全 文 ：第 21 卷　第 4 期　　　　　　　　　　　　　植　　　物　　　研　　　究 2001 年　10 月
Vol.21　No.4　　　　　　　　　　　BULLETIN OF BOTANICAL RESEARCH Oct., 　2001
双孢蘑菇 96.4菌株和单孢后代间的遗传变异
李荣春
(云南农业大学食用菌研究所 , 昆明　650201)
摘　要　本文研究了野生双孢蘑菇菌株 96.4(Agaricus bisporus)和它的 10个单孢菌株的生长发
育和遗传变异 。研究表明 ,在菌株 96.4和它的单孢菌株之间以及在 10个单孢菌株之间在菌丝生
长速度和随机扩增多态 DNA(RAPD)揭示的遗传变异等方面都存在着明显的变异。 RAPD指纹
揭示了 96.4菌株通过有性生殖产生的丰富的生物学变异。
关键词　双孢蘑菇;单孢菌株;遗传变异;RAPD
GENETIC DIVERSITY OF STRAIN 96.4
(AGARICUS BISPORUS)AND ITS SSIS
LI Rong-chun
(Institute of Edible Fungi o f Yunnan Agriculture University kunming 650201)
Abstract　The mycelial extension g row th rate on agar and the genetical diversity based on RAPDs of
ten single spore isolates(SSIs)of a wild st rain 96.4 of Agaricus bisporus and i t , which was collected
from Oxfo rdshire UK , were studied.Our studies demonst rated the signif icant differences among the
single spore isolates f rom the same basidium and between st rain 96.4 and SSIs w ere found in the
mycelial ex tension g row th rate on agar and in the genetic diversity.The abundant biomutations pro-
duced by sexual reproduction w ere revealed by RAPD markers.
Key words　Agaricus bisporus;single spore isolates(SSIs);genet ic diversity ;RAPDs
　　
1.INTRODUCTION
The but ton mushroom (Agaricus bisporus
(Lange)Imbach)is a very important crop in world.
It holds a unique posi tion among the edible fungi.In
the last five decades the mushroom product ion has
g row n into a large indust ry w hich has extended over
the world.An annual global production of fruit -
body of this basidionycete is approximately 1590 ,000
metric tons(M iles and Chang , 1997).But due to its
unusual secondarily homothallic life-cycle the breed-
ing of A.bisporus is complicated and relative diffi-
culty.The most commercial st rains are identical to o r
derived f rom only tw o hybrid st rains , HorstU1 and
Ho rstU3.The lack of genetic variation betw een the
commercial mushroom strains fo rms a severe threat to
the mushroom indust ry (De Groot , et al., 1998).
A .bisporus is a mainly secondarily homothallic
basidiomycete
[ 1～ 3] .Most basidia produce two spores
each containing tw o non-sister nuclei of four prod-
ucts of meiosis.These spores thus retain heterozy-
gosity and af ter germinat ion a fertile multinuclear
heterokaryotic mycelium was formed
[ 4～ 6] .This het-
作者简介:李荣春(1959-),男 ,副教授 ,云南玉溪人 ,主要从事食用菌育种和栽培的研究。
收稿日期:2001-05-28
erokaryot ic off spring can t be used directly inbreeding
and outbreeding.This reproductive cycle results in a
relatively low frequency of genotypic change and few
opportunity of genetic exchang betw een different
geno type.Recent observations suggested that in re-
spect of o ther characters besides mating type A .bis-
porus is a rather invariable species.[ 7] And as a result ,
the commercial strains encompassed ex tremely limited
genetic resources.[ 8 ,9 ,4] The scarci ty of genetic re-
sources among the cultivated strains and unusual sec-
ondarily homothallic life-cycle are two big barrio rs
of hybrid breeding[ 10] .This crop genetic improved
research fallen behind with o ther crops.But Kerrigen
&Ross[ 9] demonst rated that w ide genetic variability
be found among the w ild st rains.Callac , P.[ 11] re-
po rted high genetic polymorphism appeared even
w ithin each site.He had studied nine genetic differ-
ent isolates f rom the same site.This stands in sharp
contract to commercial strains and fo rms a contradic-
tion w ith secondarily homothallic life-cycle.
In this study , we have observed the biological
trains in grow th and genetic diversity of a w ild st rain
(96.4)and its singer spore isolates (SSIs)of same
basidiome.Because we thought the basidiospore bio-
logical mutation is the basis of population genetic di-
versity.Our study demonst rated the genetic diversity
exists among sing le spore isolates of the same fruit-
body.
2.MATERIALS AND METHODS
2.1　STRAINS:
The parental strain , designed 96.4 , was ob-
tained by Dr.R.Noble in Ox fordshire(UK)and by
tissue culture from wild basidiome.It belongs to A-
garicus bisporus (Lange)Imbach (A .brunnescens
Peck), which w as f riendly identified by P.Andrew s.
Sing le spore isolates(SSIs)were obtained by plating
out 0.1 ml of dilutesuspension of basidiospores(103
spores ml
-1)on to an agar plate containing complete
yeast medium (CYM).To stimulate spore germina-
tion , each plate was inverted over an A .bisporus
culture on CYM[ 2 , 12] .Plate w ere incubated at 25°C
until germination occurred , usually in 4 ～ 8 weeks.
Germinated spore w as carefully subcultured f rom the
dilution plates on to CYM agar plate using a mi-
croscalpel.The seventy germinating w ere sub-cul-
tured.Ten single spore isolates , selected at random ,
were utilised in this study.
2.2　MYCELIA GROWTH RATE:
The radial g row th rate on agar of parent and
SSIs w ere determined follow ing the inoculation of 2%
malt ext ract ag ar (MEA)[ 12] Petri dishes were inocu-
lated w ith a single 3mm diameter agar plug taken
from the leading edge of a g rowing culture.Three
replicate plates w ere prepared per isolate (parent and
10 SSIs)and incubated at 25°C fo r 5 weeks.Grow th
was measured at 4d intervals along two perpendicular
diameters of the grow ing cultures , f rom which a
mean radius w as calculated.
2.3　RAPD ANALYSIS:
2.3.1　GENOM IC DNA EXTRACTION:Mycelia
had been g row n in liquid CYM (composi tion:2%
dex t rose , 0.2% peptone , 0.2% yeast ex t ract , 0.
05% MgSO4.7H2O , 0.046%KH2PO4 and 0.1%
KH2PO4).Harvested mycelia were dried w ith filter
papers(Whatman , UK)and transferred into micro-
centrifuge tubes.The mycelia were rapidly f rozen in
liquid nit rogen vapour (-130°C)then w ere dried in
freezer drier overnight.The dried mycelia were
ground into a f ine powder in microcentrifuge tubes
using inoculated needles.DNA ex traction w as per-
formed according to Challen , M .P .et al.,[ 13] Ex-
traction buffer (0.4M KCl , 50mM EDTA pH8.0 ,
1%v/v Tri ton★RX-100)is freshly prepared on the
day of use and sterilized through a 0.2μm sy ringe fil-
ter.To each sample , 1000μl ex t raction buf fer and 0.
2μl Ribonuclease A were added and mixed thorough-
ly.The microcentrifuge tubes were incubated in w a-
ter bath at 70°C for 40 min.The required number of
QIAprep 8 strips w as placed into the manifold seats.
Postincubation , the samples were microcentrifuged
for 15 min(14000 rpm)and supernatants t ransferred
to the equilibrated QIAGEN columns that had been
wet ted w ith ext raction buf fer.Then columns were
washed , once each w ith 1ml of buffer PB and tw ice
with 1ml of buf fer PE.The vacuum should be main-
tained for a full minute af ter all of the PE had drained
through.While the w aste t ray w as replaced w ith the
QIAvac rack containing the required number of QI-
Aprep collection tubes.The DNA eluted under vacu-
606 　　　　　　植　　物　　研　　究　　　　　　　　　　　　　　　　　　21 卷
um w ith the addition of 100μl of 1mM Tris.HCl(pH
8.0).DNA concentration was compared wi th λDNA
by 0.8% agarose gel elect rophoresis and diluted to
5ngμl-1.DNA samples w ere sto red at -20℃.
2.4.2　RAPD reaction.The amplification reaction
took place in a 40μl volume which included 4μl 10x
PCR buffer , 29.85μl PCR water , 0.4μl dNTP
(mix), 0.5μl Dynazyme II (2Unitμl-1), 0.25μl a
single 10-mer primer and 5μl DNA(ca 25ng).The
reactions were perfo rmed in 0.5m l Eppendorf tubes
placed in an OmniGene thermal cycler(Hybaid , Ted-
dington , UK), under the following cycle condition:
first ini tial denaturation at 94℃ fo r 1 min , followed
by 35 cycles of 92℃ for 1min , 35℃ for 1 min , 72℃
for 2 min.
　　Table 1 Primers used for generating RAPDs
Primer
Sequence
(5 -3 )
Number of
amplified fragment
Number of
polymo rphic fragment
OP-A03
OP-A04
OP-A05
OP-A09
OP-A11
OP-A13
OP-A14
OP-A16
OP-A18
OP-A20
5 -AGTCAGCCAC-3
5 -AATCGGGCTG-3
5 -AGGGGTCTTG-3
5 -GGGTAACGCC-3
5 -CAATCGCCGT-3
5 -CAGCACCCAC-3
5 -TCTGTGCTGG-3
5 -AGCCAGCGAA-3
5 -AGGTGACCGT-3
5 -GTTGCGATCC-3
8
9
9
8
7
9
8
7
11
9
3
4
2
1
2
3
2
0
3
2
15 primers(RAPD primer Kit A from Operon Tech-
nologies Inc.)were screened with parent (96.4)
DNA sample and the ten primers(Table 1)were uti-
lized for this study .Amplification products were sep-
arated by elect rophoresis in 1.4% agarose gels run
w ith TAE buffer at 60V.Pho tographs w ere taken of
the ethidium bromide stained gel , visualized by UV
light illumination.The RAPD reactions were repeat-
ed once.
2.4.3　DATA ANALYSIS:The RAPD fragments
w ere scored as present (1)o r absent(0)for each of
the primer-isolate combinations.Estimates of genet-
ic simitarity (S)were calculated between all pairs of
parent and i ts SSIs according to Nei &Li[ 14]
Similari ty (S)=2 N xy/ (N x+N y)
Where Nx = the total number of f ragments
show n by individual “x”;Ny = the total number of
fragments show n by individual “y” and Nxy = the
number of f ragments shared by individual “ x” and
“y”.The genetic similarity matrix thus generated
w as converted to a genetic distance matrix [ genetic
distance (D)=1 - similari ty (S)] and w as used to
assess relationships among the isolates with a cluster
analy sis using Unweighted Pair-Group M ethod with
Arithmet ic Averages (UPGMA).All computations
were carried out using the computer prog ram of Phy-
logeny Inference Package version3.5.
3.RESUILT
3.1 RADIAL G ROWTH RATE
The radial g row th rate of 96.4 and ten SS Is
were show ed in Fig1.96.4.1 isolate manifested
marked different grow th rate f rom parent and sister
SSIs.It had the low est grow th rate of 0.5mmd-1.
The isolates of 96.4.5 , 96.4.7 and 96.4.13 had
lower g row th rate than 96.4.Anyhow the parent
96.4 and o ther six teen i ts single spore isolates shared
the similar grow th rate.Especially the same g row ing
pattern appeared among parent 96.4 and SSIs 96.4.
2 , 96.4.3 , 96.4.6 , 96.4.8 , 96.4.9 ,96.4.11 , 96.
4.15 and 96.4.19.Their average growing rate w as
1.49mmd-1.Experiment show ed there w as a obvi-
ous mutation in grow th rate betw een different SSI of
same frui t-body .
3.2　GENETIC DIVERSITY
RAPDs w ere performed on parent(96.4)and its
ten SSIs(96.4.1-96.4.10)and gave highly infor-
mative banding pat terns that w ere polymorphic be-
tween parent and its SSIs and w ithin SSIs.Ten
6074 期　　　　　　　　　　　　　　李荣春:双孢蘑菇 96.4 菌株和单孢后代间的遗变异
Fig 1:The radial g rowth rate
of 96.4 and ten SSI s.
F ig 2　RAPD pattern generated by a primer
OP-A04(5-AATCGGGCTG-3)
primers generated a total of 86 reproducible and
scorable amplification products , out of which 31(36.
05%)were polymo rphic.The number of products
generated by each primer varied f rom 7 to 11 w ith an
average of 8.6.The size of the amplified f ragments
ranged from 300bp to 2580bp.RAPD fingerprints of
parent and i ts ten SSIs amplif ied by primer OP-A04
are depicted in Fig 2.RAPD profiles of the two
homokary onic SSIs (96.4.1 and 96.4.7)and the
heterokaryonic parent and its other SS Is were com-
pared(Fig 2)by using primer OP -A04.This re-
sults demonst rated that 10-mers is capable of ampli-
fying polymo rphic loci in Agaricus bisporus that
readily differentiate between homokaryon and het-
erokaryon.
The genetic distance betw een parent and SS Is
ranged betw een 0 to 0.25.The genetic distance be-
tween 96.4 and three SSIs(96.4.3 , 96.4.8 and 96.
4.10)was 0 , but w as 0.25 betw een 96.4 and 96.4.
7.However genetic distance w as very small among
parent and its SSIs.
The UPGMA cluster analy sis the existence of
tw o g roups(Fig 3).The tw o isolates(96.4.1 and
96.4.7)fo rmed a small g roup , which had laborious
fruiting and w ere considered to being homokaryons.
The o ther SSIs and parent formed a large group ,
which all f ruited no rmally.
4.DISCUSSION
In this study , the marked diversities w ere found
in mycelia g row th and genetic marker polymorphism
between different single spore isolates of same basid-
iome.The ex t remely restricted grow th and labo rous
fruiting had been defined as two main characteristics
of homokaryo tic isolate of A .bisporus[ 2] .Kerrigen ,
R.W.et al[ 15] found that the mycelial ex tension rate
on malt ag ar medium was consistent ly low er fo r
homokaryotic SSIs than for heterokayotic SSIs of A .
bisporus.But Noble , R.et al[ 12] reported no signifi-
cant difference in ex tension rate betw een heterokayo t-
ic parent and its homokayo tic SSIs of A .subf locco-
sus.In our study , 96.4.1 and 96.4.13 isolate
demonstrated slow g row th.But 96.4.7 isolate had a
laborious fruiting ability and a nearly normal grow th
rate.In grow th rate , only was the lower g row th rate
mutation found in offspring.
　　In resent twenty years , many molecular markers
were used in A.bisporus research of parent and of f-
spring relat ion.Royse and M ay [ 4]studied polymor-
phism at f ive enzyme loci of A.bisporus and found
136 out of 154 single spo re progenies of heterozygous
parent were heterozygotes.Summerrbell , R.C., et
al
[ 16] , using DNA restriction f rogment leng th poly-
morphisms(RFLP), studied relation of progeny and
parent in A .brunnescens(A.bisporus).Xu , J.et
608 　　　　　　植　　物　　研　　究　　　　　　　　　　　　　　　　　　21 卷
Fig 3:UPGMA dendrog ram o f parent and its ten
SSIs based on RAPD bands derived from ten primer s.
al[ 17] studied m tDNA variation using RFLP.Loftus ,
M .G., et al[ 18] , using RFLP , studied DNA poly-
morphisms in commercial and w ild strains of A.bis-
po rus , and reported RFLPs w ere used to demonstrate
normal meiot ic segregation and to differentiate be-
tween homokaryons and heterokaryons.Khush , R.
S.et al[ 19] reported RAPD markers provide an ef fi-
cient alternative fo r strain f ingerprinting and a versa-
tile tool for genetic studies and manipulation of A .
bisporus.In our study , RAPD markers were em-
ployed to research the genetic diversities betw een par-
ent and its single spore of fspring.They ef ficiently
displayed genetic diversities between parent and of f-
spring and wi thin offspring (Fig2 , 3).It w as sur-
prised that primer OP-A04 visibly dif ferentiated la-
bo riously f rui ting isolates f rom normal fruiting isolates
(Fig2).RAPD marker is an ef ficient , quickly and
lower cost ly tool for genetic diversity research of A .
bisporus.
Our study demonstrated the w ide biomutations
exits in different single spore isolates of same fruit-
body.This biomutat ions maybe results in the gene
recombination by meiosis.This mutation had , we
thought , pravited abundant genetic source for popula-
tion evolution in nature and commercial st rains im-
provement [ 7].Greater v ariations were provided by cul-
ture of single spore.A spo re with especially valuable
hereditary factors might not express i ts full value in
multispore culture[ 20] .Selection among monospo rous
mycelia offers an oppo rtunity for isolating new
strains , while mycelia derived by a multispo rous
method or by t issue culture do no ordinarily exhibit
variations f rom the parent[ 3] .
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