Laboratory of Plant Genetics, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
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Abstract |
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Introduction |
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The alcohol dehydrogenase gene (Adh) of Arabidopsis thaliana is a single-copy gene (Dolferus and Jacobs 1984
) and was one of the first nuclear genes in this plant species to be cloned (Chang and Meyerowitz 1986
). The regulation of Adh expression in A. thaliana has been extensively studied (Hoeren et al. 1998
), and the cis-regulatory sequence elements in the Adh promoter have been identified (Dolferus, Peacock, and Dennis 1994
; Hoeren et al. 1998
). Two anaerobic response elements (AREs) similar to those in maize are present in the 400-bp region upstream of Adh. A Myb transcription factor (AtMYB2) binds to one of the AREs and is involved induction of Adh under low-oxygen conditions (Hoeren et al. 1998
).
Because induction of ADH in plants is essential for defense against environmental stress, ADH is thought to be adaptively important. DNA variation in the Adh locus of A. thaliana and related species has been analyzed from the viewpoint of population and evolutionary genetics (Hanfstingl et al. 1994
; Innan et al. 1996
; Miyashita, Innan, and Terauchi 1996
; Miyashita et al. 1998
). In a worldwide sample of A. thaliana, a high level of nucleotide variation was detected, and there were six distinct sequence types in the Adh locus. This pattern of DNA polymorphism can be explained by four intragenic recombination events between the two most divergent Adh sequence types that occurred during the evolutionary history of this plant species (Innan et al. 1996
). Two divergent sequence types (dimorphism) were also observed at several other loci (Kawabe et al. 1997
; Stahl et al. 1999
; Kawabe and Miyashita 1999
), suggesting that dimorphism could be one of characteristics of DNA polymorphism in the A. thaliana nuclear genome. However, results of neutrality tests and levels and patterns of polymorphism varied at different loci, so the origin of the dimorphism and the mechanism by which it is maintained remain to be clarified.
In this study, nucleotide sequence variation was analyzed in a 2.4-kb upstream region of the Adh locus in A. thaliana and Arabis gemmifera. This study had two purposes. One was to investigate the level and pattern of DNA polymorphism in the 5' region where regulatory sequence elements exist. It is expected that levels of polymorphism and divergence between species will be negatively correlated with functional importance (Kimura 1983
). The well-characterized 5' upstream region of A. thaliana Adh provides a good opportunity to examine this expectation, especially for a noncoding region in a plant species. The second purpose was to reveal the extent of the dimorphism detected in the transcriptional unit of Adh. If dimorphism in the Adh region is simply a consequence of neutral evolution, such as accumulation of neutral mutations in an isolated population structure, it is expected that dimorphism would extend farther into the 5' upstream region to some extent, although intensity of natural selection and recombination would influence the extent. Based on the results presented here, possible mechanisms for maintaining dimorphism at Adh are discussed.
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Materials and Methods |
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Data Analyses
Fourteen A. thaliana and five A. gemmifera sequences were used, including A. thaliana ecotype Col-0. The Adh sequences of the two species described in Innan et al. (1996)
, Miyashita, Innan, and Terauchi (1996)
, and Miyashita et al. (1998)
were included in the data set for analysis so that approximately 4.4 kb from the Adh region was analyzed. This 4.4-kb region can be divided into three parts: the 5' flanking region (2,366 bp), the transcriptional unit (1,973 bp from the transcription initiation site to the transcription termination site), and the 3' flanking region (103 bp). The first nucleotide of the transcriptional unit was assigned as coordinate position 1. Program package DnaSP, version 2.0 (Rozas and Rozas 1997
), was used to analyze intra- and interspecific variation via the estimation of nucleotide diversity (
; Nei and Li 1979
), 4Nµ (
; Watterson 1975
), the number of recombination events (Rm; Hudson and Kaplan 1985
), and 4Nc (C; Hudson 1987
) and the tests of Hudson, Kreitman, and Aguadé (1987
; HKA test), Tajima (1989a)
, and Fu and Li (1993)
. The run statistic (KR), the Kolmogorov-Smirnov statistic (DKS), and the maximum sliding G statistic (Gmax) were used to examine heterogeneity between polymorphism and divergence using the program DNA slider (McDonald 1996, 1998
).
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Results |
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Divergence in the 5' Upstream Region of the Adh Locus Between A. thaliana and A. gemmifera
A sequence alignment of the 500-bp upstream region of Adh from A. thaliana and A. gemmifera is shown in figure 4
. Although there were several gaps, sequence identity between the species was high (approximately 90%) throughout the entire upstream region. This region included the regulatory elements ARE1 and ARE2, GBOX1 and GBOX2, and the TATA box. Sequences of regulatory elements were conserved between the species. Because of the gaps, the spatial relationships between Adh regulatory elements were different in the two species. The level of divergence between the two species is summarized for functionally different regions of the Adh gene in table 4
. As observed for polymorphism in A. thaliana and A. gemmifera, the transcriptional unit had the highest level of silent divergence. The HKA test was applied to examine the correlation between polymorphism in A. thaliana and divergence between the two species for the 5' flanking region and the transcriptional unit. Significance was not detected (2 = 0.025, P = 0.87 for silent sites). None of the three statistics proposed by McDonald (1996, 1998)
detected heterogeneity in the ratio of polymorphism to divergence (data not shown). To examine peaks of divergence between the two species, sliding-window analysis was applied to the interspecific comparison (fig. 5
). The highest peak was in the first intron, which was difficult to align between the two species due to many indel changes. There were many other peaks of divergence, including a peak in the 5' region of regulatory elements and a peak in exon 4, which correspond with regions of high polymorphism in A. thaliana. When Adh sequence types 1 and 6 of A. thaliana were analyzed separately, the peaks still existed, indicating that the peaks of divergence were not due to dimorphic variations in A. thaliana.
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Discussion |
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This study also shows that dimorphism in the Adh locus does not extend into the FUR. This result seems to be inconsistent with the hypothesis that dimorphism is due to accumulation of neutral mutations in an isolated population structure, and suggests that the population structure could not be the cause of dimorphism. However, if the constraint mentioned above exists, variations would not have accumulated even in an isolated population structure. Recombination also influences the extent of dimorphism. Recombination parameters were high in the 5' region of Adh. A high level of recombination would have randomized divergent sequence types, even if two divergent sequence types had existed before recombination occurred. Because of significant deviation from neutrality and a high level of recombination detected in the 5' upstream region, the origin of dimorphism at the Adh transcriptional unit remains unclear.
Peaks of Nucleotide Variation in the A. thaliana Adh Gene
There are two regions in which peaks of variation were detected: the 400-bp upstream region, including promoter regulatory elements, and exon 4. Although these two peaks of variation were previously reported in Innan et al. (1996)
, by extending the scope of the earlier data set, this study shows that these are the only notable peaks of variation in the Adh region of A. thaliana. No clear peaks were detected in the FUR. Thus, regions of functional importance have high levels of DNA polymorphism in the Adh region of A. thaliana. This is contrary to the negative correlation between level of variation and functional importance expected from neutral mutation theory. As discussed below, it seems reasonable to consider that some kind of balancing selection had acted on Adh. Here, one could argue against the functional importance of the two regions. If the two regions are not functionally important, the two peaks of variation would be consistent with the neutral mutation theory, reflecting higher mutation rates in the regions, for example. However, it is certain that the sequence elements in the 400-bp upstream region are responsible for normal and induced expression of Adh (Hoeren et al. 1998
) and that exon 4 codes for approximately 42% of the 379 amino acids of ADH. Replacement polymorphic sites in exon 4 are associated with ADH allozyme variations, which show clear ADH activity differences (Dolferus and Jacobs 1984
). As far as ADH activity is necessary for A. thaliana in nature, it is unlikely that the two regions of high variation are free from functional constraints.
In the 400-bp region of regulatory elements, the peak is caused by polymorphic site variations located outside of the regulatory elements themselves. Two indel changes in this region could cause spatial differences between these elements and the coding region of Adh, which may have some effect on Adh expression. However, both indels are single-nucleotide changes, so the spatial change is quite small. It seems unlikely that the variations in the regulatory region have a strong influence on Adh expression. Therefore, the regulatory significance of detected polymorphic variations, both indel and nucleotide changes, and, consequently, the reason for the peak of variation are not clear.
Balancing Selection in the Adh Region from A. thaliana
There are four observations that are relevant to a consideration of the genetic mechanisms acting on the Adh region of A. thaliana. First, the dimorphic variations in exon 4, especially replacement polymorphic sites, are associated with allozyme variation in the Adh gene (Hanfstingl et al. 1994
; Miyashita et al. 1998
). Second, as shown in Innan et al. (1996)
and this study, the two peaks of variation in the Adh locus are caused by dimorphic variation in A. thaliana. This result is suggestive of balancing selection between the two sequence types (Hudson and Kaplan 1988
). Third, by investigating DNA variation at the entire genome level with AFLP analysis, Miyashita, Kawabe, and Innan (1999)
concluded that polymorphisms occurring at intermediate frequencies were rare in the A. thaliana nuclear genome. Therefore, dimorphism at an intermediate frequency in the Adh locus, especially in exon 4, could be unusual in the A. thaliana nuclear genome. Finally, this study showed drastic change in Tajima's D value along the entire region investigated. Positive peaks of Tajima's D value were detected in the regulatory region and exon 4. Because the D value in the transcriptional unit was positive (table 2 ), statistical significance of the peaks of D values can be examined by a one-tailed Tajima's test with an alternative hypothesis of D > 0 (F. Tajima, personal communication). If so, the four peak points in the transcriptional unit can be regarded as significantly larger than zero (fig. 3
). Also, considering that the present A. thaliana population has expanded its habitat recently (Price, Palmer, and Al-Shehbaz 1994
; Innan, Terauchi, and Miyashita 1997; Miyashita, Kawabe, and Innan 1999
), Tajima's D value would be expected to be even lower than that under neutrality (Tajima 1989b
). In this case, more points in the transcriptional unit would become significantly positive, although there is the problem of multiple correction.
These observations suggest that balancing selection could be invoked to explain the dimorphism at an intermediate frequency in exon 4 of Adh. A possible selection model is diversifying selection, which assumes variable direction and intensity of natural selection in subpopulations such that balancing selection is detected at the species level. However, this seems to be unlikely, because there was no association between sequence types in Adh and geographic origin (Innan et al. 1996
). If selection varies in different subpopulations, some correlation between ecotype and origin would be expected. This is not the case. Another explanation is overdominance. It may be difficult to imagine balancing selection in a selfing species like A. thaliana. In natural populations of this plant, almost complete homozygosity is detected (Abbot and Gomes 1989
; Todokoro, Terauchi, and Kawano 1996
; Bergelson et al. 1998
). However, Innan et al. (1996)
showed that there were at least four intragenic recombinations in Adh in the evolutionary history of this plant species. To have intragenic recombination, it is obvious that heterozygotes must have been formed between sequence types. Although this could be extremely rare, heterozygotes formed between sequence types might have some advantage over the parental homozygotes. An experiment to measure ADH activity and Adh expression is under way to examine the possibility of overdominance in this selfing plant.
Divergence in the Adh Region Between A. thaliana and A. gemmifera
Between the two species, the 5' flanking region has lower divergence than the transcriptional unit. Also, the regions of high polymorphism in A. thaliana, that is, the 400 bp regulatory region and exon 4, have high divergence as well, although the peak of divergence in the regulatory region is not particularly high. Unlike polymorphism in A. thaliana, the peaks of divergence were not caused by dimorphic variation in A. thaliana. However, this could be expected, since the level of divergence between species is much greater than the difference between the two most divergent sequence types in A. thaliana. Therefore, the peaks of divergence reflect solely the mechanism operating on the speciation process of the two species.
An explanation for the correlation between polymorphism and divergence in the two regions is that adaptive nucleotide changes occurred in Adh, especially in exon 4, in the divergence process to adapt to the ecological niche of each species. Drastic amino acid substitutions in exon 4 may be related to this adaptation process. There were many gaps in the sequence alignment of the regulatory region between the two species, despite a high sequence identity (>90%). This result may suggest that differences in the spatial distance between regulatory elements and the Adh coding region may be related to changes in expression of Adh in the two species to compensate the possible adaptive changes in exon 4.
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Acknowledgements |
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Footnotes |
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1 Keywords: polymorphism
Arabis gemmifera,
Arabidopsis thaliana,
Adh.
2 Address for correspondence and reprints: Naohiko Miyashita, Laboratory of Plant Genetics, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. E-mail: arabis{at}kais.kyoto-u.ac.jp
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