*Department of Botany, The University of Tennessee, Knoxville;
Department of Botany, Iowa State University
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Abstract |
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Introduction |
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Gossypium L. (Malvaceae) has become a useful model system for studying molecular evolution (Wendel, Schnabel, and Seelanan 1995
; Cronn et al. 1996
; Cronn, Small, and Wendel 1999
; Small, Ryburn, and Wendel 1999
; Small and Wendel 2000a, 2000b
) and especially for studying the molecular evolutionary consequences of allopolyploidy (Wendel, Schnabel, and Seelanan 1995
; Wendel et al. 1999
; Wendel 2000
; Liu et al. 2001
). The phylogenetic relationships of the ca. 50 diploid and 5 allotetraploid species of Gossypium are well characterized (Wendel and Albert 1992
; Seelanan, Schnabel, and Wendel 1997
; Small et al. 1998
; Wendel et al. 1999
; Cronn et al. 2002
). The five allotetraploid Gossypium species (designated AD-genome) diverged from a single recent allopolyploidization event (Wendel 1989
; Small et al. 1998
; Cronn, Small, and Wendel 1999
), and the parental diploids are represented by the extant species Gossypium herbaceum L. (diploid A-genome) and Gossypium raimondii Ulbrich (diploid D-genome); thus the two component genomes of the allotetraploids are designated A- and D-subgenomes (or A' and D') to indicate their diploid origin. This well-understood organismal history facilitates the identification and comparison of orthologous and homoeologous loci (see e.g., Cronn and Wendel 1998
; Small et al. 1998
; Cronn, Small, and Wendel 1999
; Small and Wendel 2000a
).
A previous study (Small, Ryburn, and Wendel 1999
) examined levels of nucleotide diversity for homoeologous AdhA loci in two allotetraploid species, Gossypium hirsutum L. and Gossypium barbadense L. Whereas that study revealed low diversity in both homoeologs, it also showed that the D-subgenome harbored greater nucleotide and allelic diversity than did the A-subgenome in both species. In concert with these data, a second study (Small et al. 1998
) found that for a second alcohol dehydrogenase locus (AdhC), sequences from the D-subgenome homoeologs of all five allotetraploid species were evolving at a rate significantly greater than the rate in the A-subgenome homoeologs, again suggesting differential evolutionary pressures acting on the two subgenomes. Finally, in evaluating the relative rates for the entire Adh gene family in Gossypium, we found that AdhC has higher evolutionary rates at both silent and nonsynonymous sites than AdhA (Small and Wendel 2000a
). Thus, evolutionary rates for AdhA are low, relative to those for AdhC. Because evolutionary rates and levels of nucleotide diversity are positively correlated (Hudson, Kreitman, and Aguadé 1987
), these data predict that nucleotide diversity for AdhC should be higher than for AdhA. This, in turn, suggests that the observed increase of nucleotide and allelic diversity found in the D-subgenome of the allotetraploids for AdhA might similarly be elevated for AdhC. The purpose of this study then was to test these predictions for AdhC. Specifically we asked if: (1) nucleotide diversity is elevated for AdhC relative to AdhA, as predicted by the correlation between relative rates and nucleotide diversity; and (2) the pattern of higher diversity in the D-subgenome of the allotetraploids found for AdhA is also found for AdhC.
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Materials and Methods |
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PCR Amplification and DNA Sequencing
To isolate AdhC sequences from specific duplicated genes in allotetraploid cotton, we designed two pairs of homoeolog-specific PCR amplification primers. Primer sequences were based on data from AdhC for all five allotetraploid species (Small et al. 1998
) and were designed so that the final 3' nucleotide of each primer, as well as one other nucleotide within the primer, were specific for either the A- or D-subgenome homoeolog. The forward primers span the exon 2-intron 2 boundary, whereas the reverse primers span the intron 7-exon 8 boundary (fig. 1
). To achieve homoeolog-specific amplification, a two-step procedure was used. The first step involved a 10-µl PCR amplification using 0.5 µl of template DNA, 1x Taq buffer (Promega), 200 µM each dNTP, 1.5 mM MgCl2, 0.2 µM each primer (either ADHCX2I2-D + ADHCX8I7-D to amplify the D-subgenome sequences or ADHCX2I2-A + ADHCX8I7-A to amplify the A-subgenome sequences). Cycling parameters used a touchdown approach (Don et al. 1991
) that facilitates highly specific amplification. Initial annealing temperatures are set 5°C higher than the annealing temperature of the primers, so only amplification of the specific target is accomplished (in this case, the annealing temperature of the primers was 4850°C, so the initial annealing temperature was set to 55°C). During the first 10 cycles, the annealing temperature is dropped by 0.5°C per cycle so that by the 11th cycle the programmed annealing temperature is down to the primer annealing temperature (50°C). An additional 15 cycles were then performed for a total of 25 cycles of 94°C for 1 min, 5550°C for 1 min, and 72°C for 2 min, followed by a final 5 min 72°C extension step. The second step of the amplification process used 5 µl of the PCR product from the first step as a template for a second 25 µl PCR reaction with the same reaction components as above. Cycling conditions were 25 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 2 min, followed by a final 5 min 72°C extension step. These PCR products were subjected to agarose gel electrophoresis, excised from the gel, and eluted from the gel using GeneClean (BIO 101).
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Because of the possibility that some mutations detected may be caused by nucleotide misincorporation by Taq polymerase during PCR, all singleton nucleotides were confirmed by reamplification and resequencing. In all cases, the initial sequences inferred were corroborated by resequencing. In the case of heterozygous sequences, the amplification products were cloned into pGEM-T and sequenced as described previously (Small et al. 1998
) to establish linkage relationships among polymorphic nucleotides.
Analyses
As in our previous study (Small, Ryburn, and Wendel 1999
), we assumed that for each homoeolog both alleles were amplified. In a number of cases this assumption was validated by the presence of nucleotide polymorphism in the sequencing ladder and electropherograms, indicative of two products underlying the sequence (i.e., heterozygosity). The sequence uniformity detected for most accessions is assumed to be the result of homozygosity, the predominant condition in allotetraploid cottons (Wendel, Brubaker, and Percival 1992
; Brubaker and Wendel 1994
; Small, Ryburn, and Wendel 1999
). Removing identical sequences inferred from homozygous individuals from the analyses has little quantitative effect on the results and does not change the qualitative conclusions.
The sequences generated fell into four subsets that were analyzed separately and in combination when appropriate. These data sets include the A-subgenome of G. barbadense (6 accessions, 12 alleles), the D-subgenome of G. barbadense (6 accessions, 12 alleles), the A-subgenome of G. hirsutum (22 accessions, 44 alleles), and the D-subgenome of G. hirsutum (22 accessions, 44 alleles).
Relationships among the haplotypes of the AdhC sequences from G. hirsutum and G. barbadense were inferred using the software TCS (Clement, Posada, and Crandall 2000
), which implements a statistical parsimony approach to estimating gene genealogies (Posada and Crandall 2001
). Genealogies were inferred separately for the A-subgenome sequences and D-subgenome sequences, but the G. barbadense and G. hirsutum sequences were analyzed together for each subgenome.
Descriptive statistics were calculated for each of the AdhC data sets. The two primary estimates of nucleotide diversity were (Nei 1987
, pp. 256257) and
w (Watterson 1975
), which estimate nucleotide diversity as the mean of all pairwise sequence differences and as an index of the number of polymorphic sites, respectively. A 95% confidence interval was calculated around
w using the coalescent simulation option of DnaSP v. 3.14 (Rozas and Rozas 1999
). In addition, we calculated
separately for intron, synonymous, silent (intron + synonymous), and nonsynonymous sites.
A number of statistical tests have been proposed to evaluate whether or not the distribution of nucleotide polymorphism matches that predicted by neutral theory. We performed the tests of Tajima (1989)
, Fu and Li (1993)
, Hudson, Kreitman, and Aguadé (HKA 1987)
, and McDonald and Kreitman (MK 1991)
. Additionally, we explored the extent of recombination among sequences using the approach of Hudson and Kaplan (1985)
. This analysis infers the minimum number of recombination events within a collection of sequences using the four-gamete test in all pairwise comparisons of sequences. A number of these calculations were facilitated by the software DnaSP v. 3.14 (Rozas and Rozas 1999
).
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Results |
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Gene Genealogies
Relationships among the AdhC sequences were inferred, and the resulting genealogies are depicted in figures 2 and 3
. The A-subgenome network (fig. 2
) reveals that AdhC sequences from G. hirsutum and G. barbadense are differentiated from each other by at least four mutations. Rooting this network with the diploid A-genome species G. arboreum places the root between the G. barbadense and G. hirsutum sequences. As discussed subsequently, no allelic diversity was detected in the A-subgenome of G. barbadenseall sequences were identical. Seven different haplotypes were recovered from the A-subgenome of G. hirsutum. Four of these haplotypes were found only in single homozygous individuals; the remaining three haplotypes were represented multiple times in the sample.
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However, nucleotide diversity is not equally distributed among site categories (intron, synonymous, silent, and nonsynonymous) in either AdhA or AdhC. As shown in table 1 , all nucleotide diversity in AdhA is caused by silent polymorphism (either intron or synonymous sites); no nonsynonymous mutations were detected. In contrast, for AdhC, nonsynonymous diversity contributed a great deal to the observed variation. For AdhC, nonsynonymous diversity ranged from approximately half the overall diversity per site to actually exceeding overall diversity in one case (the D-subgenome of G. hirsutum).
Among putatively silent site categories (intron and synonymous sites), we also detected variation in nucleotide diversity (table 1 ). In almost all cases, synonymous diversity was greater than intron diversity. For AdhA, only the D-subgenome of G. hirsutum contained any intron diversity at all, and in this case synonymous diversity was only slightly higher than intron diversity. For both the A-subgenome of G. hirsutum and the D-subgenome of G. barbadense, all the diversity detected was at synonymous sites. For AdhC, synonymous diversity was approximately two times the intron diversity for the D-subgenomes of both G. hirsutum and G. barbadense. The A-subgenome of G. hirsutum provided the only exception to this trend, but in this case no synonymous diversity was detected. This pattern of higher diversity and divergence at synonymous sites relative to intron sites has been noted previously both in plants and Drosophila (Moriyama and Powell 1996
; Charlesworth and Charlesworth 1998
; Vieira and Charlesworth 2001
) and is presumably caused by greater selective constraints on sites important for intron structure relative to synonymous changes in coding regions.
Nucleotide diversity values of AdhC for a given genome are consistently higher than for AdhA (except for the A-subgenome of G. barbadense which was monomorphic for both AdhA and AdhC). Values for w were 4.47.1 times higher for AdhC than AdhA, whereas
values were 1.76.6 times higher. Despite this elevation of diversity in AdhC relative to AdhA, these values are still low compared with plant nuclear genes in general. For example, the highest estimate of
in Gossypium is 0.00649 (G. hirsutum D-subgenome AdhC), whereas
in Arabidopsis thaliana has a mean of 0.00665 and ranges from 0.00300 to 0.01040 for five nuclear genes (Adh, [Innan et al. 1996
]; CAL [Purugganan and Suddith 1998
]; ChiA [Kawabe et al. 1997
]; ChiB [Kawabe and Miyashita 1999
]; CHI [Kuittinen and Aguadé 2000
]; FAH1 [Aguadé 2001
]; F3H [Aguadé 2001
]; and RPS2 [Caicedo, Schaal, and Kunkel 1999
]).
Recombination Indices and Neutrality Tests
The minimum number of recombination events per data set was inferred using the method of Hudson and Kaplan (1985)
. Recombination was detected only in the AdhC G. hirsutum D-subgenome data set. As noted above, these recombination events are restricted to a set of four closely related haplotypes (fig. 3
). The tests of neutral evolution of Tajima (1989)
and Fu and Li (1993)
were performed for each data set, including subsets of each data set (introns and exons). None of these tests revealed significant departures from neutral expectations for any data set. We also performed the HKA test (Hudson, Kreitman, and Aguadé 1987
) and the MK test (McDonald and Kreitman 1991
). For the HKA test, the data sets were partitioned as follows: the intraspecific comparison was between AdhC from the A-subgenome of G. hirsutum and AdhC from the D-subgenome of G. hirsutum; the interspecific comparison was provided by the AdhC sequences from the A- and D-subgenomes of G. barbadense. This test did not reveal any departure from neutrality (
2 = 0.84, P = 0.36). The MK test was performed by tabulating numbers of fixed and polymorphic synonymous and nonsynonymous substitutions in exons for all four data sets and performing a G-test (with Williams correction) of independence. The MK test did reveal a significant departure from neutrality (G = 6.924, P = 0.0085, fig. 5
) caused by an excess of polymorphic replacement substitutions.
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Discussion |
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Gene Genealogies and Coalescence
AdhC gene genealogies were constructed separately for the A- and D-subgenome sequences of G. hirsutum and G. barbadense (figs. 2 and 3 ). These genealogies reveal different patterns of haplotype distribution in the two subgenomes. The A-subgenome sequences reflect a simple underlying pattern (fig. 2
): G. hirsutum and G. barbadense sequences are separated on the genealogy by at least four substitutions. If this tree is rooted with an AdhC sequence from an A-genome diploid species (G. arboreum), the root falls on the branch separating the G. hirsutum and G. barbadense sequences; i.e., AdhC alleles coalesce within species. All G. barbadense sequences fell into a single haplotype. Gossypium hirsutum sequences were represented by seven different haplotypes; three of these were at intermediate frequency and four were found as homozygotes in single individuals. No recombination was detected among these sequences.
The pattern depicted in the genealogy of the D-subgenome sequences, on the other hand, is more complex (fig. 3 ). If this tree is rooted with an AdhC sequence from a D-genome diploid species (G. raimondii), the genealogy is divided into two neighborhoods. Three different G. barbadense haplotypes were recovered, all of which fall into the neighborhood above the root. The majority (8/12) of the G. barbadense sequences fell into a single haplotype; two additional haplotypes were observed as homozygotes in single individuals. The most frequent G. barbadense haplotype was also observed in two G. hirsutum accessions, once as a homozygote and once as part of a heterozygote. A number of additional G. hirsutum haplotypes are also observed in this neighborhood, each being one to four substitutions different from the most frequent haplotype in this neighborhood. The remaining G. hirsutum sequences are found in the neighborhood below the root. A long branch connects the two neighborhoods, and most haplotypes are found near the ends of these neighborhoods, although a few low-frequency G. hirsutum haplotypes are found along the branch.
D-subgenome AdhC sequences from G. hirsutum do not coalesce within the species; in fact, a number of haplotypes found in G. hirsutum are more closely related to G. barbadense sequences than they are to G. hirsutum sequences of the other neighborhood, and one haplotype is shared by G. barbadense and G. hirsutum. One explanation for the transspecies polymorphism observed is that it is caused by the inheritance of ancient polymorphism(s) from the common ancestor of G. hirsutum and G. barbadense. An alternative explanation is that these sequences have been introgressed from G. barbadense into G. hirsutum, a phenomenon previously observed between these two species (Brubaker, Koontz, and Wendel 1993
). It may be noteworthy in this respect that the G. hirsutum accessions with G. barbadense-like haplotypes all occur in the southern Mexican states of Chiapas and Guerrero or in neighboring Guatemala, the region of sympatry between G. hirsutum and G. barbadense. The pattern of introgression described by Brubaker, Koontz, and Wendel (1993)
is consistent with our data, in that they detected introgression from G. barbadense into G. hirsutum primarily in wild or feral populations in the region of sympatry. Introgression from G. hirsutum into G. barbadense, however, was generally restricted to modern cultivars.
However, the transspecies polymorphism is observed only in the D-subgenome sequences, not in the A-subgenome sequences. Introgression would not be expected to be restricted to a single locus unless strong selection was acting to promote introgression in the D-subgenome or to prevent it in the A-subgenome. No evidence of such selection pressure has been demonstrated.
Importantly, regardless of the ultimate source of these G. barbadense-like alleles, their impact on the patterns of diversity is not overwhelming. If these sequences are removed from the analyses, in the D-subgenome of G. hirsutum drops from 0.00649 to 0.00443, and
w drops from 0.00522 to 0.00507these values are still well above those observed in other Gossypium species or subgenomes. Additionally, the results of the MK test are still significant if the G. barbadense-like alleles are excluded.
Comparative Evolutionary Dynamics of AdhA versus AdhC
Our previous study demonstrated that nucleotide substitution rates are higher for AdhC than for AdhA at both silent and nonsynonymous sites (Small and Wendel 2000a
). Neutral theory predicts that evolutionary rates and nucleotide diversity will be positively correlated (Hudson, Kreitman, and Aguadé 1987
), suggesting that nucleotide diversity should be higher for AdhC than for AdhA. That expectation is confirmed by our data, where on a per genome basis nucleotide diversity is higher for AdhC in every comparison. Furthermore, allelic diversity is consistently higher for AdhC than AdhA with 26 unique haplotypes recovered for AdhC (24 in the D-subgenome of G. hirsutum) as opposed to six haplotypes for AdhA (a maximum of four in any single genome, again in the D-subgenome of G. hirsutum). In addition, haplotype diversity is more widely dispersed on the gene genealogy in the D-subgenome than in the A-subgenome. Gossypium hirsutum D-subgenome sequences differ by up to 27 nucleotide substitutions (fig. 3
), whereas the most divergent A-subgenome sequences differ by only four nucleotide substitutions (fig. 2
).
In addition to the higher overall diversity of AdhC relative to AdhA, the patterns of silent and nonsynonymous diversity for the two loci are different. Specifically, no nonsynonymous diversity was detected for the AdhA genes, whereas nonsynonymous diversity accounts for a significant portion of the diversity at AdhC (table 1
). Variation in silent versus nonsynonymous evolutionary rates has previously been described in plant genomes. For example, Gaut (1998)
examined rate variation among nine nuclear genes for a rice-maize comparison and found that synonymous rates varied over a 2.4-fold range, and nonsynonymous rates varied over a 10-fold range. More relevant to the present study, five loci of the Gossypium Adh gene family have synonymous rates that vary over a 2.9-fold range and nonsynonymous rates that vary over a 3.3-fold range (Small and Wendel 2000a
). The source of this variation in relative rates may be caused by either genomic processes that differentially affect the two loci, differential selective pressures on the two loci, or a combination of these factors. Recent evidence from extensive analyses of mammalian genomes suggests that evolutionary rates vary by genomic region, with genes from the same region showing similar synonymous rates (Matassi, Sharp, and Gautier 1999
). Alternatively, different selection pressures on the two loci may be responsible for the observed differences in silent and nonsynonymous diversity. Support for this hypothesis is provided by the results of the MK test, which reveals that the patterns of synonymous and replacement substitutions at AdhC are not in accordance with neutral expectations. Specifically, there is an excess of polymorphic replacement substitutions, the majority of which (12/14) are polymorphic in the D-subgenomes of G. hirsutum or G. barbadense (or both). This observation contrasts with the lack of replacement substitutions in AdhA, suggesting differential selective pressures on AdhA and AdhC, either purifying selection on AdhA, relaxed selection on AdhC, or a combination of the two. The lack of significant results for the Tajima or Fu and Li neutrality tests suggests that there is no disruption of the pattern of a neutral array of nucleotide substitutions, although the power of these tests is notoriously low (Simonsen, Churchill, and Aquadro 1995
), and the effect of deviations from the tests assumptions (e.g., random matingGossypium species are strongly selfing) is unknown.
Thus, our observation of consistently greater nucleotide diversity and elevated nonsynonymous substitution rates in comparing AdhA and AdhC may be accounted for either by differential genomic context of the two genes, differential selective pressures on the two genes, or a combination of the two phenomena. Evidence is presented for differential selective pressures; the influence of genomic context, however, cannot be evaluated until similar data are available for genes in the same genomic context as AdhA and AdhC.
Comparative Evolutionary Dynamics of A-subgenome versus D-subgenome Sequences
Whereas our data clearly show that evolutionary dynamics differ between loci (AdhA vs. AdhC), the data also suggest differential patterns of evolution between sequences from the A- and D-subgenomes of allotetraploid Gossypium. In all pairwise comparisons of nucleotide diversity between subgenomes within a species (e.g., G. hirsutum A-subgenome vs. G. hirsutum D-subgenome for AdhC), nucleotide diversity is consistently higher in the D-subgenome (table 1
, fig. 4
). Likewise, the number of haplotypes recovered in each data set is consistently higher in the D-subgenome, with ratios ranging from 2:1 to 3.4:1 (table 1
). Further, relative rate tests for AdhC have shown that the D-subgenome sequences are evolving at a significantly faster rate than A-subgenome sequences (Small et al. 1998
; Small and Wendel 2000a
). Finally, the excess of polymorphic replacement substitutions at AdhC, as evidenced both by the MK test and the high nonsynonymous diversity in the D-subgenome of G. hirsutum, suggests relaxed selection on the D-subgenomes of G. hirsutum and G. barbadense, at least for AdhC.
The genetic redundancy created by allopolyploidy or the large Adh gene family in Gossypium (at least seven loci in the diploid species [Small and Wendel 2000a
]) may have allowed relaxed selection in the D-subgenome, whereas purifying selection maintained a narrower array of A-subgenome sequences. This hypothesis is consistent, with respect to both AdhA and AdhC, with the elevated evolutionary rate of the D-subgenome sequences over the A-subgenome sequences (Small et al. 1998
) and the higher diversity in the D-subgenomes relative to the A-subgenomes. In addition, the presence of an intron-splice site mutation segregating in the G. hirsutum AdhC gene further suggests that, at least in G. hirsutum, this locus may be in the process of becoming a pseudogene.
Collectively, these observations might suggest an overall rate acceleration in the D-subgenome relative to the A-subgenome of allotetraploid Gossypium. Evolutionary rate analyses of 14 other nuclear loci, however, fail to reinforce this conclusion (Cronn, Small, and Wendel 1999
). The apparent contradiction between the results of Cronn, Small, and Wendel (1999)
and the present study indicates that either the Adh data are unusual, attributed perhaps to stochastic factors; the power of the relative rate tests alone are insufficient to detect subtle inequalities between subgenomes (as opposed to a combination of relative rate and nucleotide diversity analyses); or that something specific to these Adh loci promotes greater diversity in one of the two cotton subgenomes. We are currently unable to discriminate between these possibilities. Expression data and comparable studies of additional duplicated loci may provide critical clues in unraveling this conundrum.
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Acknowledgements |
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Footnotes |
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Keywords: Adh
alcohol dehydrogenase
polyploidy
cotton
Gossypium
nucleotide diversity
Address for correspondence and reprints: Randall Small, Department of Botany, 437 Hesler Biology, The University of Tennessee, Knoxville, Tennessee 37996-1100. rsmall{at}utk.edu
.
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