Laboratory of Molecular Population Genetics, Department of Biology, Graduate School of Sciences, Kyushu University, Fukuoka, Japan
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Abstract |
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Introduction |
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The Amylase (Amy) gene of Drosophila is a member of a multigene family (Payant et al. 1988
; Shibata and Yamazaki 1995
; Popadic et al. 1996
; Inomata, Tachida, and Yamazaki 1997
; Da Lage et al. 1998
; Inomata and Yamazaki 2000
). The closely linked (duplicated) Amy genes show a contrasting evolutionary pattern, involving concerted evolution in coding regions and differential evolution in the flanking regions (Hickey et al. 1991
; Popadic and Anderson 1995
; Shibata and Yamazaki 1995
; Inomata and Yamazaki 2000
; Araki, Inomata, and Yamazaki 2001
). The latter suggests that differential selection has acted on each flanking region (Shibata and Yamazaki 1995
; Okuyama et al. 1996
; Inomata and Yamazaki 2000
; Araki, Inomata, and Yamazaki 2001
). Moreover, adaptive evolution of amylase protein during speciation has also been suggested (Shibata and Yamazaki 1995
; Araki, Inomata, and Yamazaki 2001
). Other lines of evidence for differential selection come from the findings of divergent paralogous Amy genes. One is the Amyrel gene in the Sophophora subgenus, which encodes divergent proteins and shows expression patterns different from the Amy genes (Da Lage et al. 1998
). The other involves the presence of two duplication groups of Amy genes in Drosophila kikkawai and its sibling species (Inomata and Yamazaki 2000
). The first group includes head-to-head duplicated genes (Amy1 and Amy2) and the second group includes tail-to-tail duplicated genes (Amy3 and Amy4). The Amy1 and Amy2 genes cluster with the Amy genes of D. melanogaster, rather than with the Amy3 and Amy4 genes of D. kikkawai. Both coding and flanking regions are very divergent between the two duplication groups. In particular, the Amy1 and Amy2 genes have higher GC content at third positions of codons and more biased codon usage than the Amy3 and Amy4 genes. In the previous study based on the between-species comparison, we found that the Amy1 and Amy2 genes show a contrasting evolutionary pattern involving concerted evolution in coding regions and differential evolution in the flanking regions (Inomata and Yamazaki 2000
). However, we still do not know the detailed mechanism that produced such a contrasting pattern. Therefore, in this study we examined levels and patterns of intraspecific variation of the Amy1 and Amy2 genes in D. kikkawai.
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Materials and Methods |
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Genomic DNA Extraction, Polymerase Chain Reaction Amplification and Sequencing
Genomic DNA from a single fly was extracted according to a standard procedure (Ashburner 1989
, pp. 108109). Polymerase chain reaction (PCR) amplification was performed using the gene-specific primers. They were kik1f5 (5'-TAAATATCTGACCACCAAGGAG-3') and kik12f3 (5'-CTACATTATCTGCCTGAATCCCT-3') for the Amy1 gene and kik2f5 (5'-CCTAACATCGGCAGATATCAGC-3') and kik12f3 for the Amy2 gene. PCR conditions were as follows: 50 µl of the reaction mix was preheated at 95°C for 3 min. The reaction condition for 32 cycles was denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and polymerization at 72°C for 1 min; after 32 cycles, additional polymerization at 72°C for 7 min was performed. Both 5'-flanking and coding regions were sequenced. The PCR products were directly sequenced. The sequence of both strands was determined using ABI Model 377 automatic sequencer and a DNA sequencing kit (BigDye terminator cycle sequencing ready reaction, ABI) with the PCR primers and the internal primers. After direct sequencing, several PCR products were found to be heterozygous because isofemale lines were used. The PCR products of the heterozygous lines were subcloned into pGEM-T Easy Vector (promega), and the sequence of one of the alleles was determined.
Data Analysis
DNA sequences were initially aligned by the CLUSTAL W program (Thompson, Higgins, and Gibson 1994
) and then further aligned by hand. Neighbor-joining (NJ) trees (Saitou and Nei 1987
) with the bootstrap values based on nucleotide substitutions were constructed using the CLUSTAL W program. Correction for multiple hits was performed by the Jukes and Cantor's method (1969)
. The Amy genes of Drosophila lini (14028-0581.0 strain, accession numbers AB035067 and AB035068) were used as outgroup. Molecular population analyses were done using the DnaSP program, Version 3.14 (Rozas and Rozas 1999
). Nucleotide sequences obtained in this study were deposited in the DNA Data Bank of Japan with the following accession numbers: AB077388AB077436.
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Results |
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We also performed the McDonald and Kreitman test (1991)
, which examines the imbalance of the ratio of the number of replacements to synonymous substitutions between polymorphic and fixed classes. In this study, alleles from both Amy loci were pooled together and regarded as alleles of a single gene. We classified substitutions into the fixed class, which included nucleotides that were identical within alleles but differed between species, and the polymorphic class, which included other substitutions. The purpose of this classification was to take into account the effects of genetic exchanges such as gene conversion. Fixed substitutions between species should result from genetic exchanges between the duplicated genes. Assuming selective neutrality, this classification does not affect the results of the test because the ratio of the number of replacements to synonymous substitutions is the same between the polymorphic and fixed classes in any clade of the phylogenetic tree. We found that there are no fixed differences between D. kikkawai and D. bocki in replacement or synonymous classes (see table 3
). This was probably because of the close relationship between the two species. Actually, the two species are morphologically very similar (Lemeunier et al. 1986
), and females of D. kikkawai and males of D. bocki can cross and produce fertile F1 flies (Kim, Watanabe, and Kitagawa 1989
). Thus, we could not perform the test. We therefore used D. lini for a between-species comparison. The results obtained did not reject the neutral hypothesis (see table 3
).
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Linkage Disequilibrium
Linkage disequilibrium between polymorphic DNA sites was investigated (fig. 5
). We found highly significant linkage disequilibria (P < 0.001) after the Bonferroni correction for multiple comparisons; for example, a pair of sites +1332 and +1335 in the Amy1 exon and the pair +690 and +708 in the Amy2 exon. Only in one of the duplicated genes, the Amy1 gene, highly significant linkage disequilibria (P < 0.001, after the Bonferroni correction) were observed in the 5'-flanking region and in the intron. The pairs of sites with the significant linkage disequilibria were A-C and T-A at -180 and -178 immediately upstream of the glucose repressible element (Boer and Hickey 1986
) and C-T at +138, A-G, A-T, C-T, and G-A at 3rd, 9th, 18th, and 20th in the anterior part of the intron sequences. The pairs of sites, G-A, C-T, A-C, and A-C at -111, -105, -104, and -101, near the CAAT motif were also highly significant (P < 0.001), although they were not significant after the Bonferroni correction.
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Discussion |
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We found an excess of replacement polymorphic sites in the within-locus class. Furthermore, most replacement changes in this class (11 out of 12) are singletons. These results indicate that most replacement changes are deleterious. The fate of neutral variants is determined by random genetic drift. Moreover, in the case of duplicated genes, neutral variants which have occurred in one gene could spread to the other locus by gene conversion. On the other hand, advantageous variants go to fixation, whereas deleterious variants should be immediately eliminated by purifying selection. Therefore, a newly arisen deleterious variant is unlikely to be distributed to both loci. In this study, there was no supportive evidence that the shared replacement changes between the duplicated genes were adaptive.
Differential Evolution in the 5'-Flanking and Intron Regions
The Amy-coding region is suggested to have duplicated together with 450 bp of the 5'-flanking region (Okuyama et al. 1996
). Therefore, if the same evolutionary force acts on the 5'-flanking regions as on the coding regions, we should observe concerted pattern in the 5'-flanking regions. However, the 5'-flanking regions analyzed in our study have evolved divergently, indicating that they are affected by different selective forces.
We observed highly significant linkage disequilibria in the 5'-flanking region and intron of the Amy1 gene. One possible explanation is low recombination rate. However, the duplicated Amy1 and Amy2 genes are located in the center of the chromosome arm (Inomata and Yamazaki 2000
), suggesting a normal recombination rate. Furthermore, our estimate of the recombination rate is three orders larger than the mutation rate. Thus, the linkage does not appear to be responsible for the observed disequilibria. The observed linkage disequilibria may also reflect the genetic differentiation caused by the population structure because our samples come from geographically different locations. If this is true, we should observe a characteristic haplotype for each population throughout the genomic regions. However, we could not find such a tendency. For example, the highly significant pair of sites (AACG-GTTA) in the intron was found in all populations. Furthermore, most of the significant pairs of sites were observed in only one of the duplicated genes, the Amy1 gene. These observations do not support the hypothesis that population structure contributed to the observed linkage disequilibria.
Another possible explanation for the observed linkage disequilibria is epistatic selection between sites. The pairs of sites showing linkage disequilibria were found in a region immediately upstream of the putative glucose repressible element (Boer and Hickey 1986
) and within and around the CAAT motif, although most of them were not significant after the Bonferroni correction. The regulatory sequences found in the 5'-flanking region appear to be important for amylase expression (Magoulas et al. 1993
; Choi and Yamazaki 1994
). In a population cage experiment with D. melanogaster using different food environments, H. Araki et al. (personal communication) found that selection acted on the interaction between the Amy locus and its genetic background. If the interaction between the cis-regulatory sequences and trans-acting elements is necessary to optimize the levels of gene expression, a specific linkage between sites within the cis-regulatory sequences may be maintained to interact with trans-acting elements. In this case, the pairs of closely located sites may show linkage disequilibria because the cis-regulatory sequences are generally very short. Significant linkage disequilibria found in the Amy1 intron involved two haplotypes, AACG and GTTA. One GTTA haplotype in the Amy2 intron of the Nago-4 strain is probably caused by gene conversion between the duplicated genes, because we detected a gene conversion track from +138 to 20th position of the intron in the Nago-4 strain using an algorithm of Betran et al. (1997)
. Linkage disequilibria in the Amy1 intron may be caused by compensatory nucleotide changes to maintain the secondary structure of mRNAs. Linkage disequilibria observed in the Adh gene introns of Drosophila were suggested to be caused by epistatic selection maintaining precursor mRNA secondary structure rather than subpopulation structure (Schaeffer and Miller 1993
). Kirby, Muse, and Stephan (1995)
actually showed that the linkage disequilibria can be caused by epistatic selection maintaining precursor mRNA secondary structure. Furthermore, compensatory interactions between the 5' and 3' ends of the Adh mRNA were confirmed experimentally (Parsch, Tanda, and Stephan 1997
). However, at present we do not have any information on the secondary structure of amylase mRNAs. A population survey of random samples from a single natural population and molecular analyses are needed to clarify the selective significance in those pairs of sites.
Patterns of Molecular Evolution
In this study selective neutrality could not be rejected by the tests. Most of the tests for neutrality assume no recombination. Apparently genetic exchanges (recombination and gene conversion) have occurred in the Amy-coding regions of D. kikkawai. Although some researchers pointed out that the occurrence of recombination is conservative for the tests (Hudson, Kreitman, and Aguade 1987
; Tajima 1989
; Fu and Li 1993
) and its effect is also partly understood (Wall 1999
), the results of the tests may still be biased by genetic exchanges between loci. Nevertheless, our results are not likely to reject the neutrality. On the other hand, the tests should yield unbiased results when applied to the 5'-flanking regions because these regions have evolved independently like a single locus. Although the differential selection on the 5'-flanking regions was suggested (Shibata and Yamazaki 1995
; Okuyama et al. 1996
; Inomata and Yamazaki 2000
; Araki, Inomata, and Yamazaki 2001
), no significant departure from the neutrality was found by the tests. The reason is probably that the power of the statistical tests is not high in most cases (Wall 1999
).
Although the results of the sliding window plot analyses cannot be tested statistically, the within- and between-species comparisons of the pattern of substitutions along the sequences enable us to infer as to what evolutionary forces have acted on the genes. A region immediately upstream of exon 1 in the 5'-flanking regions indicated by an arrow diverged between D. kikkawai and D. lini, whereas the variation of the same region was reduced within D. kikkawai (see figs. 6A, 6B, and 7
). This region is downstream of the region where linkage disequilibria were observed. The low polymorphism and high divergence are not predicted by simple neutral hypothesis. In the corresponding region, the level of variation within D. bocki was not reduced compared with that between D. bocki and D. lini (data not shown). This is consistent with the neutral prediction. Thus, the observed pattern is specific to D. kikkawai. One of the plausible explanations for this pattern is selective sweep (Maynard Smith and Haigh 1974
; Kaplan, Hudson, and Langley 1989
). During fixation, newly arising advantageous mutations sweep neutral alleles at linked sites. This results in the reduction of polymorphism without affecting neutral divergence. Another explanation is background selection (Charlesworth, Morgan, and Charlesworth 1993
). Chromosomes with deleterious mutations are eliminated from the population and do not contribute to the next generation, resulting in a reduction of linked neutral variation. In this sense its effect is identical to that of a population size reduction. These two mechanisms have been proposed to explain low variation in low recombination regions. It is difficult to detect their effects in the regions with normal recombination rate. Recombination rate in the Amy1 and Amy2 gene regions is unlikely to be low because they are located in the center of the chromosomal arm (Inomata and Yamazaki 2000
). Even if the regions with low polymorphism and high divergence are found, the two models are not easily distinguished. However, Tajima's D is expected to be significantly negative in the selective sweep model (Braverman et al. 1995
), whereas significant Tajima's D values are not likely to be detected in the background selection model (Charlesworth, Charlesworth, and Morgan 1995
). Tajima's D in the region of the Amy1 gene was negative (-1.514), although it was unlikely to be significant (see fig. 7). Furthermore, in the Amy2 gene Tajima's D was zero. At face value, Tajima's D values obtained in the present study seem to be consistent with each model. However, we still cannot decide which mechanism is more plausible. This puzzling observation remains to be solved in future studies.
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Acknowledgements |
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Footnotes |
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Keywords: DNA polymorphism
Amylase (Amy) multigenes
linkage disequilibrium
adaptive evolution
Drosophila
Address for correspondence and reprints: Nobuyuki Inomata, Laboratory of Molecular Population Genetics, Department of Biology, Faculty of Sciences, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. ninomscb{at}mbox.nc.kyushu-u.ac.jp
.
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