DNA Polymorphism in Active Gene and Pseudogene of the Cytosolic Phosphoglucose Isomerase (PgiC) Loci in Arabidopsis halleri ssp. gemmifera

Akira Kawabe1, and Naohiko T. Miyashita

Laboratory of Plant Genetics, Graduate School of Agriculture, Kyoto University, Kyoto, Japan

Correspondence: E-mail: >akirakawabe{at}hotmail.com.


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
DNA variations in two PgiC loci were investigated in 15 strains of Arabidopsis halleri ssp. gemmifera. In a 5.5-kb region of the PgiC1 locus, 127 nucleotide substitutions and 33 length variations were observed. In a 6.0-kb region of the PgiC2 locus, 138 nucleotide substitutions and 33 length variations were observed. Frame shift, novel stop codons, and large length variations were observed in the PgiC2 coding region. These findings suggested that PgiC2 may be a pseudogene. The nucleotide diversities ({pi}) for the entire regions of both PgiC loci were approximately 0.0033. Tajima's test of both PgiC loci yielded significantly negative results. In the coding regions, the high proportions of replacement substitutions caused significant deviations from neutrality in McDonald and Kreitman's test. An excess of singletons and a high proportion of replacement polymorphic sites have been observed in the Adh and ChiA regions of A. halleri ssp. gemmifera. Thus, the A. halleri ssp. gemmifera population may not have reached equilibrium, and thus nonneutral patterns of DNA polymorphism were observed.

Key Words: PgiCArabidopsis halleri ssp. gemmifera • pseudogene • polymorphism


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Arabidopsis halleri ssp. gemmifera (synonym Arabis gemmifera [O'Kane and Al-Shehbaz 1997]) is one of the closest relatives of Arabidopsis thaliana (Miyashita et al. 1998; Koch, Haubold, and Mitchell-Olds 2000). A. halleri ssp. gemmifera is a perennial outcrossing species that grows in moist habitats, whereas A. thaliana is an annual selfing species that grows in open spaces. In A. halleri ssp. gemmifera, DNA polymorphisms in the alcohol dehydrogenase (Adh) (Miyashita, Innan, and Terauchi 1996; Miyashita 2001) and acidic chitinase (ChiA) (Kawabe and Miyashita 2002a) genes have been analyzed. The Adh region had an excess of singleton sites that were significant in Tajima's test, indicating a deviation from the test assumptions, namely neutrality and equilibrium population. In the Adh coding region, a high proportion of polymorphic replacement sites was observed in A. halleri ssp. gemmifera, and the ratio of replacement to synonymous changes in A. halleri ssp. gemmifera was significantly different from that within A. thaliana and from that between A. thaliana and A. halleri ssp. gemmifera. Miyashita, Innan, and Terauchi (1996) suggested that the different ecological niche and/or population structure of A. halleri ssp. gemmifera was responsible for these nonneutral DNA polymorphisms in the Adh gene of A. halleri ssp. gemmifera. In the ChiA region, an excess of singleton sites and a high proportion of polymorphic replacements were also observed in A. halleri ssp. gemmifera. There are two hypotheses for these patterns of DNA polymorphism in A. halleri ssp. gemmifera. One possibility is that natural selection acts similarly on the Adh and ChiA genes in A. halleri ssp. gemmifera. The other possibility is that nonneutral patterns of DNA polymorphism are not locus-specific but are instead species-specific. If the second hypothesis is true for A. halleri ssp. gemmifera, population structure and/or species history would have influenced the entire genome uniformly.

In the present study, DNA polymorphisms in the two PgiC genes were analyzed in A. halleri ssp. gemmifera. DNA polymorphism in the PgiC gene was described previously in A. thaliana (Kawabe, Yamane, and Miyashita 2000). The nucleotide diversity in the PgiC region was 0.0038, which was relatively low compared with that in other genes of A. thaliana. In A. thaliana, two divergent sequences associated with the two allozyme classes were observed. An excess of singleton sites and a high proportion of polymorphic replacements were observed in the PgiC region of A. thaliana. Newly arisen advantageous allozyme (Fast type) was proposed for the cause of these nonneutral DNA polymorphisms in A. thaliana PgiC.

The sequences of the PgiC genes in A. halleri ssp. gemmifera have been published (Kawabe and Miyashita 2002b). There are two PgiC loci in A. halleri ssp. gemmifera that are thought to be the result of a duplication after species splitting of A. halleri ssp. gemmifera and A. thaliana. One of these PgiC loci, PgiC2, was not detected by RT-PCR of cDNAs derived from whole plant during vegetative growth stages. Thus, the pattern of expression of PgiC2 should differ from those of PgiC1 and A. thaliana PgiC. The PgiC2 locus may not have a functional promoter. In the present study, the possibility that expression of PgiC2 is silenced was examined by analyzing DNA polymorphisms. The other locus (PgiC1) shares a common structure with A. thaliana and is expressed normally.

The present study had two primary objectives. The first was to identify and characterize DNA polymorphisms in the PgiC locus. DNA polymorphisms in the PgiC1 gene of A. halleri ssp. gemmifera were then compared with those in PgiC of A. thaliana and other species to evaluate gene-specific patterns in DNA polymorphism in the PgiC gene. The second objective was comparison of DNA polymorphisms between the active gene and pseudogene in A. halleri ssp. gemmifera. The rates of nonsynonymous substitutions are higher in pseudogenes in comparison with those in functional genes (Li 1981; Miyata and Hayashida 1981; Gojobori, Li, and Graur 1982), because pseudogenes are not subject to selective constraints. Thus, in pseudogenes, population structure and/or species history influence patterns and levels of DNA polymorphism directly. By comparing DNA polymorphisms between Adh and ChiA, and the active form and pseudogene of PgiC, differences in the effects of population structure and species history or selection may be revealed. If selection caused an excess of singleton sites and a high proportion of polymorphic replacements, the active PgiC gene, which is under different selection pressure from that of Adh and ChiA, may have a different pattern of DNA polymorphism. However, if population structure and species history influenced nonneutral patterns of DNA polymorphism, all genes, including pseudogenes, should show similar DNA polymorphism patterns.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Plant Materials
Fifteen strains of A. halleri ssp. gemmifera were collected from their habitats, and total DNAs were extracted. The 15 A. halleri ssp. gemmifera strains were sampled from the Kyoto (accession code: Ashibi56 and Ohara), Shiga (Taihei15), Osaka (Minou), Ibaraki (Ryujin), Niigata (Tsugawa), Yamagata (Mazawa), Iwate (Morioka), Akita (Yatate), Miyagi (Okunikkawa), Hyogo (Ohtani), Fukui (Fumuro), Gifu (Midori), Okayama (Fukiya), and Hiroshima (Uga) prefectures in Japan. For each sampling location, one plant was isolated from each local subpopulation of A. halleri ssp. gemmifera.

DNA Sequencing
Total DNAs were purified by the CTAB method as described in Kawabe, Yamane, and Miyashita (2000) and used as templates for PCR amplification of an approximately 6-kb fragment that includes the entire coding region of the PgiC gene. Two primers, AGPGIF2 (5'- GGT TTG GGT TCG TAT TAG AT 3') and AGPGIDU2 (5' ATC ATT GTG GTT CTG TCT AA-3'), were designed in the 5' flanking regions of PgiC1 and PgiC2, respectively. These primers and ATHPGI102 (5'-TTT ATG GGG TTT GGA TTA TTA G-3'), which had been designed in the 3' flanking region of the PgiC locus of A. thaliana (Thomas et al. 1993), were used for PCR amplification. PCR products were cloned into pUC18. Three clones were then mixed at equal concentrations and used as templates for sequencing reactions to avoid PCR artifacts and heterozygous sites. If heterozygous length variations were present in the sample and caused sequencing failures, the three clones were sequenced separately, and the consensus sequence was obtained. The PgiC2 locus of strain Taihei15 had two PCR bands with an approximately 500-bp difference in length. Both bands were cloned and sequenced. Twenty primers designed at approximately 500-bp intervals were used to sequence both strands of PgiC1 and PgiC2. Newly determined DNA sequences were deposited in the DNA Data Bank of Japan database under accession numbers AB100274 to AB100303.

Data Analyses
Analyzed regions included 0.8-kb and 1.2-kb of the 5' flanking regions of PgiC1 and PgiC2, respectively. In the present study, nucleotide positions were assigned relative to the translation initiation site (+1) of strain Ashibi. For the coding region of the PgiC2 gene, the original frame was considered for analyses of synonymous and replacement changes irrespective of frame shift variations. The DnaSP program version 3.0 (Rozas and Rozas 1999) was used to analyze intraspecific and interspecific variations. For A. halleri ssp. gemmifera, nucleotide diversity ({pi}) (Nei and Li 1979; Tajima and Nei 1984) and {theta} (4Neµ) (Watterson 1975) were estimated. The neutral hypothesis was assessed with the tests of Tajima (1989a), McDonald and Kreitman (1991), and Fu and Li (1993). A Neighbor-Joining (NJ) tree (Saitou and Nei 1987) was constructed based on Jukes and Cantor distances (1969) with MEGA2 (Kumar et al. 2000). Because introns could not be aligned between PgiC1 and PgiC2, only coding sequences were used for construction of a phylogenetic tree. Bootstrap probabilities with 500 replications were obtained for each internal branch.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
DNA Variations in the PgiC Loci of A. halleri ssp. gemmifera
There were 127 nucleotide substitutions and 33 indels (insertion/deletion) in the PgiC1 region of A. halleri ssp. gemmifera (fig. 1A). Sixteen nucleotide substitutions and 10 indels were observed more than once. These common variations were found across the region, and there was no clustering or clear linkage between these sites. In the PgiC1 coding region, 12 synonymous changes and 13 replacement changes were observed. Of the replacement polymorphic sites, four were observed more than once, and two of these common nonsynonymous changes yielded nonconservative amino acid changes according to Miyata, Miyazawa, and Yasunaga (1979).



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FIG. 1. Summary of DNA polymorphisms in PgiC loci of A. halleri ssp. gemmifera. (A) DNA variations in the PgiC1 region. (B) DNA variations in the PgiC2 region. At the top, positions of DNA variations are shown as vertical bars, where replacement changes and indels are indicated by open circles and arrowheads, respectively. Nonsingleton DNA variations are summarized at the bottom. DNA variations are shown after strain names, and dots indicate nucleotides identical to the consensus sequence. Positions of variations correspond to those in strain Ashibi. E and I indicate exon and intron, respectively. Synonymous sites, replacement sites, insertions, and deletions are designated as s, r, i, and d, respectively. "1" in the CONSENSUS row for PgiC2 indicates a 44-bp deletion spanning intron 14 and exon 15

 
DNA variations in the PgiC2 region are summarized in figure 1B. There were 138 nucleotide substitutions and 33 length variations. All 15 strains (16 sequences) had a 48-bp deletion containing exon 17 and intron 17. Seven long length variations (more than 10 bp) were observed in the PgiC2 region. Four were located in noncoding regions. In the 5' flanking region, a variation in the numbers of a 15-bp repeat was found. A 91-bp insertion observed in Taihei15-1 was a tandem duplication of the middle part of intron 4. A 17-bp and a 21-bp deletion were found in intron 9 of Taihei15-1 and intron 13 of Okunikkawa, respectively. The remaining three large length variations were located in part or entirely in the coding region. An approximately 1-kb insertion was found in exon 9 of the Fukiya strain. The sequence of the inserted fragment was similar to that of the reverse transcriptase of non-LTR retroposons of A. thaliana. A 15-bp repeat of As at the end of the inserted sequence suggested that this insertion occurred through mRNA-mediated gene translocation. In Taihei15-2, an approximately 500-bp deletion of intron 9 to the end of exon 12 caused the loss of three exons. The Okunikkawa and Ryujin strains both had a 44-bp deletion that spanned intron 14 and exon 15. The intron-exon junction and 24 bp of exon 15 were lost with this deletion. These changes in the coding region of PgiC2 should alter the mRNA. The other small indel variations and nucleotide substitutions may influence the mature protein structure (table 1). The existence of these DNA variations in the coding region suggests that the PgiC2 locus may not encode a functional gene. Only four sequences (Ashibi56, Minou, Ohara, and Taihei15-1) did not contain these mutations, with the exception of the 48-bp deletion at the junction of exon 17 and intron 17.


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Table 1 DNA Variations in the Coding Region of PgiC2 (1,690 bp) Causing Disruption of Protein Structure.

 
Sixteen synonymous changes, 33 replacement substitutions, and two 3-bp insertions were found in the coding region of PgiC2. The 37 replacement changes, which were present in 33 codons, created four novel stop codons, 25 nonconservative amino acid changes, and eight conservative amino acid changes. The proportion of conservative amino acid changes in PgiC2 (8/33) was lower than that in PgiC1 (7/13), although this difference was not statistically significant. The high proportion of both replacement polymorphic sites and nonconservative amino acid changes in PgiC2 also suggest that there is low selective constraint on this locus, supporting our theory that PgiC2 is a pseudogene.

DNA Variations in the PgiC Loci in A. halleri ssp. gemmifera and Between Species
The differences in the ratios of synonymous to replacement changes between polymorphism and divergence was analyzed for the PgiC loci of A. halleri ssp. gemmifera (table 2). With divergences between species, fixed synonymous differences were much larger than fixed replacement differences. The high proportions of polymorphic replacement sites in both PgiC loci of A. halleri ssp. gemmifera yielded in statistically significant results for the McDonald and Kreitman (MK) test; the results for the PgiC2 locus showed greater significance because of the higher proportion of polymorphic replacement sites. Thus, the proportion of polymorphic replacement sites was high in the PgiC loci, especially in PgiC2, in comparison with that of between-species changes. Again, the high proportion of replacements in PgiC2 and the statistical significance of these differences suggest that PgiC2 is subject to relaxed selective pressure because it is a pseudogene.


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Table 2 McDonald and Kreitman's Test for A. halleri ssp. gemmifera PgiC.

 
Levels of DNA Variation in the Two PgiC Loci
The levels of DNA variation in the PgiC loci of A. halleri ssp. gemmifera are shown in table 3. Both PgiC1 and PgiC2 had similar levels of DNA variation across their entire regions. For silent sites, synonymous sites in the coding regions showed the highest levels of variation in both loci. The level of variation in replacement sites in PgiC1 was approximately one third that of synonymous sites. The level of DNA variation in the PgiC2 locus was more than twice that of replacement sites in comparison with the PgiC1 locus. The level of DNA variation in the replacement sites in PgiC2 coding region was as high as that in noncoding regions, whereas the synonymous sites in PgiC2 had the highest level of DNA variation.


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Table 3 Summary of DNA Polymorphisms in the PgiC Regions of A. halleri ssp. gemmifera.

 
Excess of Less Frequent Alleles in PgiC Loci of A. halleri ssp. gemmifera
The results of Tajima's and Fu and Li's tests are shown in table 3. Except for the 3' flanking region of PgiC2, the statistics were all negative, indicating an excess of less frequent alleles. The excesses of less frequent alleles were observed especially in singleton sites. Of site changes, 87.4% and 90.5% in PgiC1 and PgiC2, respectively, were singletons. In PgiC1, neither test yielded significant results for coding regions. In PgiC2, most of the results were significantly negative, indicating a deviation from neutrality. The excess of singletons in both PgiC loci suggested that the test assumptions, neutrality and equilibrium population, were violated in these regions of A. halleri ssp. gemmifera. Considering that PgiC2 is most likely a pseudogene, the excess of singletons in PgiC2 may reflect population disequilibrium of A. halleri ssp. gemmifera.

The excesses of singleton sites were due to strain-specific variants, which generated a starlike phylogeny (fig. 2). The lengths of the internal branches were short for both PgiC loci of A. halleri ssp. gemmifera. Internal branches within each locus had low bootstrap probabilities. A starlike phylogeny is typically observed after a selective sweep or a population bottleneck. In either case, the starlike phylogeny may have been caused by population expansion (Kaplan, Hudson, and Langley 1989).



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FIG. 2. Phylogenetic tree of A. halleri ssp. gemmifera PgiC loci from the Neighbor-Joining method based on DNA variations in the coding region. Only bootstrap probabilities over 60% (500 replicates) are shown. A distance bar is shown

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
DNA Polymorphism in PgiC1 of A. halleri ssp. gemmifera
Levels of DNA variation in the PgiC loci have been reported to be lower than those in other regions in several organisms (Drosophila melanogaster and D. simulans [Moriyama and Powell 1996]; Dioscorea tokoro [Terauchi, Terachi, and Miyashita 1997]; Arabidopsis thaliana [Kawabe, Yamane, and Miyashita 2000]). The reasons proposed for the low level of DNA polymorphisms were not the same for all species. For example, it was suggested that a recent selective sweep occurred in A. thaliana. The level of DNA variation in the A. halleri ssp. gemmifera PgiC1 region is 0.0033, which was as low as that reported for A. thaliana PgiC (0.0038) (Kawabe, Yamane, and Miyashita 2000). The nucleotide diversities of the Adh, ChiA, and PgiC genes of A. halleri ssp. gemmifera and A. thaliana are presented in table 4 to permit comparison of the level of DNA variation in PgiC1. In contrast to the findings in A. thaliana, the level of variation in the PgiC1 locus of A. halleri ssp. gemmifera was similar to those of Adh and ChiA. The nucleotide diversity in the synonymous sites of PgiC1 was the highest among the active genic regions examined.


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Table 4 Summary of Nucleotide Diversity in A. halleri ssp. gemmifera and A. thaliana.

 
The PgiC1 locus has the lowest level of replacement variation among the A. halleri ssp. gemmifera loci examined (table 4). The nucleotide diversity in the replacement sites in PgiC1 (0.0019) was half that estimated for Adh (0.0039). The level of replacement variation in PgiC was one of the smallest among the loci analyzed in D. melanogaster, D. tokoro, and A. thaliana, although high proportions of replacement polymorphic sites were observed in the PgiC loci of these species (Moriyama and Powell 1996; Terauchi, Terachi, and Miyashita 1997; Kawabe, Yamane, and Miyashita 2000). When per-site values were compared, the replacement variation was lower than the synonymous variation in all of the species examined. In these species, reduced synonymous variation due to natural selection may be responsible for the high proportions of polymorphic replacement sites. Low level of replacement polymorphism is a DNA polymorphism characteristic in PgiC. Low level of replacement polymorphism is consistent with low replacement divergence in many plants, including species of Clarkia, Dioscorea, and Leavenworthia (Ford, Thomas, and Gottlieb 1995; Kawabe, Miyashita, and Terauchi 1997; Liu, Charlesworth, and Kreitman 1999), indicating the importance of the PGIC protein.

Normal Level of Polymorphism in the Pgic2 Locus
In the coding region of PgiC2, novel stop codons, frame shifts, and large indel variations were observed. These DNA variations may alter the mRNA or affect mature protein structures. The presence of these mutations supports our theory that PgiC2 is a pseudogene. The high proportion of replacement polymorphic sites and nonconservative amino acid changes also suggest that PgiC2 is a pseudogene. The nucleotide diversity in the PgiC2 region was 0.0033, which was similar to those of previously analyzed regions of A. halleri ssp. gemmifera (Miyashita, Innan, and Terauchi 1996; Kawabe and Miyashita 2002a). In addition, the level of variation in replacement sites of PgiC2 was similar to those of other A. halleri ssp. gemmifera loci. This result was unexpected because PgiC2 is likely a pseudogene, and pseudogenes, which are free from selective constraints, are neutral during natural selection.

Previous investigations of DNA polymorphisms in pseudogenes in D. melanogaster also did not reveal higher levels of DNA variation (Pritchard and Schaeffer 1997; Ramos-Onsins and Aguadé 1998). Background selection was suggested to explain the finding that the pseudogene of larval cuticle protein had very low polymorphisms in D. melanogaster (Pritchard and Schaeffer 1997). The pseudogenes of Ceropin multigenes had lower levels of replacement variation than synonymous variation (Ramos-Onsins and Aguadé 1998), and the Ceropin pseudogenes were suggested to be either active genes with some null alleles or young pseudogenes. In two cases of pseudogenes in Drosophila, the levels of variation in the pseudogenes were lower than those of the active counterparts and noncoding regions. However, the level of variation in the PgiC2 locus of A. halleri ssp. gemmifera is similar level to that of noncoding regions of other loci in A. halleri ssp. gemmifera (table 4).

In A. halleri ssp. gemmifera, levels of replacement variation were relatively high compared with those of synonymous variation in all genes investigated (table 4). In contrast, the level of variation was much lower in replacement sites than in synonymous sites in Drosophila (Moriyama and Powell 1996) and A. thaliana (Innan et al. 1996; Kawabe et al. 1997; Purugganan and Suddith 1998, 1999; Kawabe and Miyashita 1999; Kuittinen and Aguadé 2000; Aguadé 2001). One possible explanation for this finding is that the effective population size of A. halleri ssp. gemmifera is small. In a small population, replacement substitutions with slightly deleterious effects are not eliminated as readily as those in a large population (Ohta 1973, 1992).

Nonneutral Patterns of DNA Polymorphism in the Pgic2 Region
The PgiC2 region showed significant deviation from neutrality in the tests of MK, Tajima, and Fu and Li. The high significance in the MK test is consistent with PgiC2 being a pseudogene. In general, a high proportion of replacement polymorphism is expected in pseudogenes. In PgiC2, a high proportion of nonconservative amino acid changes was also observed. These observations suggest that PgiC2 was released from selective constraint after silencing. However, the significant results from Tajima's and Fu and Li's tests cannot be explained by silencing of PgiC2. Considering that a pseudogene is under selectively neutral conditions, polymorphisms in PgiC2 may have been influenced directly by population structure and/or species history.

In A. halleri ssp. gemmifera, DNA polymorphisms in the Adh (Miyashita, Innan, and Terauchi 1996; Miyashita 2001) and ChiA (Kawabe and Miyashita 2002a) regions were analyzed previously. An excess of singletons and a high proportion of replacement polymorphic sites were observed in all A. halleri ssp. gemmifera genes examined. The excess of less frequent alleles would be caused under strong purifying selection (Tajima 1989a), recent population bottleneck (Tajima 1989b), small population size (Tajima 1989a), and/or hitchhiking effect (Braverman et al. 1997). The significant results from Tajima's test and Fu and Li's test regarding silent sites and high level of DNA polymorphism in replacement sites suggest that strong purifying selection is not the case for these four loci in A. halleri ssp. gemmifera. If the hitchhiking effect caused the excess of singletons in A. halleri ssp. gemmifera genes, we should assume that advantageous mutations should occur frequently throughout the genome of A. halleri ssp. gemmifera because Adh, ChiA, and PgiC encode proteins with different functions and may be located in different chromosomal regions. Although the locations of these genes in the genome were not examined in A. halleri ssp. gemmifera, Adh, ChiA, and PgiC are single-copy genes located on chromosomes 1L, 5S, and 5L, respectively, of A. thaliana.

A recent bottleneck and/or small population size should be considered in the case of A. halleri ssp. gemmifera. If the mutation rates are not significantly different between A. thaliana and A. halleri ssp. gemmifera, the rather low level of DNA variation in A. halleri ssp. gemmifera (table 4) suggests that the population size of this species is small. Small populations are responsible for excesses of less frequent alleles. Similar to the two PgiC loci, starlike phylogenies were also observed for Adh and ChiA (data not shown). A starlike phylogeny is not caused by occurrence of recombination or recurrent mutations, but by strain-specific variations. This suggests the occurrence of a recent bottleneck in A. halleri ssp. gemmifera. Additionally, the extensive subpopulation structure of A. halleri ssp. gemmifera may yield an excess of singletons and a high proportion of replacement polymorphic sites. Under a subpopulation structure with a small population size, subpopulation-specific DNA variants would be fixed frequently. The high proportion of replacement polymorphic sites in A. halleri ssp. gemmifera might be explained by a subpopulation structure with a small population size of A. halleri ssp. gemmifera. In this case, replacement mutations with slightly deleterious effects would have been fixed frequently in each subpopulation. The disequilibrium population structure should have influenced DNA polymorphisms across the A. halleri ssp. gemmifera genome. Thus, the similar DNA polymorphism patterns among A. halleri ssp. gemmifera genes may be a consequence of the population structure of A. halleri ssp. gemmifera.

Small population size with low migration could explain the present results. However, these conditions might occur in highly clonal species such as selfing plants, although A. halleri ssp. gemmifera is an outcrossing species. To determine whether the population structure of A. halleri spp. gemmifera is highly fragmented, it will be necessary to analyze genome-wide variations within and between populations as A. thaliana (Abbott and Gomes 1989; Todokoro, Terauchi, and Kawano 1995; Bergelson et al. 1998).


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
We express our gratitude to T. R. Endo and T. Takano for critical reading and comments regarding early versions of the manuscript. We also thank M. Gouy and two anonymous reviewers for their comment and suggestions. A.K. was supported by a Japan Society for the Promotion of Science research fellowship for young scientists. This article is contribution number 576 from the Laboratory of Plant Genetics, Graduate School of Agriculture, Kyoto University.


    Footnotes
 
1 Present address: Laboratory of Population Genetics, National Institute of Genetics, Shizuoka, Japan. Back

Manolo Gouy, Associate Editor Back


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 Introduction
 Materials and Methods
 Results
 Discussion
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Accepted for publication February 4, 2003.





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