* Department of Biological Sciences, Stanford University
Department of Field Crops and Genetics, Faculty of Agriculture, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot, Israel
Institute of Horticulture, ARO-Volcani Center, Bet-Dagan, Israel
Correspondence: E-mail: liord{at}stanford.edu.
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Abstract |
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Key Words: duplication polyploidy segregation genome evolution diversification carp
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Introduction |
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Assuming a whole-genome duplication to explain the genome structure of intensively studied species such as yeast, Arabidopsis thaliana, and Homo sapiens raised several difficulties, mostly because of the time elapsed since the duplication event (Wolfe 2001). The low proportion of duplicated segments in these species and the shuffling in the structure of their ancestral chromosomes (Postlethwait et al. 2000; Friedman and Hughes 2001) leave the theory of genome duplication in debate. Although a substantial amount of evidence suggests a whole-genome duplication (e.g., Gu, Wang, and Gu 2002; McLysaght, Hokamp, and Wolfe 2002), many segmental duplications were also found (Bailey et al. 2002). The partially duplicated structure of the yeast and human genomes raised the possibility that this structure could also be a result of multiple independent segmental duplications (Llorente et al. 2000; Hughes, da Silva, and Friedman 2001).
A few fish species are supposed to have had an additional recent round of genome duplication late in the evolution of the teleosts that might have led to their speciation. Among these are the catastomid fishes (suckers), with an estimated duplication time of 50 MYA (Uyeno and Smith 1972); the salmonids, with an estimated time of duplication of 25 to 100 MYA (Allendorf and Thorgaard 1984); and the common carp and the goldfish (Ohno et al. 1967; Larhammar and Risinger 1994). Polyploidization has also been documented in some loaches (cobitidea) (Ferris and Whitt 1977b) and in sturgeons (Ludwig et al. 2001). The rarity of polyploidy in higher vertebrates is probably a result of genetic sex determination, and few fish serve as unique examples of polyploids with genetic sex determination (Otto and Whitton 2000).
Genome duplication in the evolution of the common carp (Cyprinus carpio) is supported by the following observations. Its chromosome number (n = 50) is twice that of other Cyprinidae, and its DNA content is higher (Ohno et al. 1967). In addition, about 52% of this carp enzymes show a pattern consistent with duplication (Ferris and Whitt 1977a). Tetraploidization of carp was suggested to take place about 50 MYA, similarly to catastomids since both express a similar proportion of enzymes in duplicates. The c-myc genes in carp gave an estimate of 58 MYA for the event of tetraploidization (Zhang, Okamoto, and Ikeda 1995). Other duplicated genes of the carp suggest a more recent divergence time of less than 16 MYA (Larhammar and Risinger 1994).
Carps of the family Cyprininae are the most cultivated species in aquaculture. Common carp is the third most cultivated species worldwide and is important in the European freshwater aquaculture, where its production has increased substantially over the last decade (FAO: http://www.fao.org/fi/default.asp). Though agriculturally important, genomic information on the carp is limited, and only 1,227 nucleotide and 606 protein sequences are currently available in GenBank (http://www.ncbi.nlm.nih.gov/). The common carp lacks a genetic linkage map, and about 100 microsatellite markers have been developed for this species (Crooijmans et al. 1997; Aliah et al. 1999; David et al. 2001).
Microsatellite markers provide a codominantly inherited tool for studying mostly noncoding regions of the genome. In a few studies, microsatellites have been useful for the study of duplications (Ludwig et al. 2001; Pyatskowit et al. 2001; Angers, Gharbi, and Estoup 2002). In this paper we use microsatellite markers to investigate the existence and extent of duplications in the common carp genome.
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Materials and Methods |
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Microsatellite Loci
Genotyping at microsatellite loci was done as in David et al. (2001). When fluorescent primers were used (family II), PCR products were separated on a 6% polyacrylamide gel (Bio Lab Ltd. Jerusalem, Israel) on an ABI PrismTM 377 DNA Sequencer (Perkin Elmer, Foster City, Calif.). Otherwise, labeling was done by adding 2.5 µCi of 32P in a total volume of 10 µl reaction. Radioactive products were denatured and separated on 5% polyacrylamide gels in TBE buffer (as in David et al. [2001]). Primer sequences and PCR conditions are detailed as follows: (1) for markers with the prefix MFW, see Crooijmans et al. (1997); (2) for markers with the prefix CCA, see Aliah et al. (1999); and (3) for markers with the prefix Koi, see David et al. (2001). Markers were first tested for polymorphism among parents of each family and for the number of fragments per individual. We used 53 microsatellite markers to genotype the four parents of family I and family II, whereas six additional markers were genotyped in only two parents of either family. A subset of 39 polymorphic markers that resulted in two or more fragments per individual was then used for genotyping the progeny. Genotypes were scored visually.
Cloning and Sequencing of Alleles
We chose seven primer pairs that represented the various segregation patterns and sequenced 36 different fragments from these loci. For each of these primer pairs, we amplified DNA of individuals that represented the whole set of observed fragments. When fragments of the same size were cloned twice from different families, their sequences were found to be identical. Six sequences of stuttered fragments (that are typical of microsatellites) were excluded, and our further analyses are based on 30 sequences. Each PCR product was cleaned using a purification kit (High Pure PCR product, Roche Applied Sciences GmbH, Mannheim, Germany). Cloning was done using pGEM®-T vector system kit (Promega, Madison, Wis.). About 50 positive colonies were chosen for each PCR reaction and cultured overnight in 100 µl of liquid LB substrate in a 96-well plate. Identification of isolated fragments was done by a radioactive PCR reaction using 0.5 µl from the cultured clone as a template for the corresponding primer pair. Products were separated as detailed above. Clones for sequencing were grown overnight in 7.5 ml of LB medium, and plasmids were isolated using the Wizard® Plus SV Miniprep DNA purification system (Promega, Madison, Wis.). Sequencing was done by ABI 3100 automated sequencer (Perkin Elmer, Foster City, Calif.) using T7 or SP6 universal primers.
Analyses of Sequences
Alignment of sequences was done using the BioEdit software (Hall 1999) and then visually refined. For each sequence, the number of microsatellite repeats was counted and their sequence was deleted. The flanking regions were used to estimate the genetic relationship between fragments using Tamura-Nei distance (Tamura and Nei 1993) in the MEGA2 software (Kumar et al. 2001). Alignment data were bootstrapped, and gene trees were constructed using the Neighbor-Joining method (Saitou and Nei 1987), as implemented in MEGA2. Scales of genetic distances and divergence times for the gene trees were calculated by this software.
For analysis of microsatellites' flanking regions, we used a nucleotide substitution rate of 3.71 x 10-9 per site per year. Since these sequences are noncoding regions, we used the highest rate found for fourfold degenerate sites based on 47 mammalian genes, assuming a divergence time of 80 Myr between human and rodent lineages. For analysis of genes, we used the rate of 3.51 x 10-9 as estimated by Li (1997, p. 90) from synonymous sites in coding regions. Time of divergence was calculated using the formula T = K/2r, where K is the number of substitutions per base between homologous sequences and r is the rate of substitution. Estimation of means, distributions, and variances, as well as statistical analyses of substitution rates, divergence times, and repeat numbers were carried out using JMP4 statistical software (SAS Institute, Cary, N.C.).
Sequences of genes were retrieved from GenBank (http://www.ncbi.nlm.nih.gov/Genbank/index.html) using the Blast tool and taxonomic information was found in the taxonomy browser (http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/).
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Results |
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Patterns of Allele Segregation in Progeny
In total, 54 segregation patterns were analyzed among 39 polymorphic primer pairs (table 1). Some primer pairs were genotyped in more than one family. For example, primer pair Koi89-90 (no. 6 in table 1) was studied in an F2 family where both parents had three fragments each, and in total four fragments were found. The segregation pattern in progeny revealed that 83% of the offspring had three fragments (table 2 and fig. 1a). All offspring had a 164-bp fragment, whereas the other three fragments segregated (fragments 194 and 184 are present in approximately half of the fish, and fragment 182 is present in 76% of them). The proportions of the genotypes in progeny approach a 1:1:1:1 ratio. Primer pair Koi53-54 amplified three fragments in parents of family II (table 2). Fragments 158 and 175 were found in about 50% of the offspring, whereas fragment 183 was detected in all fish. Four different genotypes were found in proportions approaching a 1:1:1:1 ratio; about one-quarter of the offspring had all three parental fragments. From such segregation patterns (i.e., frequencies of alleles and genotypes in progeny) and genotypes of the parents, we inferred which fragments have allelic relationships and which fragments are not alleles but duplicates (paralogs). The expected ratio of genotypes in the progeny was based on genotypes of the parents and assuming either a disomic or tetrasomic mode of inheritance. For example, four fragments were amplified in parents of family II using primer pair CCA 17 (table 2). The two paternal fragments were found in all offspring, and two of the three maternal fragments segregated in a 1:1 ratio. The segregating fragments were defined as alleles since none of the offspring had both. Fragment 187 was assumed to be in a homozygous state with fragment 150 as its duplicate since all offspring had both. These relationships predict two genotypes in progeny, with an expected ratio of 1:1 under disomic inheritance. The observed proportions of genotypes fits this postulated ratio (2 = 0.02, P = 0.89). Fourteen patterns out of 54 (25.9%) deviated significantly from expected ratios (
2 test, P < 0.05 [table 1]). All primer pairs that fitted a tetrasomic mode of inheritance also fitted a disomic model. On the other hand, some of the patterns that fitted a disomic mode of inheritance could not be explained by tetrasomic inheritance. Loci that were defined as diploid and mainly those whose alleles segregated in a 1:1 ratio could fit a tetrasomic model as well. However, disomic inheritance was used to explain segregation patterns, since it is more conservative and explains more segregation ratios at both single and duplicated loci.
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For genotyping of progeny, we chose primer pairs where segregation was expected based on the genotypes of the parents. Primer pair Koi111-112 had four fragments in both parents, and all these alleles segregated in the progeny (fig. 1b). Such loci were defined as duplicated, based on both fragment number and segregation pattern. However, some primer pairs that had no more than two fragments per parent gave rise to a segregation pattern that revealed a duplicate locus (e.g., primer pair Koi53-54 in family II [table 2 and fig. 2]). Furthermore, primer pairs where both parents appeared to be heterozygous were found to amplify paralogous loci; thus, all progeny had all the fragments from both parents (e.g., primer pair Koi89-90 in family I [table 2]). Such primer pairs, where the fragment number indicated a diploid locus while the segregation revealed paralogs, demonstrate the complex relationship between duplicates and point towards the importance of the segregation analysis.
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Of the 21 segregation patterns that fitted a disomic and duplicated model (type 1, [table 1]) only primer pair Koi111-112 in the F2 family supported two separate duplicates. All 20 of the other patterns under disomic inheritance can be equally explained as either tandem duplication or unlinked duplicates. All of these loci are either differentially fixed or have only one duplicate segregating. This monomorphism, in at least one duplicate, does not allow segregation between duplicates, which is necessary to determine whether loci are or are not linked. For example, primer pair Koi53-54 in family II can be explained as a tandem duplication if the sire had two haplotypes (;
) and the dam had two haplotypes (
;
). We could obtain the same genotypic proportions in progeny if the two duplicates were unlinked (fig. 2). A primer pair where parents had four alleles (Koi111-112 [fig. 1b]) allows the two separate loci to segregate.
Primer pair MFW 23 is the only suggested tandem duplication (tables 1 and 2) since it has two segregating haplotypes (); (
). The two duplicates that resulted in three fragments for some of the individuals appear to be completely linked. This explanation can be made because there is polymorphism both within and between duplicates.
Sequences of Cloned Alleles
For each primer pair, the range of the allelic fragment size and the repeat number is presented in table 3. Variation was found in the number of tandem repeats and in the flanking regions of the repeat, both contributing to the observed variation in fragment size. For each primer pair, we found fragments differing in the number of repeats but identical in the sequence of the flanking regions (zero genetic distance). On the other hand, fragments differing in the sequence of the flanking region differed also in the number of repeats. The maximal genetic distances between fragments of a given primer pair (table 3) can be grouped into three distinct levels: (1) 0.005 for primer pair Koi29-30; (2) 0.033-0.061 for primer pairs Koi89-90, MFW 23, and Koi3-4; and (3) 0.099 to 0.114 for primer pairs Koi35-36, Koi111-112, and Koi105-106.
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We included insertions and deletions (indels) and calculated the rate of total differences per base between all possible pairs of sequences. The distribution of these differences has three subgroups (fig. 4). There are 53 differences in two categories: 24 between alleles (diploid sets) and 29 between paralogs. At primer pairs for which segregation patterns were not fully informative, we could not define the allelic relationships between all sequences. In these cases, we used the clustering in the gene tree as an indication of allelic relationship. In figure 4, the distribution of allelic differences is skewed to the left (Shapiro-Wilk W test, P < 0.0001) and the mode is at 0.01. The average difference per base is 0.013, and the range is 0 to 0.047. The distribution of differences between duplicates has two distinct modes (fig. 4). The left distribution is normal (P = 0.27), with an average of 0.034 and a range of 0.017 to 0.050. The right distribution is also normal (P = 0.46), with an average of 0.109 and a range of 0.094 to 0.124. The range of allelic differences overlaps the left distribution, as expected from the scales for the gene trees (fig. 3) and the three groups of genetic distances (table 3).
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Discussion |
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Higher likelihood for disomic segregation was found for duplicates. However, disomic inheritance can characterize both tandem and segmental duplications (e.g., primer pair MFW 23 [table 3]). Segregation patterns of the duplicated loci, when differentially fixed or with only one segregating duplicate, can be equally explained as either tandem or unlinked duplicates (fig. 2). In fact, 20 of 21 segregation patterns of paralogs had this character. Tetraploidy however, predicts two separate duplicates rather than tandem duplication. In our study, segregation patterns strongly support the duplicated nature of loci but provide little support for a whole-genome duplication due to limited polymorphism within paralogs. However, support for tetraploidy was found in the proportion of paralogs.
Of the informative primer pairs, 60% have at least one parent with more than two fragments. We therefore offer 60% as an estimate of the extent of duplications at microsatellite loci in the common carp genome. Primer pairs such as Koi17-18 and Koi53-54 had up to two alleles in parents of one family and were found to be duplicated in the other family (table 1). Thus, 60% may be an underestimate of the proportion of duplicated loci since it is based on fragment number, although it agreed with the segregation patterns in 86% of the cases. In addition, a fraction of the noninformative loci (10%) could be found to be duplicated if studied in informative families. Our estimate is slightly higher than the estimate of 52% duplications based on expression of isozymes and allozymes from 23 loci (Ferris and Whitt 1977a), which for the same reasons may also be an underestimate.
It has been hypothesized that Homo sapiens, yeast, and Arabidopsis thaliana are paleopolyploids, having a proportion of 12.5%, 16%, and 25% duplicated genes, respectively (Wolfe 2001). Therefore, the higher proportion of duplications in common carp may suggest a whole-genome duplication rather than several segmental duplications. In the past, the hypothesis of the common carp being a tetraploid was based mainly on its chromosome number (2n = 100 to 104 chromosomes) and high nuclear DNA content (Ohno et al. 1967). Moreover, if diploidization of duplicates occurs at a similar rate among species, one can assume that the genome duplication of the carp took place much later in evolution than the duplications in these three fully sequenced species. Therefore, our survey of microsatellite loci supports the hypothesis that this carp had relatively recent genome duplication.
The hypothesis that the genome duplication in this carp resulted from allotetraploidization has two supporting pieces of karyotipic evidence: no quadrivalents have been detected in meiotic nuclei and no chromosomes were lost in the duplication event (Ohno et al. 1967). Hybridization that results in a tetraploid species is more likely between closely related species. The carp has a doubled number of chromosomes in two distinct, although similar, sets as suggested by the identification of paralogs using the PCR method. Evolution of polyploid genomes makes the distinction between allotetraploidy and autotetraploidy, based on disomic inheritance, more difficult as time elapses (Wolfe 2001). In light of the relatively short time since tetraploidization of the carp, we suggest that the disomic inheritance in carp resulted from allotetraploidization rather than by diploidization of an autotetraploid genome. In addition, the phylogeny of cyprinids fits allotetraploidy in the sense that the clades of diploid and tetraploid species coalesce before the divergence of the diploid parents of the common carp.
Higher variation between paralogs and lower variation between alleles was suggested as evidence of the duplicated nature of four tested genes in the common carp by Larhammar and Risinger (1994). We established paralogous or allelic relationships between fragments by segregation analysis and only then assessed their sequence variation. Our results confirm that sequence variation as well as variation in the number of repeats between paralogs is higher than that between alleles. The modal difference between alleles is 0.01, equivalent to 0.008 and ranging up to 0.034 substitutions per nucleotide. Thus, the frequency of potential SNPs in these regions was estimated to be 1/125 bases.
The distribution of variation between paralogous sequences formed two normally shaped distributions rather than one continuous distribution (fig. 4). The distributed values could result from different duplication times among loci and/or variation in evolutionary rate of these loci. Accordingly, two distinct distributions of duplications could be explained by (1) a continuous series of duplications over time that did not cover the gap between the observed modes due to small sample size, (2) two distinct duplication events with different evolutionary rates within each event, or (3) a combination of discrete and continuous events. The subdistribution of allelic differences implies that variation in evolutionary rate among loci exists. We suggest that the right subdistribution of differences between paralogs represents a whole-genome duplication with variation among loci, whereas the left subdistribution indicates a later surge of segmental duplications. The segmental duplications are represented by primer pairs Koi89-90, MFW 23, and Koi3-4, which show smaller genetic distances between paralogous fragments, and supported by primer pair MFW 23, whose segregation pattern suggested a tandem duplication. The normal subdistribution of recent duplications is different from the uniform distribution that would be predicted for a continuous series of segmental duplications. The apparently normal shape could be an artifact of small sample size that might prevent detection of younger or older representatives of this group. Alternatively, the segmental duplications could have occurred in bursts rather than continuously. Large-scale studies by McLysaght, Hokamp, and Wolfe (2002) and by Gu, Wang, and Gu (2002) suggest that at least one genome duplication took place in the evolution of humans (chordates) but small-scale duplications might have taken place as well. Gu, Wang, and Gu (2002) found two waves of duplications and interpreted these as evidence of an ancient genome duplication and a more recent expansion of gene families that resulted from tandem or segmental duplications. Our results (fig. 4), although on a much smaller scale, may be another example of a genome duplication followed by a more recent wave of segmental duplications.
The variance about the mean number of differences between paralogs is similar in magnitude to the corresponding variance between alleles (table 5), suggesting a single distribution of mutation rates for alleles and paralogs at microsatellite loci.
The average divergence age between microsatellite paralogs is 12 Myr, ranging between 9.5 and 13.5. Sequence comparison of the urotensin II- and urotensin II-
genes yielded a similar divergence time of 12.9 MYA, which supports the microsatellite estimate and suggests that these genes are paralogs. Our estimate based on microsatellite loci (i.e., 12 MYA) is a little lower than the previous estimate of 12 to 19 MYA based on only two genes (Larhammar and Risinger 1994). In calculating time estimates, we used substitution rates from mammals. Evidence from fish suggests lower mutation rates than in mammals (Rico, Rico, and Hewitt 1996; Krieger and Fuerst 2002) that might cause an underestimation of the times in our study. However, both divergence time and the proportion of duplicates in common carp place it among the few vertebrates in which a recent genome duplication occurred. This relatively young duplication of the genome (suggested for a few species of fish) is additional to the duplications that are proposed for all vertebrates and is also additional to that preceding the teleost fish radiation (Amores et al. 1998; Taylor et al. 2001; Wolfe 2001). These fish species, including this carp, may therefore be highly informative models for the study of genome evolution after duplication. For example, if differential fixation that was found in 28% of the microsatellite loci exists in duplicated genes, then the common carp might be a good model for the study of the functional consequences of such a state.
The region of the thyrotropin gene examined here suggests a coalescence time of 21 MYA for the Chinese carps (grass and bighead carps) that have 2n = 50 chromosomes and the common carp (goldfish) that have 2n = 100 chromosomes (fig. 5). This estimate is in agreement with the estimate of 19 MYA obtained by Larhammar and Risinger (1994). It is reasonable to assume one genome duplication for the common carp and the goldfish, which took place before their divergence at 11 MYA. Therefore, 11 to 21 MYA is the estimated interval at which genome duplication of the common carp took place. In theory, the two species that formed the ancestor of the common carp should have been close enough to be able to hybridize but different enough to generate disomic inheritance. The divergence time between paralogs provides an estimate of the time at which the two ancestral diploid species separated, and indeed 12 MYA is within the time interval that was suggested by the gene tree of thyrotropin. If the divergence of the common carp and the goldfish followed shortly after the genome duplication, as suggested by their phylogeny, then it may be an example of the role of polyploidization in speciation and diversification.
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Acknowledgements |
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Footnotes |
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