
Such a walk will conventionally extend from a flanking marker through SNT 
to a flanking marker on the opposite side of SNT. Once this has been 
achieved, the approximate location of SNT can be estimated by comparing 
the recombinational distances between the two flanking markers and SNT, 
using the map data disclosed hereinbelow, with the cumulative lengths of 
all of the cosmid inserts in the spanned interval. Based on this estimate, 
the cosmid most likely to contain SNT can be identified. The inserted DNA 
from this cosmid could be subcloned, incorporated into a suitable vector, 
such as pUC18, and introduced into hyphae or protoplasts of a recipient 
Agaricus bisporus strain. The plasmid vector construct should provide 
positive selection for retention of plasmid in host, and for insertion of 
DNA of interest into a cloning site in the plasmid sequence. A number of 
other refinements might be incorporated into such a recombinant vector, 
depending upon which strategies are ultimately successful in transforming 
Agaricus bisporus, including detection of the recombinant DNA in the 
target cells, and whether homologous or non-homologous integration, or 
maintenance of the plasmid as a self-replicating cytoplasmic component, is 
preferred. 
The recipient strain can be either a heterokaryon or a homokaryon, but will 
ordinarily carry no alleles for the "tetrasporic" trait; in other words, a 
bisporic strain will ordinarily be desired. Because the tetrasporic trait 
disclosed herein exhibits genetic dominance, the presence of the DNA of 
interest will be verifiable in any transformed strain because the 
tetrasporic trait will be expressed in mushrooms produced by a transformed 
homokaryon (or the hybrid produced by crossing a transformed homokaryon 
with another, compatible, bisporic homokaryon). This can be determined by 
fruiting the mushrooms and examining them microscopically. While this is 
an expensive and time-consuming assay, it is straightforward. If 
gene-dosage effects reduce expression of the novel DNA in a transformed 
hybrid, then homologous integration of the exogenous DNA to replace at 
least one copy of the (bisporic allele of the) SNT gene might be 
necessary. 
DNA from cosmids increasingly distant from the most likely cosmid can also 
be used to transform the recipient strain. This series of experiments will 
continue until, at some point, a cosmid clone, or a subclone, is found 
which causes expression of the trait in a transformed hybrid. 
At this point, the open reading frame (ORF) associated with the gene can be 
identified (through further subcloning and reconfirmation via 
transformation), sequenced, and studied. The subclone, in the appropriate 
vector, can be used to introduce the SNT gene controlling the trait into 
any bisporic strain of interest, without introducing any other trait from 
var. burnettii or losing any trait, other than bispory, form the target 
strain. From the sequence dam, it may be possible to understand the nature 
of the gene product and the mechanism by which it determines the pattern 
of basidiospore formation at the basidial apex. Finally, it may be 
possible to engineer new sequences which, when introduced, will have 
superior properties in terms of expression levels, the nature of the spore 
number pattern determined, or other improvements. 
Turning to gene mapping, although heterokaryotic single-spore isolate 
progeny of Agaricus bisporus individuals are inbred, they exhibit little 
of the loss of parental heterozygosity that is characteristic of 
self-fertilization in most eukaryotes. As a result, Mendelian segregation 
of alleles is not observed in most single-spore offspring of var. 
bisporus, because most spores are heterokaryotic. To carry out gene 
mapping (in other words, to locate genes that control important phenotypic 
traits), it is necessary to determine whether alleles at two or more loci 
segregate jointly or independently, for example by characterizing the 
genotypes and/or phenotypes of a sufficient number of homokaryon 
offspring. By a sufficient number, it is meant that the number be great 
enough either to permit a sufficiently powerful statistical test of 
linkage, or to furnish a representative sample of individuals in pools of 
samples which are grouped into classes by trait. This number of 
homokaryons will vary inversely with the recombinational distance 
separating two loci, approaching infinity as the recombinational distance 
approached 50 percent, or conversely, becoming as few as 12-20 homokaryons 
when two loci are absolutely linked. It is normally possible to use every 
homokaryon of a basidiomycete species to furnish data used in gene 
mapping. Therefore, any method which increases the ability to obtain 
homokaryons from Agaricus bisporus, for example by increasing the fraction 
of homokaryons above the typical 3 percent and even above the exceptional 
about 10 percent, is of corresponding value to efforts to map genes in 
this species. 
Once genes are mapped, it is possible to use linked flanking markers to 
follow them through crosses, which is of great value in efficiently 
breeding traits into or out of breeding stock and new hybrids. Thus, the 
present invention is of great value in this related aspect of mushroom 
breeding. 
An example of gene mapping in Agaricus bisporus using the present invention 
is detailed hereinbelow. Specifically, with reference to the data 
pertaining to the second-generation crosses between the 12 homokaryons of 
J102 and the five var. bisporus homokaryons, the 12 J102 homokaryons were 
characterized genotypically at two allozyme marker loci, PEP1 and BGLU, in 
accordance with the method presented in Kerrigan and Ross, "Allozymes of a 
Wild Agaricus bisporus Population: New Alleles, New Genotypes," Mycologia 
81:433-443 (1989). Genotypes for all 12 J102 homokaryons are presented in 
the Table XIII hereinbelow. 
TABLE XIII 
______________________________________ 
Genotype Data for J102 Homokaryons 
J102 PEP1 BGLU 
______________________________________ 
s11 S600-h33 JB2-s11 
s12 S600-h33 JB2-s11 
s25 JB2-s11 JB2-s11 
s30 JB2-s11 JB2-s11 
s15 S600-h33 JB2-s11 
s23 S600-h33 JB2-s11 
s31 S600-h33 S600-h33 
s40 -- JB2-s11 
s10 JB2-s11 JB2-s11 
s19 S600-h33 JB2-s11 
s22 S600-h33 JB2-s11 
s27 S600-h33 JB2-s11 
______________________________________ 
Comparison of the eleven PEP1 data and spore number trait data from crosses 
to WQ-9525 and 303-3105, and also to S381-907, show a perfect 
correspondence, as shown in the two Punnett squares hereinbelow: 
______________________________________ 
PEP1 BGLU 
JB2-s11 
S600-h33 JB2-s11 S600-h33 
______________________________________ 
SNT1 JB 2-s11 3* 0 3* 0 
S600-h33 0 8* 7 1* 
______________________________________ 
*parental classes 
The ratio of parental to recombinant genotypes for the two loci, PEP1 and 
SNT is 11:0. While the dam are too few to permit a conventional 
chi-squared test of linkage, the direct probability of obtaining this 
result by chance is only about 3% (=8/11).sup.11. This exceeds the 5% 
error threshold conventionally used in genetic linkage analysis. This 
result provides preliminary evidence of tight linkage between the two loci 
PEP1 and SNT. Conversely, for the two loci BGLU and SNT, parentals do not 
even exceed recombinants, providing no evidence of linkage between these 
two loci. 
In related experiments, it has also been determined that the loci PEP1 and 
PEP2 are tightly linked to the mating-type locus, provisionally named MAT. 
In S600, a commercial var. bisporus strain, a Punnet square for PEP2 and 
MAT gave 36 parentals:0 recombinants. This is highly significant evidence 
of tight linkage: chi-squared=36.0. From prior work, it is known that PEP1 
and PEP2 are linked at a distance of about 6 percent recombination. 
Therefore, PEP1, PEP2, MAT, and SNT are all linked, apparently fairly 
tightly. Accordingly, each locus serves as a marker for the inheritance of 
traits determined by the other, linked loci. For example, PEP1 and PEP2 
each mark both MAT and SNT, permitting these traits to be scored in 
homokaryons without necessitating the making of test-crosses, nor, in the 
case of SNT, the producing of crops of hybrid mushrooms followed by 
microscopic examination. Similarly, in a cross where both PEP1 and PEP2 
were homozygous, and therefore uninformative, in the resulting hybrid, MAT 
and SNT would still serve as predictive markers for each other. The 
confidence level associated with predictions based on linked marker scores 
depends upon the degree of linkage existing between each pair of markers, 
which may vary somewhat from strain to strain, and which, even in the best 
studied cases, is still only approximately known for these loci. 
It has been found that most hybrid intervarietal heterokaryons resulting 
from crosses between the tetrasporic var. burnettii and the bisporic var. 
bisporus of Agaricus bisporus tend to produce mushrooms in a shorter 
period of time than do isolates of var. bisporus. Specifically, after the 
casing soil layer is applied to compost which is fully colonized by the 
mushroom mycelium, mushrooms on intervarietal hybrid strains develop and 
are ready for harvest in fewer days than do mushrooms on var. bisporus 
strains. Additionally, it appears that colonization of the compost by the 
mycelium of the intervarietal hybrid strain is, in many cases, 
sufficiently vigorous to permit application of the casing layer one or 
more days earlier than what is customary for strains of var. bisporus, 
without resulting in a yield reduction. Both of these advantages may be 
combined to further shorten the duration of the crop cycle. Any such 
shortening of the crop cycle increases the number of crops that can be 
produced in a mushroom farm in a fixed period of time, which increases 
profitability of the farm. This crop cycle is, in part, under the control 
of the grower, rather than the mushroom, so the meaningful commercial 
comparison between strains must also be based upon the yield of mushrooms 
when different spawn to case intervals are imposed. The acceleration of 
the life cycle also represents an advantage in experimental breeding 
programs by permitting more rapid progress. 
A test for early fruiting was performed as indicated generally hereinabove. 
In the test, three intervarietal hybrids, J81 (JB2-s11.times.303-3105), 
J82 (JB2-s11.times.RWK 1420-9559), and J83 (JB2-s11.times.RWK 1420-s5), 
and one commercial control, S130 (commercial hybrid var. bisporus strain), 
were employed. Each of the hybrids was cased with soil except one J83 
(peat) which was cased in peat. No peat control was furnished. The results 
of this test are disclosed in Table XlV hereinbelow. 
TABLE XIV 
______________________________________ 
Results of Early Fruiting Test 
Hybrid or Spawn run Case to Yield Yield (% 
Control ID 
(days) Pick (days) 
(grams) 
of control) 
______________________________________ 
J81 8 14.0 4116 113.0 
J82 8 14.3 4282 117.6 
J83 8 14.0 3828 105.1 
S130 (Control) 
8 15.8 3642 100.0 
Intervarietal hybrid 
1.7 days 11.9% 
advantage 
J81 10 15.7 3938 85.0 
J82 10 13.2 3736 80.7 
J83 10 13.0 3869 83.5 
S130 (Control) 
10 15.3 4631 100.0 
Intervarietal hybrid 
1.3 days -16.9% 
advantage 
J83 13 12.7 4315 102.1 
J83 (peat) 
13 12.5 4734 (112.0) 
S130 (Control) 
13 15.0 4227 100.0 
Intervarietal hybrid 
2.3 days 2.1% 
advantage 
______________________________________ 
Overall speed advantage for intervarietal hybrids in all three spawn run 
durations was ca 1.7 days. Data are means of six replicates, each having 
about 0.25 square meters of cropping surface. The median yields (not 
shown) for all treatments and controls were not significantly different at 
the 95% confidence level. Thus, it can be concluded from these data that 
(1) the crop is ready for harvest almost 2 days earlier, on average, in 
the case of three intervarietal hybrids relative to the commercial var. 
bisporus control; and (2) the average yield is comparable between the 
experimental and control strains. It is also appears that the performance 
difference of the intervarietal hybrid as compared to the commercial var. 
bisporus control may tend to favor the intervarietal hybrids at the 
shortest spawn run durations. 
Furthermore, the data presented hereinbelow indicate that intervarietal 
hybrids between the tetrasporic var. burnettii and the bisporic var. 
bisporus of Agaricus bisporus have yields which are often as high as or 
somewhat higher than the best performing commercial var. bisporus hybrids. 
This is unexpected because in evaluating the results of over 3,000 
attempted matings between homokaryons of var. bisporus, the production of 
a novel hybrid capable of achieving this level of performance is almost 
unprecedented. Evidence of the frequently superior yield performance of 
the intervarietal hybrids as a group is presented in Table XIV hereinabove 
and Tables XV hereinbelow. 
TABLE XV 
__________________________________________________________________________ 
Results of First Series of J-series Hybrid Yield Tests: 
Hybrid or Case to pick 
Yield: 
Yield 
Control ID 
Cross: (days) (grams) 
(% of control) 
__________________________________________________________________________ 
First group: (chamber) 
S600 (Control) 3768 100 
A93 (Secondary control) 
3820 
J81 JB2-s11 .times. 303-3105 
2 4514 119.8 
J60 JB2-s4 .times. 303-3105 
2 4863 129.0 
J72 JB2-s8 .times. 303-3105 
1 4469 118.6 
First group: (mine) 
S600 (Control) 3658 100 
S381 (Secondary control) 
3449 
J81 JB2-s11 .times. 303-3105 
3-4 4050 110.7 
J82 JB2-s11 .times. 1420-9559 
3-4 3981 108.8 
J83 JB2-s11 .times. RWK 1420-s5 
3-4 3962 108.3 
J60 JB2-s4 .times. 303-3105 
3-4 4265 116.6 
J72 JB2-s8 .times. 303-3105 
2-3 3311 90.5 
Second group: (mine) 
S600 (Control) 3613 100 
J56 JB2-s2 .times. RWK 1420-s5 
1 3530 97.7 
J57 JB2-s3 .times. 303-3105 
1 3705 102.5 
J61 JB2-s4 .times. 1420-9559 
1 3668 101.5 
J76 JB2-s9 .times. 1420-9559 
1 3897 107.9 
J80 JB2-s10 .times. RWK 1420-s5 
2 4311 119.3 
Third group (mine): 
S600 (Control) 3650 
S381 (Secondary control) 
3888 
J75 JB2-s9 .times. 303-3105 
1 4111 112.6 
J51 JB2-s1 .times. 303-3105 
1 4062 111.3 
J71 JB2-s7 .times. RWK 1420-s5 
0 2706 74.1 
J58 JB2-s3 .times. 1420-9559 
2 4525 124.0 
Fourth group (mine): 
S600 (Control) 4310 
J52 JB2-s1 .times. 1420-9559 
1 4963 115.2 
J64 JB2-s5 .times. 1420-9559 
0-1 3212 74.5 
J79 JB2-s10 .times. 1420-9559 
1 3553 82.4 
J77 JB2-s9 .times. 1420-s5 
1 4441 103.0 
__________________________________________________________________________ 
TABLE XVI 
__________________________________________________________________________ 
Semi-finalist Yield Test Series: J-series Hybrids 
Hybrid or Case to pick: 
Yield 
Yield 
Control ID 
Cross (days) (grams) 
(% of control) 
__________________________________________________________________________ 
First group 
S130 (Control) 16.3 3400 100.0 
J90 JB2-s11 .times. S600-h1 
13.0 3787 111.4 
J105 JB2-s11 .times. S600-h2 
13.3 4295 126.3 
J104 JB2-s11 .times. S600-h3 
13.7 4708 138.5 
J114 JB2-s11 .times. S600-h20 
13.5 4635 136.3 
J116 JB2-s11 .times. S600-h31 
13.8 3905 114.9 
J154 JB2-s11 .times. S381-907 
12.8 3572 105.1 
Second group 
S130 (Control) 16.0 3571 100.0 
J108 JB2-s11 .times. S600-h12 
13.2 4689 131.3 
J117 JB2-s11 .times. S600-h35 
13.7 4678 131.0 
J119 JB2-s11 .times. S600-h37 
13.7 4582 128.3 
J120 JB2-s11 .times. S600-h38 
13.0 5018 140.5 
J121 JB2-s11 .times. S600-h42 
14.0 4514 126.4 
J125 JB2-s11 .times. S600-h49 
13.0 4740 132.7 
Third group 
S130 (Control) 15.7 4586 100.0 
J91 JB2-s11 .times. S600-h5 
13.8 3922 85.5 
J92 JB2-s11 .times. S600-h7 
13.2 4066 88.7 
J96 JB2-s11 .times. S600-h21 
13.2 4005 87.3 
J100 JB2-s11 .times. S600-h28 
13.0 4450 97.0 
J102 JB2-s11 .times. S600-h33 
13.0 4560 99.4 
Fourth group 
S130 (Control) 15.7 4594 100.0 
J95 JB2-s11 .times. S600-h17 
13.3 4663 101.5 
J97 JB2-s11 .times. S600-h22 
13.5 4454 97.0 
J109 JB2-s11 .times. S600-h13 
13.7 4783 104.1 
J113 JB2-s11 .times. S600-h19 
13.7 4876 106.1 
J118 JB2-s11 .times. S600-h36 
13.7 4586 99.8 
J89 JB2-s9 .times. 56B-4186 
13.2 4349 94.7 
J156 JB2-s11 .times. WQ-9525 
15.8 3700 80.5 
Avg. 2.5 106.6% 
__________________________________________________________________________ 
In summary, the J-series hybrids, although picked 2.5 days earlier, on 
average, yielded 6.6 percent more, on average, than the best performing 
commercial control strain. The two to three day reduction in the case to 
pick interval represents a 5.7 to 8.6 percent reduction in cycle time in a 
commercial tray-based cropping room leading to a proportional increase in 
profitability. 
Based upon the foregoing disclosure, it should now be apparent that 
crossbreeding a four-spored Agaricus bisporus mushroom, the spores of 
which are homokaryotic, and a two-spored mushroom of the same species, as 
described herein, will carry out the objects set forth hereinabove. 
Although the crossing of homokaryons is the preferred method of obtaining 
hybrids, it is also possible to cross a homokaryon with a heterokaryon or, 
in rare instances, to cross two heterokaryons. Thus, while homokaryons 
have been described throughout the specification, it should be understood 
that either homokaryons or heterokaryons, in any combination, may be 
employed (see Table XII). It is also possible to produce hybrids of 
Agaricus bisporus in less fully-defined situations in which mixtures of 
spores, protoplasts, or hyphal fragments are allowed to germinate or 
regenerate in mass cultures of uncertain ploidy, from which hybrid 
heterokaryons may subsequently be selected. 
Moreover, it is also noted that the hybrids described hereinabove were 
eminently suitable for the production of mushroom spawn and other inocula. 
Accordingly, it will be understood that mushrooms produced by the hybrid 
heterokaryons of the present invention, as well as the commercial products 
incorporating the heterokaryons or homokaryons, or other processes or 
products which utilize the heterokaryons or homokaryons, are within the 
scope of the present invention. Spawn is one such commercial product. 
Still further, it will be appreciated that the present invention does not 
necessarily relate to the intervarietal hybrid stocks. The crossing of two 
intravarietal stocks of Agaricus bisporus var. burnettii is also 
envisioned as falling within the scope of the present invention. It is 
also to be understood that any variations evident fall within the scope of 
the claimed invention and thus, the selection of specific crossbreeding 
techniques, and sources of and utilization of homokaryons or heterokaryons 
can be determined without departing from the spirit of the invention 
herein disclosed and described. Therefore, it is to be understood that 
other means of recombining genes, such as by genetic engineering, using 
recombinant DNA technology, may also be employed for the present 
invention. Thus, the scope of the invention shall include all 
modifications and variations that may fall within the scope of the 
attached claims.