The Journal of Heredity 2002:93(3)
© 2002 The American Genetic Association 93:217-221
Brief Communication |
Polymorphic Microsatellites in Antirrhinum (Scrophulariaceae), a Genus With Low Levels of Nuclear Sequence Variability
From the Institut für Tierzucht und Genetik, Veterinärmedizinische Universität Wien, A-1210 Wien, Austria (Zwettler and Schlötterer) and the Instituto de Biologia Molecular e Cellular, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal (Vieira).
Address correspondence to Christian Schlötterer at the address above or e-mail: christian.schloetterer{at}vu-wien.ac.at.
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Abstract |
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In Antirrhinum, reproductive systems range from self-compatible to self-incompatible, but the actual outcrossing rates of self-compatible populations are not known. Thus the extent to which levels of variability and inbreeding differ among Antirrhinum populations is not known. In order to address this issue we isolated nine Antirrhinum nuclear microsatellite loci. In contrast to several nuclear genes that show low levels of sequence variation, six of the microsatellite loci indicate high levels of variability within and between Antirrhinum species. The highly self-compatible Antirrhinum majus ssp. cirrhigerum population has high levels of variability and no significant deviation from Hardy–Weinberg equilibrium, suggesting substantial rates of outcrossing.
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Introduction |
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The mating system in plants is determined by many factors, including features of the reproductive system, such as self-incompatibility mechanisms and protandry (i.e., the amount of time separating anther dehiscence and the start of stigma exertion) in hermaphroditic species, pollinator behavior, selective abortion by maternal regulation of seed quality, flowering phenology (i.e., variation in floral display and structure), and population density (Shaanker et al. 1988; Marshall and Folsom 1991). The mating system affects the distribution of genetic variability, both within and between populations. For several reasons, highly inbreeding populations are expected to have low levels of variability relative to closely related outcrossing populations.
Inbreeding reduces the effective population size (Pollak 1987) and lowers effective rates of recombination due to the rarity of heterozygous individuals. Reduced recombination is associated with an increased effect of adaptive gene substitutions on neutral variability at linked sites (i.e., hitchhiking; Maynard Smith and Haigh 1974) and an increased effect of selection against deleterious alleles on neutral variation at linked sites (i.e., background selection; Charlesworth et al. 1993). Both processes tend to reduce neutral variability (reviewed in Charlesworth and Charlesworth 1998). Also, polymorphisms maintained by overdominance in outcrossing populations tend to be lost under inbreeding (Charlesworth and Charlesworth 1995; Kimura and Ohta 1971). In addition to these nonneutral effects, population structure has also been suspected to affect inbreeders. When selfing species are more likely to occur in metapopulations with high rates of extinction, this will also contribute to lower levels of variability in selfing populations (Barton and Whitlock 1997; Wade and McCauley 1988).
These theoretical predictions have been verified to a large extent by allozyme data, which consistently show higher levels of within-population variability in outcrossing than in selfing populations (Brown 1979; Hamrick and Godt 1990, 1996; Schoen and Brown 1991). While sequence variation data are still scarce, the available reports show the expected pattern of reduced diversity in inbreeders (Awadalla and Ritland 1997; Dvorak et al. 1998; Liu et al. 1998, 1999; Stephan and Langley 1998; Savolainen et al. 2000).
Recently several populations and species of Antirrhinum were characterized for their percentage of autogamy and self-fertility, and large variation was observed (Vieira 2000). However, the actual outcrossing rate is not known for self-compatible populations. In a recent attempt to correlate sequence variability with mating system, nuclear genes of the cycloidea and fil1 gene families were sequenced (Vieira and Charlesworth 2001a; Vieira et al. 1999). The low levels of sequence polymorphism observed in these studies made it difficult to correlate sequence variation with reproductive system. Furthermore, recent gene duplications at these gene families (Vieira and Charlesworth 2001a,b; Vieira et al. 1999) preclude the inference of the phylogenetic relationship between the species of this genus. Currently the phylogenetic relationships were inferred by chloroplast DNA restriction fragment length polymorphisms (RFLPs) and allozymes for a small subset of four (A. siculum, A. latifolium, A. majus, and A. tortuosum) and three (A. molle, A. lopesianum, and A. microphyllum) species (Caputo et al. 1991–92; Mateu-Andrés 1999). Additional molecular markers are required for a more refined analysis of Antirrhinum.
Length variability at nuclear microsatellite loci is generated predominantly by DNA replication slippage, a mutation process independent of nucleotide base substitutions (Schlötterer 2000). Furthermore, microsatellite mutation rates are several orders of magnitude higher than base substitution rates, ranging from 10-2 to 10-6 (Schlötterer 2000). Even if Antirrhinum species have a reduced rate of base substitution in nuclear sequences, microsatellites are expected to display high levels of variability. Therefore microsatellites are anticipated to be suitable markers to address the effect of the breeding system on overall levels of genetic diversity. Nuclear microsatellite variability has already been used to address this issue in natural plant populations of Mimulus guttatus species complex (Awadalla and Ritland 1997; Kelly and Willis 1998), Arabidopsis thaliana (Todokoro et al. 1995), and Arabis petraea and Arabis lyrata (Treuren et al. 1997).
In this study we wanted to determine if microsatellites are informative in Antirrhinum or if they show low levels of variability similar to previously analyzed genes. Furthermore, we tested the usefulness of microsatellites to infer the genealogical relationship of three Antirrhinum species. Finally, we addressed the effect of the different reproductive systems in overall genetic diversity in three Antirrhinum species.
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Materials and Methods |
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Library Construction and Screening
Genomic DNA was isolated from plants grown from a commercial Antirrhinum seed mixture using a modified Cetyltrimethyl ammonium bromide (CTAB) extraction method described in Hauser et al. (1998). Microsatellite isolation followed general protocols described in Schlötterer (1998). In brief, 10 µg of genomic DNA were digested with HaeIII. Size selected fragments (0.5–1 kb) were cloned into an SmaI cut M13mp18 vector and transformed into competent XL-1 blue (Stratagene) cells. The library was plated at a density of 200–300 plaques per petri dish to avoid rescreening of positive clones. Clones carrying a microsatellite were identified by hybridization at 37°C with (GT)7G, (CA)7C, (TC)7T, and (AG)7A oligonucleotides, which were end-labeled with
-32P. Filters were washed three times in 5x SSC, 0.1% SDS at 37°C. Positive clones were identified by autoradiography and sequenced using the BigDye sequencing chemistry. Out of 14 clones containing a microsatellite, 9 were suitable to design polymerase chain reaction (PCR) primers (Table 1).
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DNA Extractions and Plant Material
Genomic DNA was extracted from leaves of individual plants collected in Portugal in the summers of 1997 and 1998 for two A. majus ssp. cirrhigerum (cirrhigerumGala, Figueira da foz; and cirrhigerumMuel, Marinha Grande; Vieira 2000; Vieira and Charlesworth 2001a), one A. graniticum (graniticumB, Bragança; Vieira and Charlesworth 2001a), and one A. molle lopesianum (molle, Bragança; Vieira 2000), using the modified CTAB extraction (Hauser et al. 1998). The reproductive system for these populations was determined in the glasshouse using 13, 17, 5, and 9 plants for each of these populations, respectively. For each plant, five flowers were allowed to self-pollinate without manipulation to estimate the percentage of autogamy. Five additional flowers were hand-pollinated with their own pollen to estimate the seed set as a measure of self-fertility. Based on these two parameters, the reproductive system of these populations could be described as largely self-compatible (80% and 78% of the pollinated flowers set seeds in cirrhigerumGala), largely self-incompatible (7% and 5% of the pollinated flowers in cirrhigerumMuel set seeds), and self-incompatible (none of the pollinated flowers set seeds in graniticumB and molle; Vieira 2000; Vieira and Charlesworth 2001a). Microsatellite variability was surveyed for these four populations. They were typed following standard protocols (Schlötterer 1998). In brief, end-labeled (
-32P) PCR primers were used in a 10 µl reaction volume (1.5 mM MgCl2, 200 µM dNTPs, 1 µM of each primer, 50–100 ng template DNA, and 0.5 U Taq polymerase). Four minutes of initial denaturation at 94°C were followed by 30 cycles of 1 min at 94°C, 1 min at 50–56°C (depending on the primer combination), and 1 min at 72°C. We used a final extension at 72°C for 45 min to ensure quantitative terminal transferase activity of the Taq polymerase. PCR products were separated on a 7% denaturing polyacrylamide gel (32% formamide, 5.6 M urea). PCR products were sized by running a sizing ladder next to the amplified microsatellites (Schlötterer and Zangerl 1999).
Data Analysis
For the two A. majus ssp. cirrhigerum populations, FST (Wright 1965) was estimated using FSTAT version 2.9.1 (Goudet 1995), using Weir and Cockerham's (1984) method. The significance of the F statistic was calculated over all loci, not assuming random mating within samples, using the log-likelihood G statistic based on 1000 genotypic permutations (Goudet et al. 1996). Deviations from Hardy–Weinberg equilibrium were determined by a Markov chain method as implemented in an online version of the GENEPOP software (Raymond and Rousset 1995) (http://wbiomed.curtin.edu.au/genepop/genepop_op2.html).
MICROSAT software (Minch et al. 1995) was used to calculate the proportion of shared alleles. Genetic distances were converted into a tree using UPGMA as implemented in PHYLIP 3.6. Treeview (Page 1996) was used for a graphical representation of the tree of individuals. Input files for MICROSAT and GENEPOP were generated with an Excel macro provided by S. Parks. Since the macro assigns allele size 0 to missing data, the MICROSAT input file had to be adjusted accordingly.
Results and Discussion
All nine microsatellite primer pairs amplified a PCR product in a size range similar to that of the cloned microsatellite. All loci amplified in the three Antirrhinum species (A. majus ssp. cirrhigerum, A. graniticum, and A. molle). Locus AntR1.1 was highly variable, but could not be scored reliably due to extensive stutter bands. Nevertheless, allelic differences could be recognized, suggesting that the locus may be useful for mapping studies (data not shown). Primers designed for locus AntRB4.1 amplified more than two alleles in most individuals of the three species, indicating a duplication of the microsatellite locus. However, the three species could be clearly distinguished based on the amplification pattern of this primer pair (data not shown). AntRB3.1 produced too many nonspecific amplification products, which prevented reliable scoring of alleles.
The remaining six loci were polymorphic in at least one species (Table 2). This contrasts with previous results, which demonstrated that nucleotide sequences from several nuclear genes were nearly identical within and between Antirrhinum species and even between Antirrhinum and more distantly related Scrophulariaceae species (Vieira and Charlesworth 2001a,b; Vieira et al. 1999). Despite a very limited number of loci, a UPGMA tree of individuals based on the proportion of shared alleles separated most of the individuals according to their taxonomic status (Figure 1). Only a single A. majus ssp. cirrhigerum individual grouped to the wrong population (indicated by an asterisk in Figure 1). These loci can therefore be used to address the phylogenetic relationship in the genus Antirrhinum.
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The number of alleles at the six loci with an unambiguous amplification pattern varied from one (Ant11A, A. majus) to 13 (An3, A. graniticum) (Table 2). Across loci, the mean observed frequencies of heterozygotes ranged from 0.31 (cirrhigerumMuel) to 0.62 (A. molle). The low value of the first, however, is strongly affected by the lack of polymorphism at locus ANT11A in this species. Nevertheless, even if locus ANT11A is excluded, A. molle remains the most polymorphic species. Average expected heterozygosity values (He or gene diversity) vary across populations and species (Table 2). The average gene diversity using all six loci for the outcrossing species (0.47 ± 0.26; cirrhigerumMuel, A. graniticum, and A. molle) is not statistically different from that of the self-compatible population cirrhigerumGala (0.45 ± 0.25). The number of alleles averaged over outcrossing populations (4.11 ± 2.87) was greater than that for the cirrhigerumGala population (2.67 ± 1.03). However, this difference is not statistically significant. No significant deviation from Hardy–Weinberg equilibrium was observed for the self-compatible cirrhigerumGala population.
While it has to be noted that our study was based on a small number of loci and a moderate sample size, our results suggest that the self compatible cirrhigerumGala population could be highly outcrossing. This is consistent with the observation of Vieira (2000) that the two populations of A. majus ssp. cirrhigerum (cirrhigerumGala and cirrhigerumMuel) share floral features (corolla length, corolla diameter, and anther-stigma separation). Since the allocation of resources to conspicuous flowers is only advantageous for outcrossing (Barrett et al. 1996; Moore and Lewis 1965; Solbrig and Rollins 1977), the shared floral features may indicate that both populations are outcrossing. Alternatively, the similar floral features in the two A. majus ssp. cirrhigerum populations may also reflect shared history. The significant population differentiation between the two A. majus ssp. cirrhigerum populations (FST = 0.225, P < .001) suggests low gene flow between these populations. Analysis of a larger set of microsatellite loci and more Antirrhinum populations is required to obtain a complete picture of the effects of the mating system and natural variability in Antirrhinum. Such a dataset will have enough power to separate the effects of the breeding system from demographic events.
One particularly striking feature of this study is that an extremely small number of microsatellite loci allowed an almost perfect assignment of individuals to their population. The lack of statistical support for these groupings by bootstrapping is not surprising, given the small number of loci. A moderate increase to about 20 microsatellite loci should be sufficient to overcome this limitation (Schlötterer 2001). Consistent with previous results (Harr et al. 1998), our analysis also indicated that microsatellite analysis could be informative at the level of closely related species.
Most of the microsatellite loci in this study showed an amplification pattern that is consistent with no duplication of the microsatellite loci. Thus flanking sequences of microsatellites could be analyzed to determine to what extent the low nucleotide variability observed (Vieira and Charlesworth 2001a,b; Vieira et al. 1999) is limited only to gene families.
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Acknowledgments |
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We are grateful to the members of the CS lab, in particular B. Harr, for continuous support. M.-T. Hauser provided fresh plant material for the isolation of microsatellites. We thank Brandon Gaut, Deborah, Brian Charlesworth, and two anonymous reviewers for helpful comments. This work was supported by FWF grants (to C.S.).
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Footnotes |
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Corresponding Editor: Brandon Gaut
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References |
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