Copyright 0 1996 by the Genetics Society of America
Sexual Reproduction Playsa Major Role in the Genetic Structure of Populations
of the Fungus Mycosphaerella graminicola
Ruey-Shyang Chen and Bruce A. McDonald
Department of Plant Pathology and Microbiology, Texas A H M University, College Station, Texas 77843-2132
Manuscript received March 13, 1995
Accepted for publication January 10, 1996
ABSTRACT
The relative contributionsof sexual and asexual reproductionto the genetic structureof populations
can be difficult to determine for fungi that use a mixture of both types of propagation. Nuclear RFLPs
and DNA fingerprints wereusedtomake
indirect and direct measures of departures
from random
mating in a population of the plant pathogenic fungusMycosphaerella graminicola during the course of
an epidemic cycle.DNA fingerprints resolved 617 different genotypes among 673 isolates sampled from
a single field overa %month period.Only 7% of the isolates represented asexual clones that were found
more than once in the sample. The most common clone
was found four times. Genotypic diversity
averaged 85% ofits maximum possible value during the courseof the epidemic. Analysesof multilocus
structure showed that allelic distributions among FWLP loci were independent. Pairwise comparisons
of alleles at these loci were in gametic equilibrium.
between individual RFLP loci showed
that the majority
Though this fungus has the capacity
for
a significant
levelof asexual reproduction,each analysis suggested
that M . graminicola populations maintaina genetic structure more consistentwith random-mating over
the course of an epidemic cycle.
F
UNGI use a wide array of reproductive strategies in
natural populations (ANDERSON et al. 1992), often
including alternatingcycles of sexual and asexual reproduction. The genetic structure of fungal populations
will be affected by the relative contributions of sexual
and asexual reproduction to each generation. Populations that are largely asexual will exhibit a high degree
of clonality, withfew genotypes present at relatively
high frequencies. Random mating (sexual) populations
are expected to display a high degree of genotypic diversity. The balance between sexual and asexual reproduction will be affected by the availability ofcompatible
sexual strains, alternate hosts (as for some rust fungi),
and suitable climatic regimes (LEUNG
et al. 1993).
The relative contributions of sexual and asexual reproduction to the genetic structure of fungal populations often are poorly understood (KOHN1995). BURDON and ROELFS(1985) used isozymes to show that
sexual populations of Puccinia graminis f. sp. tn'tici from
the Pacific Northwest had greater genotypic diversity
than asexual populationsfromtheCentral
Plains.
BROWNand WOLFE(1990) used temporal changes in
gametic disequilibrium between pairs of virulence
genes and fungicide resistance to estimate the fraction
of an Erisyphe graminis f. sp. hordei population that originated from sexual spores. In both of these cases, the
sexual stage was known to play an important role in the
life cycle of the fungus, and the authorsconfirmed the
Cmresponding authm: Bruce A. McDonald, Department of Plant Pathology and Microbiology, Texas A&M University, College Station,
T X 778432132, E-mail: b-mcdonald@tamu.edu
Genetics 142: 1119-1127 (April, 1996)
impact of sexual reproduction on thegenetic structure
of populations. For many plant pathogenic fungi,however, it is not clear whether asexual or sexual reproduction plays the major role in determining the genetic
structure of populations. This is especiallytrue forfungi
that do not have a recognized sexual stage. In many
cases, asexual reproduction is thought to occur more
frequently than sexual reproduction because a limited
number of clones or clonal lineages are widespread
throughout agricultural fields over a wide geographical
et al. 1994; KOHLI et
range (LEVYet al. 1991; GOODWIN
al. 1995). However, repeated sampling of a few clones
that are found at high frequency because of strong selection or random drift can obscure the contribution
of a sexual cycle to the genetic structure of these populations (LEUNGet al. 1993; KOHN 1995;MCDONALDet
al. 1995). In addition,estimates of the relative degrees
of sexual and asexual reproduction may be affected
by differences in the dispersal distances of sexual and
asexual spores and by the spatial scale used for sampling. For example, if asexual spores are dispersed only
over short distances (e.g., centimeters), theasexual fraction of a populationmay not be adequately represented
if sampling is conducted at a largerscale (e.g., meters).
During the past five years, DNA-based genetic markers have become widely used to analyze the genetic
structure of fungal populations (KOHNet al. 1991; LEVY
et al. 1991; GOODWIN
et al. 1992; MILGROOMet al. 1992;
BOEGER
et al. 1993; MCDONALD
et al. 1994; KOHLIet al.
1995). These surveys have shown that fungal populations are not invariably clonal but can occupy a spectrum of population structures ranging fromhighly out-
1120
R.-S. Chen and B. A. McDonald
crossing to almost strictly clonal. RFLPs in nuclear and
mitochondrialgenomes have beenusedtoestimate
gene and genotypic diversity within and among populations. DNA fingerprinting has been used to distinguish
among different asexual lineages in a population. The
advent of DNA markers that assay genotypic variation
directly has made it possible to measure the relative
impact ofsexual and asexual reproduction using
a combination of direct and indirect measuresof the degree
of random mating in populations. The direct measures
are based o n using DNA fingerprints to estimate the
fraction of a population that is genetically identical because of asexual reproduction. Indirect measures are
based on measuring nonrandom
associations among
loci, such as gametic disequilibrium.
Mycosphaerella graminicola (Fuckel) J. Schrt. in Cohn
(anamorph Septon’a tn’tici Roberge in Desmaz.) is a haploid, ascomycete fungus that infects wheat worldwide
(KING et aZ. 1983). The fungus produces airborne
sexual
ascospores that have the potential to disperse over long
distances (kilometers). Rainsplash dissemination of the
asexual spores presumably occurs over relatively short
distances (meters). Several epidemiology studies have
suggested that ascospores were the primary inoculum
and ROYLE1987,1989;
that colonize wheatfields (SHAW
SCHUH 1990), while
asexual
pycnidiospores
were
thought tobe the main source of subsequent secondary
infection.Theteleomorph(sexualstage)hasbeen
found in several countries (SANDERSON
and HAMPTON
1978; Scorn et al. 1988; GARCIA
a n d MARSHALL 1992),
but it was not clear how sexual reproduction affected
the genetic structure of the population. Based o n results from a previous study (MCDONALDand MARTINEZ
1990a), we hypothesized that the initial infections resulted from airborne sexual ascospores
and subsequent
infections resulted fromsplash dispersal of asexual pycnidiospores. Under this hypothesis, we expected that
the genetic structure of the population would become
more clonal over the courseof an epidemic cycle. The
main objectives in this experiment were to determine
whether the genetic structure of
a M . graminicola population was more consistentwith random matingor asexual reproduction and to determine whether the effect
of asexual reproduction on genetic structure increased
over the course of an epidemic cycle.
MATERIALSANDMETHODS
Fungal isolates: Isolates of M. gruminicolu were collected
from a field experiment conducted on the Oregon State University Experimental Farm near Corvallis, OR. In October
1989, four wheat varieties that differed in resistance to M.
gruminicolu were planted in pure stands and in all possibletwo, three-, and four-way variety mixtures (15 treatments total) in
a randomized complete block designwith three replications.
The field sitewas fallow in the year before initiating the experiment. Individual plots in the field measured 1.5 X 6.1 m in
size. Natural infection was distributed uniformly through the
field by mid-December. We believe that the initial infection
came from ascospores originating from stubble in wheat fields
80 km distant from the field site. The plots were sampledon
March 6 and May 30, 1990, hereafter referred to as early and
late season, respectively. Twentyinfected leaves were chosen
randomly along two transects running across the length of
each plot. The average distance between collections was 50
cm within each plot. The infected leaf tissue was air dried at
room temperature for 2 weeks before making isolations. Only
one isolation was made from each infected leaf. A total of
149 isolates were obtained from early in the season and 562
isolates were obtained from late in the season. Sample sizes
were <900 isolates for both collections because some leaves
did not possess viable fungal fruiting bodies and limitations
in funding restricted the number of isolates that could be
assayed.
DNA extraction, probes, and hybridization: DNA wasextracted from each isolate using a CTAB extraction protocol
described previously (MCDONALDand MARTINEZ 1990b). Punfied DNA (4 pg) was digested individually with the restriction
enzymes PstI or XhoI. DNA fragments were separated on 0.8%
agarose gels and then transferred to nylon membranes using
the alkaline transfermethod (REED and MANN 1985) as recommended by the manufacturer (BioRad, Hercules,C A ).
Development of anonymous DNA probes used for RFLP
analysis was described previously (MCDONALDand MARTINEZ
1990b). The source of these probes was a partial Suu3A diges
tion of total DNA from S. tritici. DNA fragments ranging from
500-2500bpinsizeweresize-fractionated
before cloning
into a pGEM4 (Promega, Madison, WI) plasmid vector. Four
hundred randomly chosen clones
were kept for use as probes.
Based on an initial screen, 10 probes that hybridized to single
two loci
loci, and one probe that hybridized simultaneously to
located on different chromosomes (MCDONALDand MARTINEZ 1991a,b) were used. Together these probes hybridizedto
loci on 12 of the 18 chromosomes in the M. graminicola genome (MCDONALD
and MARTINEZ 1991a; B. A. MCDONALD,
unpublished results). Two probes (pSTL40 and pSTL70) that
hybridized to dispersed, repetitive DNA sequences were also
usedinthisstudy.
The repetitive probes wereshown previously to be useful for DNA fingerprinting (MCDONALDand
MARTINEZ 1991b). Probes were labeled by nick translation
following the manufacturer’s recommendations (BRL, Gaithersburg, MD).
Data analysis: Each probeenzyme combination was defined as a different RFLP locus. DNA fragments or combinations of fragments with different sizes were treated as alleles
at each RFLP locus. Only PstI was used to digest DNA from
the late-season collection to lowerthe cost of collecting data.
As a result, there are larger sample sizes for all RFLP loci that
use PstI than for RFLP loci that use XhoI. Isolates having the
samemultilocushaplotype (i.e., having the samealleles at
each of the single RFLP loci) were compared using DNA
fingerprints. Isolates having the same multilocus haplotype
and DNA fingerprint were assumedto be individual members
of the same clone (MCDONALDand MARTINEZ 1991b). Based
on frequencies of alleles at individual loci, and frequencies
of individual fragments in DNA fingerprints, the probability
that two individuals wouldhave the same DNA profile by
chance was <lo-’ on average in this population (B. A. MG
DONALD,unpublished observations). To determine whether
repeat sampling of the same clone would s e c t measures of
association among loci, a second “clonecorrected” data set
was constructed by including only one representative of each
clone for each analysis of gametic disequilibrium.
The measure of genotypic diversity (STODDART
and TAYLOR
1988) was based on the number of isolateswith different
multilocus haplotypes and DNA fingerprints in each collection. Only isolates with the full complement of single-locus
1121
Sexual
FungiReproduction in
RFLP data and fingerprints were included in this analysis.
Genotypic diversity ineach collection was measured usingthe
formula
e=
1
c CCfX)
*
WN)*I
X=O
where N is the sample size and fx is the number of genotypes
obsepled x times in the sample. The maximum possible value
for G, which occurs when each individual in the sample has
a different genotype, is the number of individuals inthe sample. To compare G in collections with different sample sizes,
we divided G from each collection by N to calculate the percentage ofmaximumpossiblediversity
that was obtained
(CHENet al. 1994).A t-test was used for statistical comparisons
between the normalized measures of G (CHENet al. 1994).
The method suggested by BROWNet al. (1980) was used to
measure multilocus associationsamong all loci based on the
variance of heterozygosity. Gametic disequilibrium between
pairs of loci was calculated using the following methods suggested by WEIR(1990).A test for significance of the disequilib
rium coefficient across all alleles for each pair oflociwas
formulated with the chi-square test statistic
for all pairs of alleles u and v, which were present at loci that
had k and 1 alleles total, respectively. n was the number of
individuals inthe sample and B,,,,was the maximum likelihood
estimator for the coefficient of disequilibrium between alleles
u and v. The observed allele frequencies for the loci were pu
and p,, respectively. This chi-square statistic had (k - 1) ( I 1) degrees of freedom for each pairof loci. We observed that
tests basedon pairs ofrare alleles at the two loci often resulted
in a significantvalue for the chi-square testas a resultof a low
expected number in the denominator. This sometimes led to
a rejection of the null hypothesis of independence when only
one or two rare allele pairs were in disequilibriumout of 6070 allele pairs intypical
a
locus-by-locuscomparison.To correct
forthisbias,allelespresent
at a frequency of <lo% were
pooled into a single category for comparisonstheinlate-season
collection ( N = 562) and alleles at a frequencyof <20% were
pooled in the early-season collection ( N = 149).
A test for the significance of the disequilibrium coefficient
between each pair of alleles at two loci was formulated with
the chi-square statistic
4
w
x;" = Pu(l- P,)P"(l - P")
This chi-square statistichad one degree of freedom. As before,
all allelespresent at frequencies of <10 and 20% in the lateand early-season populations, respectively, were pooled into
a single category. Pairs of allelesthat showed a significant( P
< 0.05) departure from random expectations weretested
further with Fisher's exact testof independence (WEIR1990).
The two-tailed exact test for independence between pairs of
alleles at different loci was calculated with the computer program FISH 6, version 1.001(ENGELS1988). In each exact test,
all alleles except for the tested allele pairs were pooled into
a single category.The level of significance usedfor the exact
test was P < 0.05.
RESULTS
To make direct measures of the asexual fraction present in each sample, only the 673 isolates that had com-
"
'
1
"
-
4
"
,
.
I
."
d
9 .
FIGURE
1.-Examples of
DNA fingerprints and RFLPs in
nuclear DNA of Mycosphaerelkz graminicokz isolates sampled
from a single plot late inthe epidemic. The same isolatesare
shownin each panel. All DNA was digested with PstI. (A)
Probe pSTL70 hybridizesto a dispersed repetitive
DNA family.
Arrows indicate pairs of isolates with the same DNA fingerprints. (B) Probe pSTS192 hybridizes to two loci located on
different chromosomes. The upper bands represent alleles at
one locus and the lower bands represent alleles at a second
locus. (C) Probe pSTS43 hybridizes to one locus. (D) Probe
pSTL2 hybridizes to one locus.
plete multilocus haplotypes were compared by DNA
fingerprints to differentiate clones among isolates.
There were several casesin which isolateshad thesame
multilocus haplotypes but different DNA fingerprints.
In these cases, the isolates with different DNA fingerprints represented differentclones. All isolates that had
the same DNA fingerprint had thesame multilocus h a p
lotype. The frequency distributions of isolates having
the same multilocus haplotypes or DNA fingerprints in
the early- and late-season populations are shown in Table l. There were two cases where isolates withthe same
DNA fingerprints were found both early and late in the
season (CHENet al. 1994). In one of these cases, the
clones were found in different, neighboringplots, demonstrating that asexual spores can move 2 1 m over the
course of an epidemic. In theother case, the same clone
was sampled four times from the same plot, twice early
and twice late in the season. Based on DNA fingerprints,
617 different genotypes were found among673 isolates
sampled. Only 7% of these genotypes were found more
andR . 4 . Chen
1122
TABLE 1
Frequency distributionof genotypes in early and late season
populations of Mycosphaere22a graminicola in the Oregon
field experiment
Multilocus
haplotypes"
DNA fingerprintsb
Late Early Late Early
X" season
season
season
season
1
2
3
4
5
Total number of
genotypes
Total number in
sample ( N )
e.
( G / N )%"
80
14
4
1
112
1
387
42
14
4
1
0
1
0
100
448
120
497
129
78.1
60.5%
544
384.3
70.6%
129
111.7
86.6%
544
449.8
82.7%
7
459
30
7
1
0
Multilocus haplotypes were differentiated by combining
the alleles present at 11 and 12 individual RFLP loci in the
early- and late-season populations, respectively.
bDNA fingerprints were based on hybridization of probe
pSTL40 and pSTL70 to DNA from each isolate.
Number of times each genotype is present in the population.
Index of genotypicdiversity G (STODDART
and TAYLOR
1988).
"The percentage of maximum possible value for G.
than once in the sample. Two measures of genotypic
diversity were made from these data. From the multilocus haplotype data, 60.6 and 70.7% of the maximum
possible valueswere found in theearly- and late-season
populations, respectively. These valueswere not significantly different ( P = 0.54). Based on the DNA fingerprint data, 86.6 and 82.7% of maximum possible
values were found in the early-and late-season populations, respectively ( P = 0.72). Though the DNA fingerprintsdetectedmore
genotypic diversity thanthe
multilocus haplotypes, the difference between the two
values was not statistically significant ( P = 0.14 for lateseason haplotypes us. fingerprints).
Table 2 summarizes multilocus associations among
RFLP loci in the M . graminicola population based on
the method of BROWNet al. (1980). Allsix subsets of
the data had an
adjusted value of S; that exceeded
p(2), supporting independence of loci. Statistically significant increases in multilocus associations (SE exceeding L) were notfound in any population, suggesting that allele distributions among RFLP loci were
independent. \'slues of X(2), the measure of intensity
of multilocus association, were low ranging from 0.016
to 0.095 among populations.Values of X(2) were higher
in the clonecorrected data, but there were no significant differences among any data sets.
WEIR'Smeasures of gametic disequilibrium for the
B. A. McDonald
entire population are summarized in Table 3. Only the
sixRFLP loci common to both early- and late-season
collections were used in this analysis. Pairwise comparisons for gametic disequilibrium between RFLP loci were
measured for all isolates (uncorrected) included in the
data set or only one representative of each clone (clonecorrected). For the 15 pairwise comparisons among loci
in the uncorrected data,one pair of loci was in disequilibrium (5% level). In the clone-corrected data, all of
the loci were at gametic equilibrium. Allele-by-allele
comparisons were significant in 12 of 132 cases in the
uncorrected and 14 of 132 comparisons in the clonecorrected data. However, only nine cases (7%) in the
uncorrected data and nine cases (7%) in the corrected
were significant at the 5% level with Fisher's exact test.
In the early-season population, eight out of 55 pairwise comparisons from the uncorrected and seven comparisons for the corrected data were significant at the
5% level (Table 4). In the uncorrected samples, 43 of
306 allele-by-allele comparisons were significant at 5%
level, but only nine (3%) of these comparisons were
significant with Fisher's exact test. Inthe clone-corrected samples, 37 of 306 allele-by-allele comparisons
were significant at 5% level, but only five (2%) of these
comparisons were significant with Fisher's exact test.
Table 5 shows the measures of gametic disequilibrium
in the late-season population. Significance at the 5%
level was found among six of the 66 possible painvise
combinations from the uncorrected data,
and only four
of these comparisons were significant in the clonecorrected data. Allele-by-allele comparisons showed that
64 and 54 of 623 cases in the uncorrected and clonecorrected data, respectively, were significant at the 5%
level and 37 (6%) and 24 (4%), respectively, were significant with Fisher's exact test.
DISCUSSION
DNA fingerprints and tests for departures from gametic equilibrium among single copy RFLPprobes were
used to made direct and indirect assessments of the
relative importance of sexual and asexual reproduction
on thegenetic structureof a M. graminicola population.
Direct measures of the amountof asexual reproduction
based on DNA fingerprints showed that a low degree
of clonality existed in the M . graminicola population
during the course of an epidemic cycle. This suggested
that the genetic structure
of this M. graminicola population was dominated by outcrossing. Despite the potential for a significant amount of asexual reproduction,
617 differentnuclear
DNA genotypes were found
among 673 isolates. Many M. graminicola isolates had
the same multilocus haplotypes, formed by combining
11- 12 individual RFLP loci at one time, while their
DNA fingerprints were different. An average of 20 fragments was found for the DNA fingerprints, suggesting
that more loci were being sampled by the probes that
in
Sexual Reproduction
1123
Fungi
TABLE 2
Multilocus association among RFLP loci in an Oregon population of MycaSphaereUu gmminicola
Population
Uncorrected data
Whole year
Early season
Late season
Corrected data''
Whole year
140
Early season
Late season
rn
n
h
P(2)
L
s:
342)
6
11
12
71 1
149
562
0.373
0.344
0.438
1.095
1.843
2.505
1.205
2.253
2.672
1.113
1.983
2.633
0.016
0.076
0.051
6
11
12
655
0.370
0.349
0.436
1.094
1.844
2.51 1
1.204
2.268
2.814
1.143
1.934
2.760
0.045
0.054
0.099
515
From BROWNet at. (1980). rn = number of RFLP loci;
?z
=
number of isolates sampled; h = mean single-locus diversity; p ( 2 )
= expected central moment, under Ho; L = upper 95% confident limit; Sz = observed variance of the number of heterozygous
comparisons; X(2) = nleasures of multilocus structure.
Data corrected for clonal fraction in the population based on DNA fingerprints.
(1
hybridized to dispersed, repetitive sequences. These results confirmed our previous observations (MCDONALD
and hlARTINEZ 1991b; BOECERet al. 1993) that DNA
fingerprints have greater resolution to identify clones
than multilocus haplotypes.
Based on DNA fingerprints, 571 (93%) of the 617
different genotypes were unique. No clone was observed at a high frequency and themost common genotype was present only four times in either the early- or
late-season populations. The clonal fraction was similar
early and late in the season. By STODDART'S
measure,
genotypic diversity was 85% of' its maximum possible
value on average and there was not a significant difference between the amount of' diversity found early and
late in the epidemic. The findings of a large number
of different genotypes and a high degree of genotypic
diversity suggested that these M . graminicola isolates
originated from a sexually reproducingpopulation.
The finding that theclonal fraction did not significantly
increase from the early to late season suggests that asexual reproduction did not have a significant impact on
the genetic structure of the population over the course
of the epidemic cycle.
In a large random-mating population, neutral, un-
linked loci are expected to be at gametic equilibrium.
Statistical measures of departures from gametic equilibrium may provide indirect indicators of the relative importance of sexual and asexual reproduction. To determine the effect of repeated sampling of the same clone
on measures of disequilibrium, we analyzed the data
using either all isolates, called the uncorrected sample,
or only a single representative of each genotype, called
the clone-corrected sample. The purpose of using a
clonecorrected sample was to eliminate artificial associations among loci that result from multiple sampling
of the same clone. In Neissm'u meningztidis, MAYNARD
SMITHet al. (1993) found that there was a significant
degree of gametic disequilibrium when688isolates
were compared, but when the 331 electrophoretically
distinct genotypes were used, they appeared to be
nearly indistinguishable from a panmictic population.
In a California population of M . graminicola, 22 different clones were present in a sample of 93 isolates originating from 19 leaves (MCDONALDet al. 1995). Gametic
disequilibrium was found among 76% of pairwise combinations of 12 RFLP loci when all 93 isolates wereused
in the analysis. In the clone-corrected sample of22
isolates, significant disequilibrium was found for only
TABLE 3
Measures of gametic disequilibrium among pairs of RFLP loci in whole-year population of Mycosphaerella gmminicola
collected from a wheat cultivar mkture field experiment
pSTS192-PstIA
pSTS192-PstIB
pSTS192-PstIA
pSTS192-PstIB
pSTS 14PstI
pSTS2-PstI
PSTLlO-PstI
pSTL53PstI
0/4 NS
0/6 NS
2/6 (1) NS
2/6 (0) NS
2/10 (2) NS
pSTS14PstI
PSTLlO-PstI
pSTS2-PstI
pSTL53-Pd
0/4 NS
0/6 NS
0/6 NS
0/6 NS
1/6 (0) NS
0/6 NS
1/10 (0) NS
(2)
2/9 (2) NS
0/9 NS
3/15
(3)
NS
2/6 (1) NS
0/6 NS
2/9
NS
1/6 (0) NS
1/10 ( 1 ) NS
0/6 NS
0/10 NS
0/9 NS
4/15 (4)*
1/9 (0) NS
0/15 NS
(1) 1/15
NS
1/15 (1) NS
0/9 NS
0/15 NS
Numbers below the diagonal are measures of disequilibrium calculated using only a single representative of each clone (clonecorrected). Numbers above the diagonal were calculated with all isolates (uncorrected). The top entry shows the number of
significant (P< 0.05) chi-square tests between individual alleles at different RFLP loci per the total number of tests made with
the number of tests that were also significant with Fisher's exact test in parentheses. The bottom entry shows the results of a
chi-square test for the significance of association between all alleles at the two loci: NS, not significant; * P < 0.05.
1124
R . 4 . Chen and B. A. McDonald
TABLE 4
Measures of gametic disequilibrium among pairs of RFLP loci in early-season population of Myco+huerella graminicola
collected from a wheat cultivar mixture field experiment
pSTS192XhoIA
pSTS192-
pSTS192PstIA
pSTS192PstIB
pSTS14
PstI
4/4 0/4
(0)
0/4
0/6
NS
0/4
NS
0/4
NS
0/4
NS
2/4 (0)
NS
0/4
NS
4/4 (0)
**
XhnIA
pSTSl92XhnIB
pSTS192PstIA
pSTS192PstIB
pSTS14
PstI
pSTSI96Xhd
pSTS2PstI
pSTL2XhoI
pSTL10PstI
pSTL.53.
XhoI
pSTL.530/4
PstI
pSTS192XhnIB
4/4 (0)
**
0/4
NS
0/4
NS
0/4
NS
0/6
NS
0/6
NS
1/6 (1)
*
0/6
NS
2/40/4
(0)
NS
**
3/4 (0)
**
0/4
NS
2/4 (0)
NS
2/6 (0)
NS
2/6 (0)
NS
1/6 (0)
NS
4/6 (0)
**
0/4
NS
0/4
NS
NS
4/4 (0)
NS
0/4
NS
0/4
NS
0/6
NS NS
0/6
NS
0/6
NS
2/6 (0)
NS
NS
0/4 0/4
NS
*
4/4 (0)
*
0/6
NS
0/6
NS
1/6 (0)
NS
O/fi
NS
014
NS
0/4
NS
0/6
NS
0/6
NS
0/6
NS
0/6
NS
0/4
NS
NS
pSTS196XhnI
NS
0/6
NS
0/6
NS
0/6
NS
0/6
NS
1/9 (0)
NS
0/9
NS
0/9
NS
0/6
NS
0/6
NS
pSTS2PstI
pSTL2XhnI
pSTL10PstI
pSTL53XhoI
pSTL53PstI
0/6
NS
2/6 (0)
NS
0/6
NS
0/6
NS
2/6 ( 1 )
0/6
NS
4/6 (0)
2/4 (0)
NS
0/4
NS
0/4
NS
0/4
0/4
4/6 (4)
NS
1/9 (0)
0/9
NS
0/9 (0)
NS
0/6
NS
0/6
NS
*
1/6 (0)
NS
0/6
NS
1/6 (0)
NS
0/6
NS
0/9
NS
0/9
NS
*
2/6 (0)
NS
0/6
0/4
NS
0/6
NS
0/9
NS
0/9
NS
2/9 (0)
*
2/9 (0)
*
0/6
NS
2/6 (1)
NS
0/6
NS
0/6
NS
NS
0/4
NS
0/6
NS
0/6
NS
0/6
NS
1/6 (0)
NS
NS
0/4
NS
0/4
NS
NS
0/4
NS
0/6
NS
0/6
NS
3/6 ( 1 )
*
0/6
NS
4/4 (3)
**
4/4 (3)
**
Numbers belowthe diagonal are measuresof disequilibrium calculated using only
a single representativeof each clone (clonecorrected).Numbers above the diagonal were calculated withall isolates (uncorrected).The top ently row shows the number of
significant ( P < 0.05) chi-square tests between individual alleles at different RFLP loci per the total number of tests made with
the number of tests that were also significant with Fisher’sexact test in parentheses. The bottom ently shows the results of a chisquare test for the significance of association between all alleles at the two loci: NS, not significant; * P < 0.05; ** P < 0.01.
12% of the painvise combinations. On average, nearly
five isolations were made from each leaf in the California population. The majority of isolates that had the
same DNA fingerprint were sampled fromdifferent
fruiting bodies in a single lesion or different lesions on
the same leaf. This analysis of the California population
showed how the scale of sampling can affect the interpretation of the contribution of asexual reproduction
to a population. In the case of M. gruminicolu, asexual
reproduction may be significant on a small spatial scale
(<1 m) as a result of the limited spread of the splashdispersed conidia.
For the Oregon population described in this experiment, the difference in disequilibrium between the uncorrected and the clone-corrected samples was not significant. This probably is due to the fact that only a few
clones were present in the Oregon population relative
to the total number of isolates sampled. When collecting isolates in Oregon, only one isolate was taken from
each leaf, which eliminated the possibility of sampling
the same clone from the same leaf. We believe that the
sampling method used in this Oregon experimentgave
a more accurate measure of the influence of random
mating on thegenetic structureof M. gruminicolu populations than the California population described previously (MCDONALDet ul. 1995).
LENSKI (1993), who summarized several studies of
the genetic structure of bacterial populations, pointed
out that subdividing data on population structure into
meaningful subsets based on electrophoretically distinct genotypes or geographic scales can reveal useful
information. In R Zeguminosurum, subdivided data obtained on geographic basesshowed that both allelic
diversity and linkage disequilibrium increased markedly
with geographic distance (SOUZAet al. 1992). LENSKI
(1993) also suggested that one must be cautious in accepting the joint inference that subsets are in linkage
equilibrium where data sets as a whole exhibit clonality
because a sufficiently subdivided data set may lose the
statistical power to reject the null hypothesis of panmixia. In this study, the whole-year population and its
two temporalsubpopulations, early- and late-season
populations, were tested for gametic equilibrium. The
results showed that temporal subdivision made no significant difference to the analysis, though more RFLP
lociwere used in the analysis of the early- and lateseason populations than in the whole-year population.
The data were also subdivided based on geographic
separation (replications of complete blocks) and based
on host treatment (wheat mixtures from which the fungal isolates originated). A comprehensive discussion of
the differences among host treatments is beyond the
scope of this paper. What is relevant here is that there
were no significant differences in measures of genotypic
1125
Sexual Reproduction in Fungi
TABLE 5
Measures of gametic disequilibrium among pairs of RFLP loci in late-season population of Mycosphmrella gmmimcola
collected from a wheat cultivar mixture field experiment
PstI
PsdB
pSTS192pSTS192PstIA
pSTS14
pSTS196
pSTS2pSTL2-
pSTLlO
pSTL53-
pSTS194
pSTS197pSTL31pST.543PstI
Psa
PstI
pSTS192PstIA
pSTS192PstIB
pSTS14
PstI
pSTS196
PstI
pSTSBPstI
pSTL2-Pstl
pSTLl0PstI
pSTL53PstI
pSTS199PstI
pSTS1907PSlI
pSTL31PrtI
~
~~~
Numbers below the diagonal are measures of disequilibrium calculated using only a single representative of each clone (clonecorrected). Numbers above the diagonal were calculated with all isolates (uncorrected). The top e n t ~shows the number of
significant ( P < 0.05) chi-square tests between individual alleles at different RFLP loci per the total number of tests made with
the number of tests that were also significant with Fisher’s exact test in parentheses. The bottom e n t shows
~
the results of a chisquare test for the significance of association between all alleles at the two loci: NS, not significant: * P < 0.05; ** P < 0.01.
diversity or gametic disequilibrium among blocks or
host treatments used in this experiment. There was no
evidence for population subdivision among blocks or
treatments, suggesting that subdivision by location or
host treatment did not affect the analysis of disequilibrium.
The hypothesis that allelic distributions among RFLP
lociwere independent was not rejected ( S z did not
exceed L ) for any of the samples tested (Table 2), suggesting gametic equilibrium in these populations. One
drawback to this measure of multilocus structure is that
it ignores the behavior of particular allelic combinations in the sample. It is possible that gametic disequilib
rium only occurs for particular allele combinations as
a result of selection. Therefore, we further tested the
association between pairs of RFLP loci in the populations using chi-square tests. On average, -90% of locus
pairs were at equilibrium for both uncorrected
and
clone-corrected data sets. The allele-by-allele comparisons also showed a low level (-3%) of disequilibrium
among these RFLP loci. These results showed that the
great majority of alleles at RFLP loci were randomly
associated. All of these findings supported the hypothesis that these isolates originated from a random mating
population. We conclude that genetic recombination
must occur very often and that it plays a major role in
the genetic structure of M. graminicola populations.
Selection driven by host genotype is thought to play
a dominant role in establishing the genetic structure of
many plant pathogen populations. This selection may
be especially strong in agroecosystems because genetically uniform resistant hosts areplanted over large
areas. Under these conditions, particular combinations
of virulence genes in the pathogen are expected
to
increase in frequency as a result of strong selection to
match corresponding resistance genes in the host. This
selection will lead to high levelsof disequilibrium
among particular combinations of virulence genes as
frequently described in the rusts and mildews (e.g.,
WOLFE and ~ O T 1982;
T
HOWaLLER and Q ) S T E R G h
1991; KOLMER1992). Inthese cases, it is often assumed
that the associations among virulence loci result from
strong selection to match particular combinations of
resistance genes in host populations. It is also possible
that much of this disequilibrium is maintained over
extended periods of time and at arbitrary levels due to
asexual reproduction rather than because of selection
among particular loci (MCDONALDand MCDEFWOTT
1993).
In a pathogenwith low levels ofsexual reproduction,
selection will increase the frequency of particular clonal
lineages having combinations of virulence genes that
match resistance genes in the host. Under these conditions, clonal lineages can persist for many generations
and plant pathologists are usually correct when they
assume that isolates with the same virulence spectrum
are identical by descent. When pathogens undergo limited sexual reproduction, plant breeders can exploit
andR.S.Chen
1126
the limits on recombination of virulence genes by deploying new combinations of resistance genes into a
single host genotype. This is the strategy that has been
used successfully to control many of the cereal rusts in
North America for several decades.
In a pathogen with a high level of sexual reproduction, such as M. paminicola, every sexual generation
produces new combinations of virulence genes that can
be selected out by the corresponding host resistance
genes. Under this scenario, fungal isolates having the
same combinations of virulence genes may not have
recent common ancestors and the assumption that a
population is composed of a limited number of clonal
lineages will be incorrect. A breeding program oriented
toward pyramiding resistance genes intocommon varieties is likely to fail because the pathogen has the potential to rapidly recombine new combinations of matching virulence genes. In this case, plant breeders should
pursue nonspecific resistance that often is inherited as
a quantitative trait.
Before deciding on a gene deployment strategy, plant
breeding programs should consider the genetic structure of the pathogen population. In this manuscript,
we have described methods that can be used to assess
the relative contributions of sexual and asexual reproduction to the genetic structure of pathogen populations. In thecase of the wheat pathogen M. graminicoh,
it appears that resistance gene pyramids wouldnot r e p
resent an appropriate deployment strategy. Resistance
genes may prove to be more durable if they are deployed in variety mixtures.
The authors gratefully acknowledgethe assistance ofC. C. MUNDT
and M. SCHMITT,
who collected much of the infected leaf tissuefrom
which the isolatesused in thisstudy originated. This project was
supported in part by National Science Foundation grant DEE
9306377.
LITERATURE CITED
J. B., L. M. KOHN and J. F. LESLIE, 1992 Genetic mechanisms in fungal adaptation, pp. 73-98 in The Fungal Community:
Its Organixation and Role in the Ecosystem, edited by G. C. CARROLL
and D. T. WICKLOW.
Dekker, New York.
BOEGER,
J. M., R. S. CHENand B. A. MCDONALD,
1993 Gene flow
between geographic populations of Mycosphuerella gruminicola
(anamorph Sgbtm'u hitici) detected with RFLP markers. Phytopathology 83 1148-1154.
BROWN,A. H. D.,M.W.
FELDMAN and E. NEVO,1980 Multilocus
structure of natural populations of Hordeum spontaneum. Genetics
96: 523-536.
BROWN,
J. K. M., and M. S. WOLFE,1990 Structure and evolution of
a population of Eqsiphe gruminis f. sp. hordei. Plant Pathol. 39:
376-390.
BURDON,J. J., and A. P.ROELFS, 1985 The effect of sexual and
asexual reproduction on the isozyme structure of populations of
Puccinia gruminis. Phytopathology 7 5 1068-1073.
CHEN,R. S.,J. M. BOEGERand B. A. MCDONALD,
1994 Genetic stability in a population of a plant pathogenic fungus over time. Mol.
EcoI. 3: 209-218.
ENGELS,
B., 1988 FISH6. Version l.001.UniversityofWisconsinGenetics Department, Madison, WI.
GARCIA, C.,
and D. MARSHALL, 1992 Observations on the ascogenous
stage of Septm'a tn'tici in Texas. Mycol. Res. 96: 65-70.
ANDERSON,
B. A. McDonald
GOODWIN,
S. B., L. J. SPIELMAN,
J. M. MATLJSZAK.S. N. BERGERON
and
W. E. FRY,1992 Clonal diversity and genetic differentiation of
Phytophthol-a infatans populations in Northern and Central Mexico. Phytopathology 8 2 955-961.
GOODWIN,
S. B., B.A. COHEN
and W. E. FRY,1994 Panglobal distribution of a single clonal lineage of the Irish potato famine fungus.
Proc. Natl. Acad. Sci. USA 91: 11591-11595.
HOVMBLLER, M. S., and H.@STERG&,
1991 Gametic disequilibria
between virulence genes in barley powdery mildew populations
in relation to selection and recombination. 11. Danish observations. Plant Pathol. 40: 178-189.
KING, J. E., R. J. COOKand S. C. MELVILLE,1983 A review of Septoria
diseases of wheat and barley. Ann. Appl. Biol. 103: 345-373.
KOHLI,Y., L. J. BRUNNER,
H. YOELL,M. G. MILGROOM,
J. B. ANDERSON
et ul., 1995 Clonal dispersal and spatial mixing in populations
of the plant pathogenic fungus, Sclerotinia sclerotzmm. Mol. Ecol.
4 69-77.
KOHN,L.M., 1995 The clonal dynamicin wild and agricultural
plant pathogen populations. Can. J. Bot. 73(Suppl. 1): S1231S1240.
KOHN,L. M., E. STASOVSKI,
I. CARBONE, J. ROIERand J. B. ANDERSON,
1991 Mycelial incompatibility and molecular markersidentify
genetic variability in field populations of Sclerotiniu sclootimm.
Phytopathology 81: 480-485.
KOLMER,J. A., 1992 Virulence heterozygosity and gametic phase
disequilibria in two populations of Puccinia reconditu (wheat leaf
rust fungus). Heredity 68: 505-513.
LENSKI,
R. E., 1993 Assessing the genetic structure of microbial p o p
ulations. Proc. Natl. Acad. Sci. USA90: 4334-4336.
LEUNG, H., R. J. NELSON
and J. E. LEACH,
1993 Population structure
of plant pathogenic fungi and bacteria, pp. 157-205 in Advances
in Plant Pathology, Vol. 10, edited by J. H. ANDREW and I. C.
TOMMERUP.
Academic Press, New York.
LEVY,
M., J. ROMAO,
M. A. MARCHETTI andJ. E. HAMER,
1991 DNA
fingerprinting with a dispersed repeated sequence resolves
pathotype diversity in the rice blast fungus. Plant Cell 3: 95102.
MAYNARDSMITH,
J., N. H. SMITH,M. O'ROURKE
and B. G. SPRATT,
1993 How clonal are bacteria? Proc. Natl.Acad.Sci.USA90:
4384-4388.
~,
Population genetics
MCDONALD,
B. A,, and J. M. M C D E R M O1993
of plant pathogenic fungi. Bioscience 4 3 311-319.
MCDONALD,
B. A., and J. P. MARTINEZ, 1990a DNA restriction fragment length polymorphisms among Mycosphaerellu gruminicola
(anamorph Septon'a hitici) isolates collected from a single wheat
field. Phytopathology 80: 1368-1373.
MCDONALD,
B. A,, and J. P. MARTINEZ, 1990b Restriction fragment
length polymorphisms in Septmia hiticioccur at a high frequency.
Curr. Genet. 17: 133-138.
MCDONALD,
B. A., and J. P. MARTINEZ, 1991a Chromosome length
polymorphisms in a .9@toriu tn'tici population. Curr. Genet. 1 9
265-271.
MCDONALD,B.A,, and J. P. MARTINEZ, 1991b DNA fingerprinting
of the plant pathogenic fungus Mycosphmlla gruminicola (anamorph Sqbtm'a hitici). Exp. Mycol. 15: 146-158.
MCDONALD,
B. A., J. MILES,L. R. NELSONand R. E. PETIWAY,1994
Genetic variability in nuclear DNA in field populations of S t a p
nosporu n o d m m . Phytopathology &I: 250-255.
MCDONALD,
B. A.,R. E. PETIWAY,
R. S. CHEN,J. M. BOEGERand
J. P. MARTINEZ, 1995 The population genetics of Septm'a tn'tici
(teleomorph Mycosphaerella gruminicola). Can. J. Bot. 73(Suppl
1): S292-S301.
MILGROOM,M. G., S. E.LIPARI and W. A. POWELL,
1992 DNA fingerprinting and analysis of population structure in the chestnut
blight fungus, CTphonechiu parasitica. Genetics 131: 297-306.
REED, K. C., and D. A. MANN, 1985 Rapid transfer of DNA from
agarose gels to nylon membranes. Nucleic Acids Res. 13: 72077221.
SANDERSON,F.R., and J. G. HAMITON, 1978 Roleof the perfect
states in the epidemiology of the common Sqbtoria diseases of
wheat. N. Z. J. Agric. Res. 21: 277-281.
SCHUH,W., 1990 Influence of tillage systems on diseaseintensity
and spatial pattern of Septoria leaf blotch. Phytopathology 8 0
1337-1340.
Sexual Reproduction in Fungi
Scorn,P. R.,F. R.SANDERSON
and P. W. BENEDIKZ, 1988
Occurrence
of M y c o s p h l h gmminicola, teleomorph of
hitici, on
wheat debris in the UK. Plant Pathol. 37: 285-290.
SHAW,M. W., and D. J. Row. 1987 Spatial distributions of sgtmia
nodorum and sq?toria tritici within crops of winter wheat. Plant
Pathol. 3 6 84-94.
S m w , M. W., and D. J. Ram, 1989 Airborne inoculum as a major
source of Septm'a tn'tici ( M y c o s p h l h gmminicolu) infections in
winter wheat crops in the UK. Plant Pathol. s8: 35-43.
SOUZA.
V., T. T. NGUYEN,
R R.HUDSON,
D. F'INERO and R. E. LENSKI,
1992 Hierarchical analysisoflinkage disequilibrium in Rhim
1127
bium populations: Evidence for sex? Proc. Natl. Acad. Sci. USA
8 9 8389-8393.
STODDART,
J. A., and J. F. TAILOR,1988 Genotypic diversity: estimation and prediction in samples. Genetics 118 705-711.
WEIR,B. S., 1990 Gcnctic Data Analysis. Sinauer Associates, Sunderland, M A .
WOLFE,M. S., and D. R. mom, 1982 Populations of plant path*
gens: some constrains on analysis of variation in pathogenicity.
Plant Pathol, 31: 79-90.
Communicating editor: A. H. D. BROWN