Download Marker-based inferences about fecundity genes contributing

Marker-based inferences about fecundity genes contributing to inbreeding
depression in Mimulus guttatus
YONC-BIFU' A N D KERMIT
RITLAND
Department of Botany, University of Toronto, Toronto, ON M5S 3B2, Canada
Corresponding Editor: R.S. Singh
Received January 1 1, 1994
Accepted July 28, 1994
K. 1994. Marker-based inferences about fecundity genes contributing to inbreeding
Fu, Y.-B., and RITLAND,
depression in Mimulus guttatus. Genome, 37: 1005- 10 10.
Eight unlinked isozyme loci were used as genetic markers to characterize fecundity genes contributing to inbreeding
depression in two selfed progeny arrays of Mimulus guttatus. Five fecundity traits were measured. Six of eight
marked chromosomal segments were significantly associated with the expression of these traits. The number of
genes detected for five traits in two progeny arrays varied, with an average of 2.8 genes per trait. Individual segments
explained 1.44-9.29%, and together accounted for 3.85-1 1.32%, of phenotypic variation. Of 20 significant
associations, 10 could be interpreted as exhibiting partial dominance, 7 overdominance, 3 partial recessivity, and 0 underdominance. Significant pairwise epistasis was rare. The results of this study suggest that inbreeding depression is caused
by many deleterious genes of relatively small, partially dominant effects.
Key words: linkage, isozymes, QTLs, inbreeding depression, Mimulus guttatus.
Fu, Y.-B., et R I T L A N DK., 1994. Marker-based inferences about fecundity genes contributing to inbreeding
depression in Mimulus guttatus. Genome, 37 : 1005- 10 10.
Huit loci isoenzymatiques non lies ont kt6 employes comme marqueurs ginktiques afin de caractiriser les gknes
de fkconditi qui contribuent B la perte de vigueur observke chez deux populations autoficondies de Mimulus
gutratus. Cinq traits de fkconditi ont kt6 mesuris. Six des huit segments chromosomiques marquis montraient une
association significative avec l'expression de ces traits. Bien que variable, le nombre de gknes ditectis pour les
cinq traits chez les deux populations itait en moyenne de 2,8 gknes par trait. Pris individuellement, ces segments contribuaient de 1,44 B 9,29% de la variation phknotypique et, pris globalement, de 3,85 B 11,32% de la variation.
Des 20 associations significatives, dix demontraient une dominance partielle, sept une surdominance, trois ktaient
partiellement ricessives et aucune ne montrait de sousdominance. Lorsque comparis deux a deux, ces segments
ont rarement montrk de l'kpistasie. Les resultats de cette etude suggkrent que la perte de vigueur associke a la consanguiniti est le produit de plusieurs gknes dilitkres aux effets partiellement dominants et de faible envergure.
Mots cle's : linkage, isoenzymes, QTLs, consanguiniti, Mimulus guttatus.
[Traduit par la redaction]
Introduction
Inbreeding depression, the reduced fitness of progeny produced by inbreeding, has been extensively studied because
of its importance in many areas of biology (Darwin 1876;
Charlesworth and Charlesworth 1987). Many attempts have
also been made to explain this phenomenon and its inverse,
heterosis ( e . g . , see Sprague 1983; Charlesworth and
Charlesworth 1987; Srivastava 1991 ; Crow 1993). Among the
explanations proposed for inbreeding depression, the dominance and overdominance hypotheses are predominant
because they are experimentally testable. The dominance
hypothesis proposes that inbreeding depression is due to
expression of recessive or nearly recessive deleterious genes
in homozygotes, whereas the overdominance hypothesis
states that inbreeding depression is caused by the loss of
heterozygosity upon inbreeding because of heterozygote
superiority at specific loci (Crow 1952). The empirical evidence collected over a century seems to favor the dominance
hypothesis, although overdominance is not fully rejected
(Wright 1977; Simmons and Crow 1977; Charlesworth and
Charlesworth 1987; Barrett and Charlesworth 199 1 ; Stuber
et al. 1992; Crow 1993; Mitton 1993). However, most empirical data come from quantitative genetics studies, which
'present address: Biotechnology Laboratory, University of
British Columbia, #237-6174 University Blvd., Vancouver,
BC V6T 123, Canada.
Pr~ntedIn Canada 1 l m p r ~ m eau Canada
usually characterize only the average effects of genes in the
entire genome, but not those of individual genes (Wright
1977; Mather and Jinks 1982). Thus the molecular genetic
basis of inbreeding depression is still not well understood.
Inbreeding depression is a complex quantitative character,
presumably controlled by many deleterious genes of different magnitudes of effect (Lande and Schemske 1985;
Charlesworth et al. 1990). Recent marker-based studies have
been successful in characterizing individual genes (or specific
marker-linked chromosome regions) affecting quantitative
characters (QTLs) in cultivated plants (e.g., Paterson et al.
1988; Stuber et al. 1992). Studies using codominant markers
in F, populations have further revealed a wide range of
marker-associated gene actions, from additivity to overdominance (e.g., Edwards et al. 1987). These marker-based
methods raise the prospect that the genetic basis of inbreeding
depression in plants can be studied at a resolution not attainable by classical quantitative genetics methods. However,
inbreeding depression is difficult to measure directly (Lewontin
1974) and is usually expressed at different stages of the life
cycle (Schoen 1983). Different analytic methods are required
to characterize genes affecting various fitness-related components such as viability and fecundity.
To study viability genes, we recently developed a markerbased, graphical method for inferring the relative importance
of different modes of viability gene expression (Fu and
Ritland 1994). Applying this method to data of 2577 selfed
1006
GENOME, VOL. 37, 1994
500 plants in population
&
Parent 1
&
8
t
Maximization of
15 isozyme loci
for homozygotes
Parent 2
&
t
for homozygotes
&
t
for heterozygotes
again to select the most heterozygous plants. One F, plant in
each cross, heterozygous for most of marker loci, was selfed to
produce the F, progeny. For clarity in reporting, these two selfed
progeny arrays are denoted as F,, and F,,, respectively.
A total of 1080 F, seeds (540 per cross) were sown in 15 trays
in a 50:50 mixture of ProMix and Pearlite. In each 72-cell tray,
36 F, seeds from each cross were randomly chosen and each
sown at random (one seed per cell with ca. 1 in.3). The trays
were randomly placed in a growth room (2.5 X 3 m2) and watered
once a week. All F, plants were grown at 18"C/16 h light and
10°C/8 h dark with relative humidity of 80% for 2 months from
sowing on April 21, 199 1. The germination rates and seedling
mortality rates were 82.4 and 5.4% for F,, and 80.2 and 4.4% for
F2b.
(selfed)
(Phenotyping F2 for five tramits)
(Genotyping F2 for eight loci)
FIG. 1. Diagram of experimental procedures for constructing
single crosses in M. guttatus.
progeny of 31 plants of Mimulus guttatus, we found that,
in the chromosomal segments identified by isozyme markers,
partial dominance played a predominant role in inbreeding
depression (Fu and Ritland 1994). Less attention has been
paid to fecundity genes in wild plants. Fecundity genes
should play an important role in expression of inbreeding
depression since strong inbreeding depression usually occurs
in later stages of the life cycle in many plants (e.g., see
Schoen 1983 and Charlesworth and Charlesworth 1987).
In this paper, we report results of a marker-based study
of fecundity genes contributing to inbreeding depression in
t w o selfed p r o g e n y a r r a y s o f Mimulus g u t t a t u s D C
(Scrophulariaceae), the common yellow monkeyflower. This
plant (2n = 28) occurs throughout western North America in
moist areas such as wet meadows, streambanks, or roadsides, and has many bee-pollinated, self-compatible flowers
(Vickery 1978). This species exhibits a wide range of selfing rates in populations (with an average of 30-40%) and
significant inbreeding depression in selfed progeny, with
an average of 40-60% (Ritland and Ganders 1987; Ritland
1990; Dole and Ritland 1993; Willis 1993; Latta and Ritland
1994). This study was conducted using isozyme markers to
infer (i) the minimum number of genes affecting five fecundity traits, (ii) the magnitude of the gene effects, (iii) the
mode of the gene action, and (iv) pairwise epistasis of genes.
Materials and methods
Plant materials and trait measurements
Experimental procedures for constructing single crosses in
M. guttatus are given in Fig. 1. Seeds and corollas were collected
in May 1989 from each of 500 plants along a linear transect
through a population near Hough Springs, Lake Co., California.
In this population, the selfing rate and inbreeding depression
were inferred to be 0.29 and 0.06, respectively (Table 1 in Ritland
1990). On the basis of an isozyme survey of adult plants,
95 plants were selected. Their seeds were sown and several
progeny (5-10) from each plant genotyped. Alternative homozygous plants were then selected for controlled crosses. Two crosses
were made in Fall 1990 and their F, progeny were genotyped
Fecundity traits used in this study were (i) relative growth
rate, RT; (ii) number of nodes in main stem, NN; (iii) total number of flowers, NF; (iv) height, HT; and (v) aboveground dry
weight, WT. During the experiment, the dates of both opening the
first leaf and the first flower were recorded for each F, plant.
When the experiment was terminated, NN, NF, and HT were
scored and all F, plants were cut at the soil level for later measurement of WT. RT was determined by dividing 60 (days) by
days from opening of the first leaf to the first flower. These
traits showed significant levels of inbreeding depression in this
experiment, ranging from 8% for NN to 25% for NF (data not
shown).
Electrophoresis
While 15 isozyme loci could clearly be scored in this study,
only eight loci were heterozygous in the F,s. These loci were
aconitase 2 (Aco), diaphorase (Dia), esterase (Est), glutamicoxaloacetate transaminase (Got2), malic enzyme (Me),
6-phosphoglucose dehydrogenase 1 and 2 (6Pgdl and 6Pgd2), and
triosephosphate isomerase (Tpi). Seven loci (Dia, Est, Got2, Me,
6Pgd1, 6Pgd2, and Tpi) were polymorphic in F,, and five loci
(Dia, Est, 6Pgd2, Aco, and Tpi) in F,,. Fresh corollas were collected for electrophoresis using procedures described in Ritland
and Ganders (1987). Linkage analyses of these isozyme data
demonstrated that these eight loci are unlinked (data not shown)
and thus individually represent specific chromosomal segments.
Statistical analysis
An analysis of variance with the fitness data was conducted
using the SAS GLM procedure (SAS Institute 1988). Differences
among trays were significant and were removed for subsequent
analyses. For each trait, a test for normality was conducted using
the Shapiro-Wilk's statistic (Royston 1982). For the traits with
highly significant skewness, logarithm transformations were
made to ensure the normality.
Associations of marked chromosomal segments with QTLs
were examined using the SAS GLM procedure. A one-way
ANOVA (one trait on one locus with three genotypic classes in
F, progeny) was computed for each segment. A significant
F-test indicated segregation of genotypes of at least one QTL
linked to the segment, and its R' value gave the proportion of total
phenotypic variance explained by this segment, i.e., the magnitude
of gene effect. The cumulative gene effect associated with all
the significant segments for a trait was determined using the
SAS GLM procedure with main-effects models. The resulting
multiple R2 value gave the proportion of phenotypic variance
by these significant segments.
Following Edwards et al. (1987), the additive (a) and dominant
(d) effects were calculated for each QTL as follows:
a = 12[f,2%2
-fIl~I111
d = 2[fl,xI, - (f22X22 + fllX11)I
where
f is the observed frequency of each marker genotype and
x is the observed phenotypic mean of a trait, conditioned on
marker genotype. The ratio of these effects (d/a) was used to
estimate the degree of dominance for the segment. For the
1007
FU A N D RITLAND
TABLE1. Significant associations between chromosomal segments and loci affecting five traits in two
selfed progeny arrays of M . guttatus
F2 progeny
F2,
Marked
chromosomal
segment
Frequency of
marker genotypes
AIAl
AlA2
Observed means for
marker genotypes
A2A2
Trait
AIAl
A,A2
A2A2
F-test
Dia
Got2
Me
Tpi
Dia
6Pgd2
Aco
Tpi
TABLE2. Estimates of gene effects and d/a ratios of QTLs affecting five traits
in two selfed progeny arrays of M . guttatus
F2 progeny
Trait
Marked
chromosomal
segment
Gene effect"
Single
Multiple
Gene action
d/a
ode^
Dia
Tpi
Me
6Pgd2
Tpi
6Pgd2
Tpi
Me
6Pgd2
Tpi
Got2
Me
6Pgd2
Tpi
Dia
Tpi
Aco
6Pgd2
6Pgd2
6Pgd2
"The magnitude of gene effect is determined by the proportion of the total phenotypic variance explained
by single and all the significant chromosomal segments, respectively.
he mode interpreted is based on the observed d/a ratio. The observed d/a ratios for four modes are
as follows: d/a > 1 , OD (overdominance); 1 2 d/a > 0, PD (partial dominance); 0 2 d/a > - 1 , PR (partial
recessivity); and d/u I - I , UD (underdominance).
interpretation of gene action, these estimates were classified into
four types: d/a > 1 for overdominance (OD), 1 2 d/a > 0 for
partial dominance (PD), 0 2 d/a > - 1 for partial recessivity
(PR), and d/a I - 1 for underdominance (UD).
Pairwise epistasis (i.e., interactions between two marked
chromosomal segments in their associated effects on each trait)
was tested using a two-way ANOVA on each trait in both F2s,
with the two marker loci as main effects.
GENOME, VOL. 37. 1994
T A B L E3. Significant pairwise epistasis between
chromosomal segments for five traits in two selfed
progeny arrays of M. guttatus
Nonsignificant association
F2 progeny
Trait
Pair of segments
R' X 100"
F2n
RT
NF
WT
NF
WT
Got2 X TpiT*
MeX6Pgd2?*
Dia X 6 P g d l *
DiaX6Pgd2?**
DiaXEst*
Dia X 6 P g d 2 t **
2.47
2.67
3.90
4.4 1
4.32
3.84
F,,
NOTE: *, significant interaction at the 0.05 level; t. significant main
effect at the 0.05 level; **, significant interaction at the 0.01 level.
"R' represents the proportion of the phenotypic variation explained only
by the interaction term.
,
,
O
!
I
I
I
1
Dominant/additive ratio
FIG. 2. Frequency distribution of 60 observed d/a ratios.
Results and discussion
Number of QTLs detected
All the traits studied showed continuous variation, typical
of quantitative traits, and most of them exhibited approximately normal distributions (data not shown). Significant
associations of six chromosomal segments with traits examined in two F,s are given in Table 1. Of the 6 0 possible
marker-trait combinations in two F2s, 20 (33%) showed significant associations, of which 11 were significant at the
1.0% level or lower (note that three of these significant
associations, 5% of 60, may be due to chance alone). Among
the six segments, three (Dia, Got2, and Aco) were significantly associated with only one trait and three (Me, 6Pgd2,
and Tpi) with more than one trait in the two F,s. Table 1
also shows the significant associations with the traits in
each of the F,s: 14 for F,, with the seven loci employed
(i.e., 4 0 % of the associations tested) and six for F,, with
the five loci (i.e., 24% of the associations tested).
The number of QTLs detected for RT, NN, NF, HT, and
W T were 2, 3, 2, 3, and 4, respectively, with an average of
2.8 QTLs per trait in the two F,s (Table 2). However, the
number of QTLs detected for each trait (except for RT) varied between the two F2s (Table 2). For WT, for example,
four QTLs were detected in F,, and one in F,,.
These results imply that, as expected, a large number of
fecundity genes are involved in the expression of inbreeding
depression. However, the number of QTLs found for each
trait here should be considered a minimum estimate. We
examined only a small portion of the whole genome (1 1.2%
based on calculations following Bishop et al. 1983) so that
genes in the other regions of the genome were not included.
Also, even in these marked regions, it is quite possible that
genes of small effect and (or) multiple genes were undetected.
Phenotypic variation explained by QTLs
T h e phenotypic variation of a trait explained by single
chromosomal segments was small, ranging from 1.44 for
HT to 9.29 for RT in R2 X 100 in the two F2s, and differing
between the two F,s (Table 2). For example, the value of
R2 X 100 owing to Tpi for RT was 9.29 in F2a and 2.06 in
F,, (Table 2). For the segments (Me, 6Pgd2, and Tpi) that
were significantly associated with the expression of more
than one trait, the proportions of phenotypic variation that
they explained varied for different traits, even in the F,
progeny of one cross (Table 2). For the segment marked by
Tpi in F,,, for instance, the values of R' X 100 were 9.29 for
RT and 2.83 for NN (Table 2).
Phenotypic variation explained by several significant segments was relatively large, ranging from 3.85 for RT to
11.32 f o r N F (in R' X 100) in both F2s. It a l s o varied
between the two F2s (Table 2). For RT, the values of total
R2 X 100 owing to both Dia and Tpi were 11.22 in Fla and
3.85 in F,, (Table 2).
Gene effects of the detected QTLs influencing the five
fecundity traits are relatively small, because individual significant chromosomal segments explained a small proportion
of total phenotypic variation. This seems to suggest that
fecundity genes contributing to inbreeding depression have
small effects. However, the proportion of phenotypic variation
explained by all significant segments can be large ( u p to
11.32%). Considering that about 11.2% of the M. guttatus
g e n o m e was marked by the seven markers in F,,, these
results suggest that a saturated marker m a p may explain
100% of the phenotypic variance, or that these traits have
100% heritabilities. This could be due to fortuitous sampling
of QTLs o r to nonadditive genetic and (or) genotype-byenvironmental variances (Edwards et a]. 1987).
Gene action of QTLs
The estimates of d/a for the significant associations are
given in Table 2. These estimates ranged from -0.41 to
7.27 for the five traits in both F,s. Of 2 0 significant associations, 10 could be interpreted as showing partial dominance, 7 overdominance, 3 partial recessivity, and 0 underdominance (Table 2). To examine the distribution of d/a
ratios, all the observed d/a ratios including nonsignificant
associations in the two F,s are plotted in Fig. 2. It becomes
clear that the d/a ratios f o r the significant associations
showed a bimodal distribution, consisting of either partial
dominance and partial recessivity, or overdominance.
Examination of the m o d e s of g e n e action within a n d
between t h e F,s s e e m s to indicate s o m e heterogeneity
(Table 2). For example, while partial dominance was observed
for almost all the associations with the segment by Tpi, the
modes for the associations with the segment by 6Pgd2 were
different between the F,s. Overdominance was observed for
all the associations with the segments by Dia, Me, Got2,
FU A N D RITLAND
and Aco, but only the associations with the segment by Dia
were consistently found in both F,s.
The distribution of d a values in Fig. 2 suggests that partial
dominance is relatively predominant over the other modes in
the expression of the traits in the selfed progeny. This is
particularly true because these overdominant loci may include
those of pseudooverdominance, i.e., the complementary
action of linked recessive or nearly recessive loci in repulsion
(Crow 1952; Charlesworth 1991). One possible way to determine true overdominance is to examine these loci in the F,
progeny of many crosses. If the overdominant effects were
due to true overdominant loci segregating in the populations
studied, one could find these loci in most crosses, or at least
more often than partially dominant loci. Unfortunately, this
pattern cannot be determined from only two crosses, but at
least, less agreement for the cases of overdominance than
those of partial dominance was observed between these two
crosses (Table 2). Thus while the number of overdominant
loci should be fewer than estimated, the number of partially
dominant loci is at least as many as observed.
However, the finding of the predominance of partially
dominant loci, such as those found for viability genes (Fu and
Ritland 1994), is not consistent with either the dominance or
overdominance hypotheses proposed to account for inbreeding
depression. The dominance hypothesis described above
strictly refers to only the genes of complete or partial recessivity and does not include those of complete or partial
dominance (Crow 1993), because neither complete nor partial
dominance contributes to reduction in fitness of selfed progeny (i.e., the mean of the homozygote fitnesses exceeds that
of the heterozygote). The reason for this unexpected finding
remains unknown. Further study is certainly needed for
correctly interpreting these partially dominant loci.
The advantage of such an inference about gene actions
over the quantitative genetics methods lies mainly in the
ability to estimate d/a ratios at specific loci or at least specific chromosomal segments. With these estimates, one could
distinguish between the modes of selection operating at specific loci, allowing more precise distinction between the
dominance and overdominance hypotheses of inbreeding
depression. However, these estimates of d/a could be biased
either downward by loose linkage or upward by linkage of
multiple QTLs. Linked markers are required to resolve this
bias.
Pairwise epistasis between chromosomal segments
Epistatic interaction was tested between pairs of marked
chromosomal segments for each trait. Of 155 pairs for the
five traits in the two F,s, only six (3.9%) were significant,
less than would be expected by chance (Table 3). Of 31 pairs
for each trait, one significant interaction was found for RT,
two for NF, and three for WT; no significant interactions
were observed for the traits NN and HT. Also, all these
interactions explained small proportions of phenotypic
variation, ranging from 2.47 to 4.32% (Table 3).
These results provide little evidence that pairwise epistasis
is an important factor in the expression of inbreeding depression, because few significant interactions were detected and
little variation was explained by these interactions. However,
these results do not rule out the importance of multiple gene
interactions in the expression of inbreeding depression. We
have performed analyses of multiple gene interactions for
these data and found that multiple gene interactions in deleterious homozygotes are largely multiplicative in nature,
1009
although some synergistic interactions could not be fully
ruled out (Y.-B. Fu and K. Ritland, in preparation).
Marker-based churucterization of genes influencing
inbreeding depression
This study represents the first use of genetic markers to
infer the nature of gene action at specific chromosome
regions affecting fecundity in a wild plant. The results of
this study clearly demonstrate that fecundity genes contributing to inbreeding depression can be characterized in
terms of gene effects, gene actions, and gene interactions
in M. guttatus. Thus this approach seems to be suited for
characterizing fecundity genes contributing to inbreeding
depression in wild plants.
One obstacle to the more widespread use of this approach
to investigate the genetic basis of inbreeding depression is
the limited number of genetic markers available for most
wild plants. However, recent development of molecular technology has provided researchers with numerous molecular
genetic markers such RFLPs, RAPDs, and microsatellites
(Botstein et al. 1980; Williams et al. 1990; Herne et al.
1992). These new types of markers supersede in many ways
the limitations of isozymes and thus make this approach
feasible for studying wild plants. As more easily assayed
markers are used, more families are analyzed in different
environments, and specific experimental designs are combined, some trends may become apparent in selfed progeny.
These include areas of the genome that consistently have
an effect on a particular trait, types of gene action generally exhibited for fecundity traits, and patterns of gene interaction. Studies of this nature will allow better understanding
of the genetic basis of inbreeding depression in later stages
of the life cycle in wild plants.
Acknowledgments
We thank Lloyd Smith for technical assistance and Spencer
Barrett, Scott Reinhart, Bob Latta, and Neal Straus for their
comments on this project. We also thank Jeffery Dole and
Sean Graham for their critical reading on the early version
of this manuscript and Deborah Charlesworth for her helpful
comments that added greatly to the final version. This work
was supported by grants from the Natural Sciences and
Engineering Research Council of Canada to K.R. and a
University of Toronto Open Doctoral Fellowship and a Mary
H. Beatly Fellowship to Y.B.F.
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