Construction of a genetic linkage map in
the wild plant Mimulus using RAPD and
isozyme markers
Jing-Zhong Lin and Kermit Ritland
Abstract: As a first step to mapping quantitative trait loci for mating system differences, a genetic linkage map
was generated from an interspecific backcross between Mimulus guttatus and Mimulus platycalyx. The linkage
map consists of 99 RAPD and two isozyme markers. Eighty-one of these markers were mapped to 15 linkage
groups, spanning 1437 contiguous centiMorgans, and covering 58% of the estimated genome. The genome length
of Mimulus is estimated at 2474 + 35 cM; bootstrapping indicates that only ca. 40 markers are needed to give an
accurate estimate of genome length. Further statistical analyses indicate that many RAPD markers cannot be
ordered with certainty and that uncertain linkage groups tend to map nonlinearly even under commonly used
mapping functions. Strategies for speeding up the mapping process for a wild species and possible applications
of a partial linkage map in evolutionary studies are discussed.
Key words: linkage map, mating system, Mimulus, RAPD.
RCsumC : Comme premikre Ctape en vue de l'identification de loci de caractkres quantitatifs associCs au mode de
reproduction, une carte gCnCtique a CtC Ctablie au moyen d'un rktrocroisement interspkcifique entre le Mimulus
guttatus et le Mimulus platycalyx. La carte comprend 99 marqueurs RAPD et deux marqueurs isoenzymatiques.
Quatre-vingt-un de ces marqueurs sont situCs sur l'un des 15 groupes de liaison qui totalisent 1437 centiMorgans
contigus et couvrent environ 58% du gCnome. La taille du gCnome du M. guttatus est estimke h 2474 + 35 cM.
Des analyses de rCCchantillonage (<< bootstrapping >>)indiquent qu'environ 40 marqueurs suffisent pour donner
un bon estimC de la taille du gCnome. Des analyses statistiques additionnelles montrent que plusieurs marqueurs
RAPD ne peuvent Ctre ordonnCs avec certitude et que certains groupes de liaison incertains tendent h Ctre nonlinCaires et ce mCme en utilisant des fonctions cartographiques communCment employkes. Des stratCgies
permettant d'accC1Crer le processus de cartographie chez une espkce sauvage et des applications possibles de
cartes incomplktes dans le cadre d'Ctudes sur l'kvolution sont discutkes.
Mots cle's : carte gCnCtique, mode de reproduction, Mimulus, RAPD.
[Traduit par la RCdaction]
lntroduction
Recent years have seen considerable research interest in
the genetic basis of plant mating system evolution
(Holsinger 1988; Macnair and Cumbes 1989; Shore and
Barrett 1990; Holtsford and Ellstrand 1992; Latta and
Ritland 1993; Fenster and Ritland 1994). The parameters of
primary interest are the number of genes controlling a mating system character (Macnair and Cumbes 1989; Shore
and Barrett 1990; Holtsford and Ellstrand 1992; Fenster
and Ritland 1994), their relative effects (Fenster and Ritland
1994), and dominance level (Charlesworth 1992). Classical
analyses of segregation in crosses between closely related
plant species have revealed a moderate to large number
of genes responsible for most mating-system character differences (Macnair and Cumbes 1989; Holtsford and
Ellstrand 1992; Fenster and Ritland 1994). However, these
I
Corresponding Editor: R.S. Singh.
Received June 5, 1995. Accepted September 22, 1995.
J.-Z. Lin and K. Ritland. Department of Botany,
University of Toronto, 25 Willcocks Street, Toronto, ON
M5S 3B2, Canada.
Genome, 39: 63-70 (1996). Printed in Canada 1 ImprimC au Canada
analyses gave only minimum estimates of the number of
genes and their average effects. The questions of whether
genes controlling mating system divergence are dominant
or recessive, and whether these genes are of large or small
effect, remain largely unanswered (Fenster and Ritland
1994). With the advent of quantitative trait loci (QTLs)
mapping techniques, these questions can be answered in
a more precise way, if a genetic linkage map can be constructed for the species in question.
The genus Mimulus (Scrophulariaceae) consists of about
150 species (Vickery 1978), which display a considerable
diversity of mating systems, ranging from predominantly
selfing to predominantly outcrossing (Ritland and Ritland
1989). Many taxa are intercrossable (Vickery 1978), providing an excellent model system for studies of the genetic
basis of mating system differences.
Random amplified polymorphic DNA (RAPD; Williams
et al. 1990) is the most accessible class of molecular markers for geneticists working with wild species; such DNA
markers are far more easily obtained than are restriction
fragment length polymorphisms (RFLPs) or microsatellite
markers (Rafalski and Tingy 1993). We have initiated a
project to determine more precisely, via QTL mapping
Genome, Vol. 39, 1996
with RAPD and isozyme markers, the number of loci, their
individual effects, and level of dominance for mating system
differences in Mimulus. As a first step to this end, we have
generated a genetic linkage map with 99 RAPD and two
isozyme markers using an interspecific backcross between
M i m u l u s p l a t y c a l y x and M i m u l u s g u t t a t u s . Herein, we
report this map and address the following specific questions.
1. How reliable are RAPD markers for genetic mapping,
particularly for wild species? Reports on the reliability of
RAPD markers have been mixed. Some consider RAPDs
reliable in most mapping applications (e.g., Weeden et al.
1992; Williams et al. 1993), while others are concerned
with the transferability of RAPD markers between laboratories (e.g., Penner et al. 1993). We note that mistyping
usually inflates the estimate of recombination frequency
(Weeden et al. 1992; Kesseli et al. 1994); hence, estimated
distances between markers may be nonadditive in that twopoint distances will be shorter than multipoint distances. As
a result, markers may map "nonlinearly," a situation analogous to a circular chromosome. Chiasma interference also
contributes to the "nonlinearity" of the map in that it
depresses the double-crossover class thus expanding the
two-point map distance over a given region. The net effect
of chiasma interference would be larger for multipoint distance than for two-point distance of the same region.
However, use of a mapping function that takes into account
chiasma interference (e.g., the Kosambi mapping function;
Kosambi 1944) should remove this contribution to "nonlinearity" of the map. Thus, we test our data for "nonlinearity" of the map by comparing two-point distances with
multipoint distances inferred by both Haldane and Kosambi
mapping functions; deviations from linearity indicate
misty ping of markers.
2. How reliable is the genetic map that is obtained? The
estimated linkage order may not reflect the true order
owing to either inadequate sample sizes or nonreliability of
RAPD amplification. We test our data for accuracy of linkage orders by examining the "stability" (or constancy of
gene order) among bootstrapped data sets.
3. How many markers are needed to provide a reasonably
accurate estimate of genome length? This enables us to
estimate the proportion of the genome covered by a partial
linkage map and, therefore, to extrapolate estimates of
QTL number from the partial linkage map to the entire
genome. This is important for studies of QTLs in wild
species, where saturated genetic maps are not available
owing to the great experimental effort required. Using the
maximum likelihood estimator of genome length of
Chakravarti et al. (1991), we estimate with our data the
number of markers needed for genome length estimation.
This has not been examined previously.
Materials and methods
Plant materials
Two taxa with contrasting mating systems are used in the present studies. The large flowered M. guttatus is a predominantly
outcrossing taxon (t (outcrossing rate) = 0.6-0.9), while the
smaller flowered M. platycalyx is a predominantly selfing taxon
(t = 0.1-0.2) (Ritland and Ritland 1989). Both taxa are annual
yellow monkeyflowers and have the same haploid chromosome
number of n = 14 (Vickery 1978).
Population 138 of M. guttatus and population 606 of
M. platycalyx were chosen for this study. Population 138 was
collected at 1.2 miles (1 mile = 1.6 krn) east of Bingen, Oregon,
U.S.A. by K.R. and population 606 was collected at the intersection of Joy Road and Bodega Road, Sonoma County, Calif.,
U.S.A., by Jeffery Dole of Illinois State University.
One individual from each population was chosen to make the
cross and the Fl was then backcrossed to one of the selfed
progenies of the M. platycalyx parent. Two hundred and fortyseven BC, progeny were grown in a growth chamber at 14 h
light (18°C) : 10 h dark (10°C).
DNA isolation
After the 247 BCl plants were measured for six mating system
characters for QTL mapping (J.-Z. Lin and K. Ritland, in preparation), 1-2 g of leaf tissue was harvested, ground to fine powder in liquid nitrogen,
and then stored at 280°C in a 50-mL
Falcon tube.
Ninety-six individuals with extreme phenotypes in two noncorrelated characters (flower width and stigma-anther separation)
were chosen for DNA isolation and RAPD and isozyme assays.
This selective genotyping (Lander and Botstein 1989) allows us
to build a marker linkage map while increasing the power of
detecting QTLs for mating system differences. We show elsewhere (J.-Z. Lin and K. Ritland, submitted for publication)1
that selective genotyping (as opposed to a random sample) will
cause only limited bias to estimates of map distances (2-10%)
between markers linked to QTLs of large effects, and negligible
bias to marker order, when 40% (as in this study) of the individuals are selected.
The frozen sample was thawed with 10 mL of hexadecyltrimethylammonium bromide (CTAB) buffer (100 mM
Tris-HC1 (pH 8.0), 1.4 M NaCl, 20 mM EDTA, 2% CTAB,
0.2% 2-mercaptoethanol) and DNA was isolated according to
the procedures of Doyle and Doyle (1987) with the following
modifications: an additional chloroform extraction was performed; after incubation with RNAse A, nuclear acids were
reprecipitated using 100% ethanol with 0.15 M NaCl; and the
pellet was washed with 70% ethanol.
There was not enough DNA from the first isolation to complete the study, owing to degradation. Thus, we re-isolated DNA
from a pooled sample of at least 50 selfed progenies (3-week-old
seedlings) of the 96 BCl individuals. This pooled sample ensures
the same RAPD banding profile as that of the BCl individuals,
since the selfed (F,) progeny of a heterozygous BCl plant will
give a 3: 1 ratio of bands : no bands, while that of a homozygous
recessive BCl plant will show no bands.
RAPD primers and PCR protocols
One hundred and eighty RAPD primers from Operon Technologies (Operon Technologies, Alameda, Calif., U.S.A.) were
initially tested for polymorphisms using the two parents and
six BCl individuals. Fifty-one primers yielded polymorphic
markers and were assayed for the rest of the 96 BCl individuals.
Except for primers M04, M 11, M 13, M 15, M 16, and M20,
PCRs were performed in a Perkin-Elmer Cetus DNA thermocycler (Perkin-Elmer Corp., Connecticut, Mass., U.S.A.) with
a 15-pL reaction mixture consisting of 10 mM Tris-HC1, 50 mM
KCl, 1.5-2.0 mM MgCl,, 0.01% (wlv) gelatin, 200 pM each of
dATP, dCTP, dTTP, and dGTP, 7.5 pmol (0.5 pM) primer,
0.5-0.6 units of Ampli-Taq (Perkin Elmer Cetus), and 15-30 ng
DNA. A mastermix was prepared and aliquoted into individual
J.-Z. Lin and K. Ritland. The shrinkage of estimated map
distances due to selective genotyping for a quantitative
trait. Submitted for publication.
Lin and Ritland
Fig. 1. Segregation of three RAPD markers revealed by primer L17. Lane M, ADNA digested
with EcoRI-HindIII; lanes P1 and P2, M. guttatus and M. platycalyx parents, respectively;
lanes 1-22, BC, progeny. Segregating markers are indicated by arrows.
tubes before DNA was added and the total mixture was then
covered with one drop of mineral oil. The reaction was carried
out with an initial denaturing at 94°C for 1 min, followed by
45 cycles of 94°C for 1 rnin, 36°C for 1 min, 55°C for 30 s, and
72°C for 2 min, and finished with an extension at 72°C for
10 min; the reaction mixture was then held at 4°C until recovery.
PCRs with primers M04, M 11, M 13, M15, M16, and M20 were
performed in a Coy-60 thermocycler according to Williams et
al. (1990) using Taq supplied by BioICan (BioICan Scientific
Inc., Ont., Canada) and a 50 pL reaction volume. All PCR
products were separated by electrophoresis on 1.4% agarose
gels, detected by ethidium bromide staining, and scored as
present versus absent.
Isozyme survey
Protein extracts from corollas were assayed for 15 enzyme systems following the procedures of Ritland and Ganders (1987).
Genotypes of the 96 individuals were recorded for three polymorphic loci: malic enzyme, phosphoglucose isomerase, and
esterase.
Linkage analysis
Prior to linkage analysis, a X2 test was used to examine the
goodness of fit to a 1: 1 ratio for each polymorphic locus.
Twelve loci deviated significantly from a 1: 1 ratio and thus
were excluded from further analyses.
Linkage analysis was carried out using MAPMAKER (v. 3.0)
(Lander et al. 1987; Lincoln et al. 1992). A two-point recombination frequency of 0.37 (67.4 cM) was determined by X2
test as the linkage criterion at 99% confidence level using
96 BC, individuals. Markers were placed into linkage groups
using the order and compare functions of MAPMAKER,
first with
LOD 3.0 and then with LOD 2.0. Map distance was estimated
using the Haldane mapping function (Haldane 1919).
Linearity of markers
To examine the linearity of markers, we plotted the pairwise
two-point distance against the multipoint distance inferred by
MAPMAKER using the Haldane mapping function for markers
in each linkage group. We also plotted these distances after
converting them into Kosambi map distances as (cf. Crow
1990)
where DK is Kosambi map distance and D H is Haldane map
distance.
Ten pairs of markers in linkage group 1 and one pair in
linkage group 6 have a recombination frequency 0 2 0.5 and thus
were excluded from the plot. All other pairs were included
even though some of the two-point linkages were not significant.
A regression line was then fitted for each of the plots.
Nonlinearity of markers is evidenced by differences between the
regression line and the expected line of 45°C (slope = 1). In this
case, however, a significance test is not appropriate because
the data points are not independent.
Stability of linkages
Stability of linkages was examined using bootstrap data sets
from one representative linkage group (group 2). Replicate
data sets were created by resampling individuals in this linkage
group with replacement from the original data set. Only markers
ordered with LOD 2 3.0 were used. This group was chosen
since it had the highest number of loci with LOD 2 3.0. For
each bootstrap sample, the maximum likelihood order of all
possible marker triples was found via three-point analysis in
MAPMAKER.
If the markers on the ends of the triples are linked
(determined by two-point analysis), and if the order of the
triples remains among replicated samples, then standard deviations were found for the map distances and the frequency of
marker triples recorded.
Genome length estimate
Genome length was estimated using the maximum likelihood
method of Chakravarti et al. (1991). To examine the effect of
number of markers on genome length estimate, the first 10,
15, ..., 100, and 101 marker loci were chosen from the original
101 marker data set. For each set of markers, bootstrapping
was carried out to resample individuals from the original
96 individuals. Standard deviations for genome length estimated
for each set of markers were found based on 100 bootstraps.
Results
Level of polymorphisms
Twenty-eight per cent (511180) of the RAPD primers
revealed polymorphism between the two parents. One hundred and ten easily scorable polymorphic marker loci were
produced with the 51 RAPD primers, giving an average
66
Genome, Vol. 39, 1996
Fig. 2. A genetic linkage map of Mimulus. The 15 linkage groups are arranged on the basis of their length. Map distances in
centiMorgans (cM) are listed to the left and loci to the right of each linkage group. RAPD locus names include the name of the
primer and the approximate size of the fragment in kilobases. The broken line indicates that the distance between markers could
not be determined with certainty (LOD < 2.0) and an asterisk indicates that markers or linkage groups were ordered with LOD
2.0. Unlinked loci are also listed.
1
2
.
16.5
mll-0.3
ml6-1.6
3
12.7
21.5
111-1.3
6*
7
8
Unlinked loci:
e01-0.9
b18-0.7
e04-0.4
c19-0.54 *
e04-1.0
el6-1.2
f b12-o' 78
10
17.9
0010-1.0
20.9
5
bO6-0.5
9*
19.4
4
112-0.3
11 7-1.75
114-0.72
f c19-1'0
11
i
f"j"5
12
f --O"
c19-0.3
TO2-'"
e12-0.45
e19-1.1
41.5
44.3
13
EST
11 7-0.45
39.9
115-0.6
dl5-1.3
of 2.2 loci per primer. An example of multiple markers
revealed by one primer is shown in Fig. 1. Among the
15 isozyme systems tested, three (20%) were polymorphic,
each with one scorable locus.
The reproducibility of RAPD markers was examined
by re-amplifying and rescoring all 96 progenies for four
primers (AA12, L17, M 16, and T15). The discrepancy was
found to be about 1.5%. This is primarily due to degradation
of template DNA, since most of the discrepancy occurred
between fragments longer than 1.0 kilobase (kb), and we
have also observed a few failed reactions (data not shown).
Linkage map
Eighty-one of the 101 polymorphic loci were mapped to
15 linkage groups, covering 1437 contiguous centiMorgans
(Fig. 2). Twenty loci could not be placed into any linkage
group with two-point linkage criteria of LOD 3.0 and
recombination frequency 0.37 (67.4 cM). Some loci were
placed at LOD 2.0 and there are also a few loose linkages
among the mapped loci.
Linearity of markers
Figure 3 shows the plots of two-point versus multipoint
map distance for markers in linkage groups 1-9. Plots for
linkage groups 10-15 are not shown, since the number of
markers in these groups is small.
Linkage groups 3, 4, 5, and 6 do not deviate from linearity in any significant manner, but some markers in
groups 1, 2, 7, 8, and 9 appear not to map linearly. Multipoint distances are generally longer than two-point distances.
Interestingly, most of these nonlinearly located markers
were ordered with LOD < 3.0 in the linkage map.
Figure 3 also shows that markers appear to be more
linearly mapped with the Kosambi mapping function than
with the Haldane mapping function, probably because the
14
f%ii
m04-1.75
I
15
29.6
b11-o'6
bll-1.1
g17-0.97
108-0.9
118-1.25
PC1
101-0.56
101-1.3
105-0.45
Kosambi mapping function corrects for chiasma interference.
However, differences between the regression lines of
Haldane and Kosambi map distances are small, indicating
that nonlinearity of the map is mainly due to mistyping.
Stability of linkages
The triples given for linkage group 2 were observed from
37 to 85 times in 100 bootstrap samples and the map distances have rather large standard deviations (Table 1).
A comparison of the log-likelihood ratios indicate that the
most-likely order is only slightly more probable than the
next-most-likely order for most linkage groups (data not
shown). These results show that the linkage groups are
not very stable.
Genome length
The genome length of Mimulus is estimated to be
2474 + 35 cM. The 1437 contiguous centiMorgans in the
linkage map (Fig. 2) thus cover 58% of the estimated
genome, and the average distance between adjacent loci
is 2 1.8 cM. The relationship between the genome length
estimate and number of markers is shown in Fig. 4. This
figure indicates that genome length tends to be over estimated and to have rather large standard deviations with a
small number of markers. A more reliable estimate is
achieved with 40 or more markers. So, our estimate via
101 markers is near the asymptotic efficiency. This result
is in accordance with what was found with simulated data
by Chakravarti et al. (1991).
Discussion
Owing to their technical simplicity, low cost per data unit,
and high levels of polymorphisms, RAPDs are the obvious
choice of molecular markers for genetic maps in wild
species (Rafalski and Tingy 1993; Mitchell-Olds 1995).
Lin and Ritland
Fig. 3. Plots of two-point distance versus multipoint distance for pairwise markers with recombination frequency
8 < 0.5 in linkage groups 1-9. Deviation of the observed regression lines (solid) from the expected line (dotted)
indicates nonlinearity of markers. Squares and K are the data points and regression line, respectively, of Kosambi
map distances and circles and H are the data points and regression line, respectively, of Haldane map distances.
Group 1
Group 3
Group 2
200
Group 5
Group 4
F
0
V
Q,
80
Group 6
120
120
60
0
Group 7
100
Group 8
Group 9
80
80
Multipoint distance (cM)
Our mapping of the wild plant Mimulus using RAPD and
isozyme markers lays the basis for ultimately mapping
QTLs for mating system differences and demonstrates the
practicality of molecular mapping techniques in evolutionary
studies in wild species. Although our statistical analyses
have revealed a few undesirable features of RAPDs in
genome mapping, they can be avoided by practical alternatives, which are discussed below.
Difficulties in ordering RAPD markers (instability of
linkage groups) is indicated by the large proportion of
markers (44%) not ordered with certainty (LOD < 3.0,
Fig. 2) in multipoint analyses and by the low frequency
at which the same order within a linkage group is inferred
in three-point analyses across bootstrapped data sets
(Table 1). The former result is similar to those found in
Eucalyptus grandis and Eucalyptus urophylla (44% and
55%, respectively; Grattapaglia and Sederoff 1994) but is
lower than that found in Arabidopsis (60%; Reiter et al.
1992). In lettuce, Kesseli et al. (1994) found that RAPDs
are generally ordered with lower certainty than RFLPs;
Genome, Vol. 39, 1996
Table 1. Map distances and frequency of marker triples observed in 100 bootstrap
samples for markers ordered with LOD 3.0 in linkage group 2.
Marker
triplesa
a
b
Two-point distance (cM)
c
a-b
b-c
a-c
Three-point
distance (cM)
a-c
Frequency
of triple
(%)
Note: Numbers in parentheses are standard deviation.
"Markers: 1 , b18-0.7; 2, b20-1.15; 3, g16-0.6; 4 , c19-0.54; 5 , e16-0.88; 6 , 108-0.8; 7 , 111-0.5
Fig. 4. Relationship between number of loci and genome
length estimates. Error bars are standard deviations based
on 100 bootstrap samples.
Number of loci
they concluded that the dominant expression of RAPD
markers caused this (Kesseli et al. 1994), although this
should be less important in a backcross population since
there are only two genotypes. The efficiency in ordering
RAPD markers can be increased significantly by introducing codominant anchor markers into the linkage groups
(Kesseli et al. 1994).
The nonlinearity of RAPD linkage groups detected here
indicates that mistyping can be significant for RAPD markers (Fig. 3). We found that ambiguity of genotyping occurs
most often with incomplete amplification owing to template
DNA degradation. This problem can be avoided by aliquoting DNA samples into a few tubes to reduce repeated thawing and freezing. Alternatively, fresh DNA can be re-isolated
from leaf samples previously frozen in liquid nitrogen and
stored at -80°C. However, the rate of mistyping in RAPD
markers can only be determined when other types of markers not prone to mistyping (e.g., RFLPs or microsatellites)
are available in the same mapping population. A comparison of RAPDs and RFLPs in lettuce showed that the
chance of mistyping RAPDs is similar to the chance of
mistyping RFLPs (Kesseli et al. 1994), indicating that
RAPDs are reliable molecular markers in genetic mapping
applications, particularly for studies with wild species.
In our map, the occurrence of some loose linkages and
unlinked markers indicates that significant portions of the
genome are not covered by our map. This can be due to
either nonrandom distribution of markers or to insufficient
numbers of markers used. It is hard to predict how many
more markers would be needed to coalesce these linkage
groups and unlinked loci into the 14 chromosomal linkage
groups. In lettuce, Kesseli et al. (1994) found that nearly
doubling the number of markers did not reduce the number
of linkage groups. Bulk segregant analysis methods
(Michelmore et al. 1991) can be employed to efficiently
increase the marker density in a specific region (Michelmore
et al. 1991; Reiter et al. 1992; Kesseli et al. 1994), particulary about the ends of linkage groups, in order to coalesce
the linkage map.
Lin and Ritland
The estimated genome length of Mimulus (n = 14) was
2474 k 35 cM, which is among the largest so far recorded
for plants. By contrast, some other estimated genome
lengths are maritime pine (haploid chromosome number
n = 12), 2000 c M (Gerber and Rodophe 1994); lettuce
(n = 9), 1950 cM (Kesseli et al. 1994); sugarcane (2n =
64), 2550 c M (Al-Janabi et al. 1993); and E. grandis,
1620 cM (Grattapaglia and Sederoff 1994). Our estimate of
the Mimulus genome length is probably slightly inflated
owing to violations of the assumption of random distribution
of markers (Chakravarti et al. 1991; Kesseli et al. 1994).
Linkage maps with moderate marker density, such as
the one presented here, have applications in several areas
of evolutionary biology. One such application is the examination of the mode of gene action of QTLs for quantitative
traits of evolutionary significance. In many cases, models
that require only flanking markers are available to estimate
the additive, dominant, and epistatic effects of QTLs (Haley
and Knott 1992; Martinez and Curnow 1992; MorenoGonzalez 1992). A linkage map with an average marker
distance of 25 cM or less contains several such flanking
markers. We are currently building models using flanking
dominant markers (RAPDs) to estimate .the additive, dominant, and epistatic effects of QTLs that contribute to the
evolution of inbreeding.
Another such application is the examination of QTLs
for quantitative traits of interest. The presence of QTLs
and their individual contribution (large vs. small effect)
to genetic variation of the quantitative traits can be determined by either interval mapping (for which the minimum
requirement is the existence of flanking markers) or onemarker methods. The results given by these two classes
of methods have been found to differ little in either theoretical (e.g., Darvasi et al. 1993) or empirical studies (e.g.,
Doebley and Stec 1991, 1993; Stuber et al. 1992). Other
applications include examining selected linkage groups
for evidence of introgression and comparing recombination
rates between congeneric species using selected pairs of
marker loci.
Finally, we note that studies in crop species provide
only indirect information about evolutionary changes in
nature. The domestication process causes many changes
in the genetic composition, the nature of selection, and
the environment of the species. Proper understanding of
evolutionary processes in nature requires detailed studies of
wild plant populations, including construction of genetic
linkage maps. To our knowledge, linkage maps of wild
plants have been used in only two species: to examine the
genetic basis of morphological differences in Microseris
(Vlot et al. 1992; van Houten et al. 1994; Hombergen and
Bachmann 1995) and to study speciation processes of
Helianthus anomalus (Rieseberg et al. 1993). The ability to
rapidly generate a linkage map using RAPD markers will
greatly facilitate evolutionary studies in other wild species,
such as Mimulus.
Acknowledgements
We thank D. Schoen, J. Anderson, Y.-B. Fu, J. Shore,
J. Dole, S.C.H Barrett, and an anonymous reviewer for
comments on an early version of the manuscript. This work
was supported by operating grants from the Natural Sciences
and Engineering Research Council (NSERC) of Canada to
K.R. and NSERC Postgraduate Scholarships to J.Z.L.
References
Al-Janabi, S.M., Honeycutt, R.J., McClelland, M., and
Sobral, B.W.S. 1993. A genetic linkage map of Saccharum
spontaneum L. 'SES 208'. Genetics, 134: 1249-1260.
Chakravarti, A., Lasher, L.K., and Reeder, J.E. 1991. A maximum
likelihood method for estimating genome length using genetic
linkage data. Genetics, 128: 175-1 82.
Charlesworth, B. 1992. Evolutionary rates in partially fertilizing
species. Am. Nat. 140: 126-148.
Crow, J.F. 1990. Mapping functions. Genetics, 125: 669-67 1.
Darvasi, A., Weinreb, A., Minke, V., Weller, J.I., and Soller, M.
1993. Detecting marker-QTL linkage and estimating QTL gene
effect and map location using a saturated genetic map.
Genetics, 134: 943-95 1.
Doebley, J., and Stec, A. 1991. Genetic analysis of the morphological differences between maize and teosinte. Genetics,
129: 285-295.
Doebley, J., and Stec, A. 1993. Inheritance of the morphological
differences between maize and teosinte: comparison of
results for two F, populations. Genetics, 134: 559-570.
Doyle, J.J., and Doyle, J.L. 1987. A rapid DNA isolation procedure for small amounts of fresh leaf tissue. Phytochem.
Bull. 19: 11-15.
Fenster, C.B., and Ritland, K. 1994. The quantitative genetics
of mating system divergence in the yellow monkeyflower
species complex. Heredity, 73: 422-435.
Gerber, S., and Rodophe, F. 1994. An estimation of the genome
length of maritime pine (Pinus pinaster Ait.). Theor. Appl.
Genet. 88: 289-292.
Grattapaglia, D., and Sederoff, R. 1994. Genetic linkage maps
of Eucalyptus grandis and Eucalyptus urophylla using a
pseudo-testcross: mapping strategy and RAPD markers.
Genetics, 137: 1 1 21-1 137.
Haldane, J.B.S. 1919. The combination of linkage values, and
the calculation of distance between the loci of linked factors.
J. Genet. 8: 299-309.
Haley, C.S., and Knott, S.A. 1992. A simple regression method
for mapping quantitative trait loci in line crosses using
flanking markers. Heredity, 69: 3 15-324.
Holsinger, K.E. 1988. Inbreeding depression doesn't matter:
the genetic basis of mating-system evolution. Evolution,
42: 1235- 1244.
Holtsford, T.P., and Ellstrand, N.C. 1992. Genetic and environmental variation in floral traits affecting outcrossing rate
in Clarkia tembloriensis (Onagraceae). Evolution, 46:
2 16-225.
Homberger, E.-J., and Bachmann, K. 1995. RAPD mapping of
three QTLs determining trichome formation in Microseris
hybrid H27 (Asteraceae: Lactuceae). Theor. Appl. Genet.
90: 853-858.
Kesseli, R.V., Paran, I., and Michelmore, R.W. 1994. Analysis
of a detailed genetic linkage map of Lactuca sativa (Lettuce)
constructed from RFLP and RAPD markers. Genetics, 136:
1435- 1446.
Kosambi, D.D. 1944. The estimation of map distance from
recombination values. Ann. Eugen. 12: 172-175.
Lander, E.S., and Botstein, D. 1989. Mapping Mendelian factors
underlying quantitative traits using RFLP linkage maps.
Genetics, 121: 185-199.
Lander, E.S. Green, P., Abrahamson, J., Barlow, A., Daly, M.J.,
Lincoln, S.E., and Newburg, L. 1987. Mapmaker: an interactive computer package for constructing primary genetic
Genome, Vol. 39, 1996
linkage maps for experimental and natural populations.
Genomics, 1: 174-181.
Latta, R., and Ritland, K. 1993. Models for the evolution of
selfing under alternative modes of inheritance. Heredity,
71: 1-10.
Lincoln, S., Daly, M., and Lander, E. 1992. Constructing genetic
maps with MAPMAKERE 3.0. Whitehead Institute Technical
Report. 3rd ed.
Macnair, M.R., and Cumbes, Q.J. 1989. The genetic architecture
of interspecific variation in Mimulus. Genetics, 122: 21 1-222.
Martinez, O., and Curnow, R.N. 1992. Estimating the locations
and the sizes of the effects of quantitative trait loci using
flanking markers. Theor. Appl. Genet. 85: 480-488.
Michelmore, R.W., Paran, I., and Kesseli, R.V. 1991. Identification of markers linked to disease resistance genes by
bulked segregant analysis: a rapid method to detect markers
in specific genomic regions by using segregating populations.
Proc. Natl. Acad. Sci. U.S.A. 88: 9828-9832.
Mitchell-Olds, T. 1995. The molecular basis of quantitative
genetic variation in natural populations. Trends Ecol. Evol.
10: 324-328.
Moreno-Gonzalez, J. 1992. Genetic models to estimate additive
and non-additive effects of marker-associated QTL using
multiple regression techniques. Theor. Appl. Genet. 85:
435-444.
Penner, G.A., Bush, A., Wise, R., Kim, W., Domier, L.,
Kasha, K., Laroche, A., Scoles, G., Molner, S.J., and
Fedak, G. 1993. Reproducibility of random amplified
polymorphic DNA (RAPD) analysis among laboratories.
PCR Meth. Appl. 2: 341-345.
Rafalski, J.A., and Tingy, S.V. 1993. Genetic diagnostic in
plant breeding: RAPDs, microsatellites and machines. Trends
Genet. 9: 275-280.
Reiter, R.S., Williams, J.G.K., Feldmann, K.A., Rafalski, J.A.,
Tingey, S.V., and Scolnik, P.A. 1992. Global and local
genome mapping in Arabidopsis thaliana by using recombinant inbred lines and random amplified polymorphic
DNAs. Proc. Natl. Acad. Sci. U.S.A. 89: 1477-1481.
Rieseberg, L.H., Choi, H., Chan, R., and Spore, C. 1993.
Genomic map of a diploid hybrid species. Heredity, 70:
285-293.
Ritland, C., and Ritland, K. 1989. Variation of sex allocation
among eight species of the Mimulus guttatus complex. Am.
J. Bot. 76: 1731-1739.
Ritland, K., and Ganders, F.R. 1987. Crossability of Mimulus
guttatus in relation to components of gene fixation. Evolution,
41: 772-786.
Shore, J.S., and Barrett, S.C.H. 1990. Quantitative genetics of
floral characters in homostylous Turnera ulmifolia var.
angustifolia Willd. (Turneraceae). Heredity, 64: 105-1 12.
Stuber, C.W., Lincoln, S.E., Wolff, D.W., Helentjaris, T., and
Lander, E.S. 1992. Identification of genetic factors contributing to heterosis in a hybrid from two elite maize inbred
lines using molecular markers. Genetics, 132: 823-839.
van Houten, W., van Raamsdonk, L., and Bachmann, K. 1994.
Intraspecific evolution of Microseris pygmaea (Asteraceae,
Lactuceae) analyzed by cosegregation of phenotypic characters (QTLs) and molecular markers (RAPDs). Plant Syst.
Evol. 190: 49-67.
Vickery, R.K. 1978. Case studies in the evalution of species
complexes in Mimulus. Evol. Biol. 11: 405-507.
Vlot, E.C., van Houten, W.H.J., Mauthe, S., and Bachmann, K.
1992. Genetic and nongenetic factors influencing deviations
from five pappus parts in a hybrid between Microseris
douglasii and M. bigelovii (Asteraceae, Lactuceae). Int. J.
Plant Sci. 53: 89-97.
Weeden, N.F., Timmerman, G.M., Hemmat, M., Kneen, B.E.,
and Lodhi, M.A. 1992. Inheritance and reliability of RAPD
markers. In Applications of RAPD technology to plant breeding. Joint Plant Breeding Symposia Series. Crop Science
Society of America - American Society for Horticultural
Science - American Genetic Association, Minneapolis,
Minn. pp. 12-17.
Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A.
and Tingey, S.V. 1990. DNA polymorphism amplified by
arbitrary primers are useful as genetic markers. Nucleic
Acids Res. 18: 6531-6535.
Williams, J.G.K., Hanafey, M.K., Rafalski, J.A., and Tingey, S.V.
1993. Genetic analysis using random amplified polymorphic
DNA markers. Methods Enzymol. 218: 704-740.