J. Microbiol. Biotechnol. (2010), 20(4), 683–692
doi: 10.4014/jmb.0906.06031
First published online 10 February 2010
Efficiency of RAPD and ISSR Markers in Differentiation of Homo- and
Heterokaryotic Protoclones of Agaricus bisporus
Mahmudul, Islam Nazrul1,2 and Yin-Bing Bian1*
1
Institute of Applied Mycology, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China
On-Farm Research Division, Bangladesh Agricultural Research Institute, Gazipur-1701, Bangladesh
2
Received: June 16, 2009 / Revised: November 18, 2009 / Accepted: November 23, 2009
Morphologically, nine different slow-growing protoclones
were screened from regenerated protoplasts of heterokaryotic
Agaricus bisporus. As such, the present study is the first
report on differentiating homo- and heterokaryotic protoclones
using random amplified polymorphic DNA (RAPD) and
inter-simple sequence repeat (ISSR) markers. Among 80
primers tested, the seven ISSR and seven RAPD primers
selected for the analysis generated a total of 94 ISSR and
52 RAPD fragments, respectively. The ISSR fingerprinting
also detected more polymorphic loci (38.29%) than the
RAPD fingerprinting (34.61%). A principal coordinate
analysis (PCA) was employed to evaluate the resolving
power of the markers as regards differentiating protoclones.
As a result, the mean polymorphism information content
(PIC) for each marker system (i.e., 0.787 for RAPD and
0.916 for ISSR) suggested that ISSR is more effective for
determining polymorphisms. The dendrograms constructed
using RAPD, ISSR, and an integrated RAPD and ISSR
marker system were highly correlated with one another as
revealed by a high Mantel correlation (r= 0.98). The
pairwise similarity index values also ranged from 0.64 to
0.95 (RAPD), 0.67 to 0.98 (ISSR), and 0.67 to 0.98 (RAPD
and ISSR), whereas the mean similarity index values of
0.82, 0.81, and 0.84 were obtained for the RAPD, ISSR,
and combined data, respectively. As there was a good
correspondence between the RAPD and ISSR similarity
matrices, ISSR would appear to be an effective alternative
to RAPD in the genetic diversity assessment and accurate
differentiation of homo- and heterokaryotic protoclones
of A. bisporus.
Keywords: Agaricus bisporus, homokaryon, RAPD, ISSR,
protoclones
*Corresponding author
Phone: +86-13507116085, 86-27-87282221; Fax: +86-27-87287442;
E-mail: [email protected], [email protected]
Agaricus bisporus is a widely cultivated and economically
valuable mushroom worldwide [44], as it has abundant
nutritional and medicinal functions [37]. However, despite
its value and importance, progress in directed strain
improvement for A. bisporus has been limited owing to the
difficulty of producing homokaryons and the dearth of genetic
markers for proving. The recovery of self-sterile, presumably
homokaryotic strains by the micromanipulation of spores
from tetrasporic basidia [14] or screening of random singlespore isolates [46] is time consuming and very laborious,
as the process generally involves screening monospore
cultures for slow growth rates and then subjecting selected
strains to a multiple locus molecular marker analysis or
fruiting tests. Basidiospores homoallelic at all loci that
were heteroallelic in the parental strains are considered
homokaryons [25]. The more marker loci used, the more
robust the results. In fruiting tests, self-sterile cross-fertile
spores are considered homokaryons [17]. However, this
method is less robust than using molecular markers.
Recently, an alternative method for obtaining homokaryons
through protoplasts was reported [3, 10, 45]. When protoplasts
are produced from a heterokaryon and allowed to regenerate
mycelia, approximately 1 in 10 is homokaryotic. Traditionally,
the primary differentiation of homo- and heterokaryotic
strains is based on morphological attributes that have been
mapped by a somatic incompatibility (SI) test [53]; these
attributes are rooted in the concept of “the Buller
phenomenon” described by Buller [6], who showed that
heterokaryons can denote a nucleus to homokaryons.
Although the Buller phenomenon occurs in A. bisporus, so
far there is no evidence of nuclear migration in this species
[36]. In most basidiomycetes, one of the morphological
characteristics that identify heterokaryotic mycelia is the
presence of a clamp [1]. Yet, distinct from other members
of Basidiomycota, heterokaryotic A. bisporus does not
form clamp connections [22]. Therefore, distinguishing
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Mahmudul and Bian
heterokaryons (he) from homokaryons (ho) for population
and other genetic studies of A. bisporus remains a challenge.
Until just a decade ago, homokaryotic and heterokaryotic
isolates of A. bisporus could only be potentially differentiated
by qualitative assessments that combined colony characteristics
with growth rate and isozyme differences, which could be
easily affected by the environmental factors of the culture
[27, 39]. Thus, an alternative method, like molecular
markers, that can accurately assess the relationship of the
colony morphology for differentiating homokaryons and
heterokaryons is needed for A. bisporus. Over the years,
the methods used for detecting and assessing genetic
diversity have extended from the analysis of discrete
morphological traits to biochemical and molecular traits.
Several DNA marker systems are now commonly used in
diversity studies of plants and fungi. RAPD and ISSR
markers have both been used for DNA fingerprinting [5,
13, 18, 31], and population and phylogenetic studies [20].
There are also some reports in which the capacity of
RAPD and ISSR markers to detect polymorphisms and
discriminate among taxa has been compared [15, 32].
Although RAPD markers can be applied to assess genetic
variation in single-spore progeny from different Agaricus
species or to identify homokaryons derived from a
heterokaryotic species [26, 51], their poor consistency and
low reproducibility limit their utilization [50].
The marker system called inter-simple sequence repeats
(ISSRs) is also a PCR-based technique [52] that has been
successfully applied to genetic analyses of citrus [16],
potato [33], cutgrass [43], and fungal classification [48]. In
particular, ISSR markers can be highly variable within a
species and have an advantage over RAPDs in utilizing
longer primers that allow more stringent annealing
temperatures [8, 49]. Thus, it was in this context that the
current authors first utilized these technologies (viz.,
RAPD and ISSR) to evaluate the usefulness of molecular
markers in assessing and analyzing the nature and extent of
molecular variation among slow-growing protoclones of A.
bisporus, and distinguishing homokaryons from heterokaryons.
MATERIALS AND METHODS
Strains
The commercially cultivated strain of A. bisporus (ACCC50659) used
in this investigation was obtained from the Mushroom Spawn Center,
Huazhong Agricultural University, China. Moreover, it has been
deposited in the Agricultural Culture Collection Center of China.
Protoplast Production, Regeneration, and Isolation
Protoplasts were produced for obtaining protoclones, as described
by Amin et al. [2] with some modifications. The mycelia were
cultured in a PDYA liquid medium for 5-7 days. The cultures were
harvested by filtration through a nickel sieve, and washed twice
with a sterilized 0.6 M mannitol solution. The harvested mycelia
Fig. 1. Microscopic digital view of isolated protoplasts of A.
bisporus used in this experiment: two protoplasts are indicated
by arrows.
were then dried on presterilized absorbent paper, suspended in a
filter-sterilized 2% Lywallzyme solution (0.02 g/ml in a 0.6 M mannitol
solution; Guangdong Institute of Microbiology, China), and mixed
with 7% snail enzymes (Sinopharm Chemical Reagent Co., Ltd,
China). Digestion of the cell walls was allowed to proceed for 3 h in
a hot water bath at 32oC. The hyphal fragments were removed by
filtration through a column of cotton wool packed up to the 0.5-ml
mark of a small syringe. The protoplasts were then collected by
centrifugation (10 min, 1,500 ×g) and washed twice by centrifugation
in 0.6 M mannitol. After resuspending the pellets in 0.6 M mannitol,
the protoplast yield was determined using a hemocytometer. A drop
of the protoplast suspension was placed on a slide, covered with a
cover slip, and the sample examined under a microscope (Olympus
BX51). The microscopic digital view of the protoplasts is provided
in Fig. 1. For regeneration, 0.5 ml of the protoplast suspension (about
104/ml) was plated on a PDYA [4] regeneration medium including
0.6 M mannitol as an osmotic stabilizer. After the germination started,
the protoclones were randomly isolated, transferred individually to
PDYA slants according to Magae et al. [28] and Zhao and Chang
[56], and incubated at 24oC for further observation.
Screening of Putative Homokaryons
After 25 days of incubation, the protoclones of A. bisporus were
isolated on a PDYA slant, and their growth compared with that of
the parental heterokaryotic strain (Fig. 2). Forty slow-grown protoclones
were screened from among 500 protoclones, and their colony
morphology and mycelial growth rate compared with those of the
parental heterokaryotic strain [24, 26]. Among the 40 selected
protoclones, those that exhibited similar colony characteristics to the
parental heterokaryotic strain - stranded, fluffy, and good aerial
growth, with or without sectoring - were discarded (data not shown).
The remaining nine protoclones that had a slower growth rate
than the parental heterokaryons and showed either appressed or
strandy hyphae morphologies in each of five subcultures were
selected as putative homokaryons [21, 54] and numbered from 1 to
9. These slow-growing isolates were then subjected to a growth rate
study on a PDYA medium. Spawn-run and fruiting trials were also
conducted on a composted substrate, and RAPD and ISSR analyses
were performed to confirm their homokaryotic status.
IDENTIFICATION OF AGARICUS BISPORUS HOMOKARYOTIC PROTOCLONES
685
tested protoclones, including the parental heterokaryotic control, was
extracted using the cetyltrimethylammonium bromide (CTAB) protocol
[40]. The integrity of the obtained genomic DNA was detected by
electrophoresis in a 1% agarose gel, stained with ethidium bromide,
and its concentration was determined using a Bio Photometer 6131
(Eppendorf, Germany).
Fruiting Trial
A small-scale fruiting trial for the putative homokaryotic protoclones
obtained from a heterokaryotic strain (ACCC50659) of A. bisporus
was carried out in plastic boxes with a surface area of 475 cm2 each.
Three replications including a parental heterokaryotic control were
maintained in the fructification trial of the putative homokaryotic
protoclones. Spawn run, case run, primordial initiation, and maturation
of fruit bodies all occurred under the controlled conditions [7].
RAPD Reaction
Forty random primers obtained from Operon Technologies (Alameda,
CA, U.S.A.) were used for the polymorphism test of the nine
putative homokaryotic protoclones of A. bisporus. Among these,
seven primers produced clear and unambiguous bands, and were
used in this investigation (Table 1).
The amplification was performed in a 25-µl volume containing 1×
PCR buffer, 400 µM dNTP (Sigma-Aldrich, St. Louis, MO, U.S.A.),
0.2 µM random primer (Operon Technologies Inc, Alameda, CA,
U.S.A.), 1 unit Taq DNA polymerase, and 50 ng of DNA. The PCR
amplifications were performed in a thermal cycler using heated lid
technology and the following conditions: (1) initial denaturation at
95oC for 4 min, followed by (2) 45 cycles of denaturation at 94oC
for 1 min, (3) primer annealing at 36oC for 1 min, (4) DNA
amplification at 72oC for 2 min, and (5) a final primer extension at
72oC for 8 min. The amplicons were loaded on a 1.3% agarose gel
and separated by electrophoresis at 75 V for about 3 h. The DNA
marker DL 2000 (TaKaRa, Japan) was used as the size marker. The
RAPD fragments were stained with ethidium bromide, and the
profiles recorded using a Syngene Gel Documentation System with
GeneSnap software. All the RAPD reactions were performed more
than twice to test the reproducibility of the amplicon profiles.
Extraction of Mycelial Genomic DNA
The mycelia for the total genomic DNA extraction were maintained
in a liquid medium, as described by Yang [55]. The DNA of all the
ISSR Reaction
Forty ISSR primers purchased from Sangon (Shanghai, China) were
tested on the nine putative homokaryotic protoclones of A. bisporus.
Fig. 2. Differences in mycelial growth between homo- and
heterokaryons of A. bisporus: 1-3, putative homokaryons; 4-5,
progeny heterokaryons; 6, parental heterokaryon.
Table 1. Efficiency of individual primers for RAPD and ISSR differentiation in nine putative homokaryotic protoclones of A. bisporus.
Primer type
Sequence (5'-3')
RAPD primers
OPA02
TGCCGAGCTG
OPA15
TTCCGAACCC
OPA19
CAAACGTCGG
OPA20
GTTGCGATCC
OPA24
GTGCCTAACC
OPA30
TGCCCGTCGT
OPA35
CTGTTGCTAC
Total
Average per primer
ISSR primers
P3
(GA)8T
P8
(CT)8AGA
P14
(CT)8T
P23
(CA)8RC
P30
(GA)8C
P38
(CTC)6
P39
(CAAGG)3
Total
Average per primer
Size range (bp)
200-1,800
245-950
350-1,000
250-1,200
250-1,800
340-2,000
400-1,700
100-2,100
400-2,000
360-1,900
250-1,500
250-1,500
250-2,000
350-2,400
Total amplicons
5
6
9
9
7
10
6
52
12
19
10
17
14
8
14
94
Polymorphic markers
2
2
4
3
2
4
1
18
5
9
4
8
6
2
2
36
% Polymorphism
PIC value
40.00
33.33
44.44
33.33
28.57
40.00
16.66
0.711
0.872
0.790
0.912
0.682
0.932
0.615
34.61
0.787
41.66
47.36
40.00
47.05
42.85
25.00
14.29
0.939
0.960
0.891
0.910
0.873
0.895
0.948
38.29
0.916
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Mahmudul and Bian
Among these 40, seven ISSR primers that showed a clear and
reproducible band pattern were chosen for this study (Table 1).
The PCR amplification was performed in a 20-µl volume containing
30 ng of template genomic DNA, 30 ng of primer (AuGCT
Biotechnology, China), 0.15 mM dNTPs, 2.0 mM MgCl2, 1 U Taq DNA
polymerase (Dingguo Biotechnology, China), and 1× PCR buffer.
The mixture was overlaid with mineral oil and subjected to a
PCR in a Perkin Elmer 480 thermal cycler programmed for an
initial step of 5 min at 94oC, followed by 40 cycles of 30 s at 94oC,
45 s at 52oC, 90 s at 72oC, and a 7-min final extension step at 72oC.
The PCR products were analyzed by electrophoresis on a 1.3%
agarose gel (BioWest, Spain) containing 0.5 µg ethidium bromide/ml.
The DNA marker DL 2000 (TaKaRa, Japan) was used as the size
marker. Similar to the RAPD, the ISSR reactions were also performed
more than twice to test the reproducibility of the amplification profiles.
Data Analysis
A marker index was calculated for the RAPD and ISSR markers to
characterize the capacity of each primer to detect polymorphic loci
among the protoclones. As such, the marker index was the sum of
the polymorphism information content (PIC) values for all the
selected markers produced by a particular primer. The PIC value
was calculated using the formula PIC=1-Σp 2, where P is the
frequency of the i allele [42].
The data obtained by scoring the presence (1) or absence (0) of
amplified fragments from the RAPD and ISSR profiles with different
individual primers, as well as collectively, were subjected to the
construction of a similarity matrix using Dice’s [12] and Jaccard’s
[23] coefficients of similarity. Among the various similarity indices,
those of Jaccard and Dice were chosen as the most appropriate ones
for dominant markers, like ISSR and RAPD, since they do not
attribute any genetic meaning to the coincidence of band absence.
The similarity values were then used for a cluster analysis. Sequential
agglomerative hierarchical nonoverlapping (SAHN) clustering was
performed using the unweighted pair group method with arithmetic
averages (UPGMA), and the results were summarized as dendrograms.
The data analysis was conducted using NTSYSpc software 2.02 [38].
i
i
The goodness-of-fit of the dendrograms to the original GS matrix
was calculated by computing the cophenetic value (rcoph) using the
cophenetic (COPH) and matrix comparison (MXCOMP) modules of
NTSYSpc. The COPH module computes a symmetrical matrix of
cophenetic (ultrametric) similarity or dissimilarity values in the form
of a tree matrix from the set of nested clusters produced by the
SAHN clustering module. The product-moment correlation (r) based
on the Mantel Z-value [29] was computed to measure the degree of
relationship between the similarity index matrices produced by any
two-marker system. A comparison of the RAPD and ISSR-based
GS matrices was also performed using the MAXCOMP routine of
NTSYSpc, and the COPH subroutine of NTSYSpc was used to
determine the cophenetic correlation between different clusters. An
rcoph ≥ 80% is generally considered a good fit [38].
Finally, a principal coordinate analysis was performed to highlight
the resolving power of the ordination.
RESULTS
Colony Growth Rate and Spawn Run
The appressed colonized protoclones with growth rates of
less than 0.96 mm/d exhibited a slow spawn run and failed
to complete the spawn-running process, even after 45 days.
These results also agreed well with the findings of Singh
et al. [41]. In this study, all the appressed protoclones
produced structures like primordia in one or two out of
three replicates. Although the strandy protoclones produced
fruits, the number was lower than that produced by the
parental control (Table 2). A previous study reported that
appressed colonized single spores tend to be less vigorous
and grow more slowly than strandy heterokaryons, resulting
in an inability to produce fruit bodies [54]. Fruit-body
formation in A. bisporus is considered an indication of
heterokaryosis [14].
Table 2. Mycelial growth rate, colony characteristics, spawn run, and fruit bodies on composted substrate of slow-growing single-spore
isolates of A. bisporus.
In 2 kg composted substrate
Isolates
Growth rate (mm/day)a
Colony characteristics
Spawn runb
Fruit bodiesc
1
2
3
4
5
6
7
8
9
C
0.79±0.012
0.98±0.026
0.80±0.033
0.84±0.025
0.88±0.025
0.78±0.009
0.79±0.016
0.80±0.028
0.96±0.026
1.64±0.085
Appressed
Strandy aerial
Appressed
Appressed
Appressed
Appressed
Appressed
Appressed
Strandy aerial
Strandy aerial fluffier
+
++
+
+
+
+
+
+
++
+++
±
5.00±1.6
±
±
±
±
±
±
4.66±1.29
6.34±1.44
The values are the means (± standard errors).
a
Colony growth rate was calculated from the results of three separate experiments with four replications on a PDYA medium incubated at 24oC.
b
Spawn-run process in substrate: +, slow; ++, moderate; +++, fast; ±, primordial-like structures; C, parental control.
c
Number of fruit bodies calculated from the results of one set of experiments with three replications.
IDENTIFICATION OF AGARICUS BISPORUS HOMOKARYOTIC PROTOCLONES
Fruiting Trial
Nine putative homokaryons and one parental heterokaryon
were tested for haploid fruiting in (15×25×12 cm) plastic
boxes, and the number of fruit bodies was determined after
three picking weeks. Among the nine, two homokaryotic
protoclones (2, 9) produced fruit bodies with a similar
morphology to the parental heterokaryotic control in all
three replicates, yet the number of fruit bodies was lower
than that produced by the parental control. None of the
appressed colonized protoclones produced any fruit bodies
during the whole picking period (Table 2).
RAPD Analysis
Forty primers were tested, and 7 were chosen for their
clear and reproducible band patterns (Table 1). The seven
selected primers generated 52 RAPD fragments, an average
of 7.42 bands per primer, and the size of the amplified
products ranged from 250 to 2,000 bp. The total number of
polymorphic markers and percentage of polymorphism
were 18 and 34.61%, respectively (Table 1). The RAPD
marker profile produced by primer OPA20 is depicted in
Fig. 3. The PIC values, a reflection of the allele diversity
and frequency among the protoclones, were not uniformly
higher for all the RAPD loci tested. The PIC values ranged
from 0.615 (OPA35) to 0.932 (OPA30) with a mean of
0.787, and the similarity coefficients based on 52 RAPD
amplicons ranged from 0.64 to 0.95. The mean similarity
index was 0.82. A cluster analysis was performed based on
both Dice’s [12] and Jaccard’s [23] similarity coefficient
matrices, calculated from the RAPD markers, to generate
two dendrograms for the nine putative homokaryotic
protoclones of A. bisporus. The dendrograms separated the
protoclones of A. bisporus into two clusters. The first
cluster was formed by the parental heterokaryotic control
(C) and included two protoclones (2, 9), whereas the
second cluster was further divided into three subclusters
(Fig. 5A and 5B).
Fig. 3. RAPD marker profile of nine putative homokaryotic
protoclones of A. bisporus generated by primer OPA20 in 1.3%
agarose gel and stained with ethidium bromide.
Lanes 1-9 are putative homokaryotic protoclones, lane C is the parental
control, lane N is the negative control, and the rightmost lane M contains
the 2-kb size marker.
687
Fig. 4. ISSR marker profile of nine putative homokaryotic
protoclones of A. bisporus generated by primer P8 in 1.3%
agarose gel and stained with ethidium bromide.
Lanes 1-9 are putative homokaryotic protoclones, lane C is the parental
control, lane N is the negative control, and the rightmost lane contains the
2-kb size marker.
ISSR Analysis
Among the 40 primers tested, 7 were chosen for their clear
and reproducible band patterns (Table 1). The other ISSR
primers did not produce any bands or clear bands for
scoring in the experiments (data not shown). Among the
primers tested, the most polymorphic pattern was produced
when using ISSR P8, (CT)n, whereas the poly(AT)n primers
gave no amplification products. The seven selected primers
generated 94 fragments, an average of 13.43 bands per
primer. The size of the amplified products ranged from 100
to 2,400 bp, and the total number of polymorphic markers
and percentage of polymorphism were 36 and 38.29%,
respectively (Table 1). The ISSR marker profile produced
by primer P8 in a 1.3% agarose gel is shown in Fig. 4. In
the case of the ISSR analysis, the mean PIC value was
0.916, and the lowest and highest PIC values were 0.873
(P30) and 0.960 (P8), respectively. The similarity coefficients
for the nine protoclones of A. bisporus based on the 94
ISSR fragments ranged from 0.67 to 0.98. The mean
similarity index was 0.81. A cluster analysis of the ISSR
markers separated the nine putative homokaryotic protoclones
of A. bisporus into two distinct clusters in both Dice’s [12]
and Jaccard’s [23] similarity coefficient matrices. The first
cluster included only two protoclones (2, 9) with the parental
heterokaryotic control (C), and the second cluster included
the remaining seven protoclones and was further divided
into four subclusters (Fig. 5C and 5D).
Combined RAPD and ISSR Analysis
For each primer, two strandy hyphae protoclones (2 and 9)
displayed identical amplification products similar to the
parental heterokaryotic control, whereas the rest of the
appressed hyphae protoclones displayed a loss of polymorphic
bands when compared with the parent dikaryon. A similar
phenomenon was also observed when using RAPD markers
to differentiate the genotypes of A. bisporus progenies and
identify homokaryons derived from a specific heterokaryon
[26].
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Mahmudul and Bian
Fig. 5. UPGMA dendrograms of nine putative homokaryotic protoclones of A. bisporus based on Dice and Jaccard genetic indices
when using RAPD and ISSR markers: A, RAPD (Dice); B, RAPD (Jaccard); C, ISSR (Dice); and D, ISSR (Jaccard).
The similarity coefficients of the nine putative homokaryotic
protoclones of A. bisporus based on 52 RAPD markers and
94 ISSR markers ranged from 0 .67 to 0.98, and the mean
similarity index value of the combined RAPD and ISSR
was 0.84. The individual DICE similarity coefficient matrix
and cophenetic value matrix derived from the phenetic
analysis of the RAPD and ISSR profiles were compared
using the Mantel matrix correspondence test. The Mantel
test between the two Dice and between the two Jaccard
similarity matrices gave r=0.981 and r=0.991, respectively,
showing for both indices a high correlation between the
RAPD- and ISSR-based similarities.
A cluster analysis performed based on combining the
data for both markers generated two dendrograms (Dice
and Jaccard), which separated the putative homokaryotic
protoclones of A. bisporus into two distinct clusters. The
first cluster included only two protoclones, 2 and 9, and the
parental heterokaryotic control (C), whereas the second
cluster was further divided into three subclusters. Among
these subclusters, the first included protoclone 3, the
second contained protoclones 1, 6, 4, and 7, and the third
included protoclones 5 and 8 that showed a 100% similarity
between each other (Fig. 6A and 6B).
The UPGMA dendrograms, obtained from a cluster analysis
of each of the four index × marker similarity matrices, gave
similar results, identifying two main clusters corresponding
to the nine putative homokaryotic protoclones. In all the
dendrograms, protoclones 2 and 9 were closer to the
parental heterokaryotic control than the other protoclones.
To estimate the correlation level between the dendrograms,
a new set of cophenetic matrices was calculated and
compared using the Mantel test. A cophenetic correlation
of r=0.99 was obtained from the MXCOMP program for
the RAPD, ISSR, and integrated (RAPD and ISSR) matrix,
respectively, indicating a good fit between the original
similarity matrix and the resulting clustering analysis of
the RAPD, ISSR, and integrated RAPD and ISSR [38].
A three-dimensional ordination confirmed the cluster
analysis results, showing that the fertile and non-fertile
protoclones were sharply separated (Fig. 7A-7C).
DISCUSSION
To enable population and other genetic studies of A. bisporus,
a molecular-based method for accurately distinguishing
homokaryons from heterokaryons is required. Nuclear
ribosomal DNA (rDNA) is frequently used for taxonomic
and phylogenetic studies of many different species of
edible fungi. The RAPD technique has already been used
in studies of both wild and cultivated strains of A. bisporus
fungi [26, 47] and the delineation of homokaryons [51].
IDENTIFICATION OF AGARICUS BISPORUS HOMOKARYOTIC PROTOCLONES
689
Fig. 6. UPGMA dendrograms of nine putative homokaryotic
protoclones of A. bisporus based on Dice and Jaccard genetic
indices when using combined RAPD and ISSR markers: A,
RAPD-ISSR (Dice); and B, RAPD-ISSR (Jaccard).
Meanwhile, the ISSR has been utilized in research on
several kinds of edible fungi (viz., Auricularia auricular
[48] and Lentinula [35]), and has been proven useful in
assessing genetic variations among commercial Agaricus
strains in China [19].
In the present experiment, the ISSR markers demonstrated
more polymorphisms than the RAPD markers. Fang and
Roose [16] also previously mentioned that ISSR markers
revealed more polymorphic fragments than RAPD.
Accordingly, this study compared the applicability of
RAPDs and ISSRs as genetic markers in terms of
characterizing nine slow-growing putative homokaryotic
protoclones of A. bisporus. The results showed that the
markers exhibited a similar potential to discriminate between
the nine protoclones. In all the UPGMA dendrograms, the
same sample distributions were obtained that indicated a
sharp definition of the nine protoclones, and that protoclones
2 and 9 were closer to the parental heterokaryotic control
(C) than the other protoclones. These results were also
consistent with some of the morphological and fruiting
characteristics. For instance, protoclones 2 and 9 were
fertile and had a strandy hyphae morphology, whereas the
other protoclones were non-fertile appressed hyphae. In a
previous study, Yadav et al. [54] reported that appressed
Fig. 7. Principal coordinate analysis using RAPD, ISSR, and
integrated RAPD and ISSR markers with Jaccard similarity
index: A, RAPD (Jaccard); B, ISSR (Jaccard); and C, RAPDISSR (Jaccard).
colonized homokaryotic single spores are commonly less
vigorous and grow more slowly than strandy heterokaryons,
and are unable to produce fruit bodies. The efficiency of a
molecular marker technique depends on the amount of
polymorphism and PIC values (a parameter associated
with the discriminating power) of the two marker systems.
Despite the great and similar discriminating power of both
marker systems, some differences between the two were
detected, such as higher PIC values for the ISSRs when
compared with the RAPDs, and a higher number of total
690
Mahmudul and Bian
amplicons and higher percent of polymorphism for the
ISSRs than for the RAPDs. This was because of the
polyallelic nature of the ISSR markers.
When a cluster analysis was carried out on three sets of
marker profiling data, based on the RAPD, ISSR, and an
integrated RAPD and ISSR, the results based on the entire
three DNA marker profiles broadly grouped the nine
putative homokaryotic protoclones of A. bisporus into two
clusters. In the experiments, a high reproducibility was
obtained for the ISSR markers when compared with the
RAPD markers; only very faint fragments were not
reproducible and such fragments were discarded. Qian et
al. [34] in their studies also found that RAPD bands were
less reliable and reproducible, plus Nagaoka and Oigihara
[32] mentioned that ISSR primers produced more reliable
and reproducible bands when compared with RAPD primers.
Furthermore, the UPGMA dendrograms and 3D PCA plot
clearly separated the non-fruiting homokaryotic protoclones
from the fertile heterokaryotic protoclones and parental
control.
As previously pointed out, during the ISSR primer
screening, good amplification products were obtained from
the primers based on (GA)n and (GT)n repeats, whereas
the (AT)n primers gave no amplification products, despite
the fact that poly(AT) dinucleotide repeats are thought to
be the most abundant motif in plant species [11]. Similar
results have also been obtained with chestnut [9], wheat
[32], and Nothofagus species [30]. A possible explanation
for these results is that ISSR primers based on AT motifs
are self-annealing, owing to sequence complementarity,
and thus form dimers during PCR amplification [5].
Consequently, the results of the present study can be seen
as a starting point for future research aimed at defining
the level of intra- and interspecific genetic diversity, and
detecting homokaryotic isolates of A. bisporus.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Acknowledgments
The authors would like to thank Dr. Wang Zesheng of the
Agricultural Academy of Fujian Province, Fujian, China,
for his helpful cooperation. We are also grateful to the
Mushroom Spawn Center, Huazhong Agricultural University,
China, for supplying the strains and for their excellent
technical assistance during the spawn run and fruiting trials,
without which this study could not have been completed.
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