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Genetic analysis of two common carp broodstocks by RAPD and microsatellite markers
Aquaculture 219 (2003) 157 – 167 www.elsevier.com/locate/aqua-online
Genetic analysis of two common carp broodstocks by RAPD and microsatellite markers Richa´rd Ba´rtfai a,b, Sa´ndor Egedi b, Gen Hua Yue a, Bala´zs Kova´cs b, Be´la Urba´nyi c, Gizella Tama´s d, La´szlo´ Horva´th c, La´szlo´ Orba´n a,b,* a
Reproductive Genomics Group, Temasek Life Sciences Laboratory, 1 Research Link, The NUS, Singapore 117 604, Singapore b Laboratory of Aquatic Molecular Biology, Agricultural Biotechnology Center, POB 411, Go¨do¨llo , H-2101 Hungary c Department of Fish Culture, Faculty of Agricultural and Environmental Sciences, Szent Istva´n University, Pa´ter K. u.1, Go¨do¨llo , H-2103 Hungary d Dinnye´s Fish Farm, Dinnye´s H-2485 Hungary
Received 29 November 2001; received in revised form 28 April 2002; accepted 12 September 2002
Abstract The whole broodstock of two Hungarian common carp farms—80 and 196 individuals—was analyzed by using random amplified polymorphic DNA (RAPD) assay and microsatellite analysis. Ten polymorphic RAPD markers and four microsatellites were selected to genotype both of the stocks. As expected, microsatellite analysis revealed more detailed information on genetic diversities than RAPD assay. Results obtained with both types of DNA markers showed lack of major differences between the genetic structure of the two stocks: heterozygosity values and allele frequencies were very similar. Dendrograms created from both sets of data did not show grouping of individuals according to stocks. Genotypes from the two stocks were also compared to those from a limited number of samples collected from other hatcheries and two rivers. Allele frequencies in the groups were similar, with the exception of wild carps. An interesting observation was that three private microsatellite alleles were found in the eight wild carp individuals, compared to the seven detected in the rest of the samples tested (372 individuals). D 2003 Elsevier Science B.V. All rights reserved. Keywords: Cyprinus carpio; Cyprinid; Genotyping; Fish breeding; Landrace
* Corresponding author. Reproductive Genomics Group, Temasek Life Sciences Laboratory, 1 Research Link, The NUS, Singapore 117 604, Singapore. Tel.: +65-6872-7413; fax: +65-6872-7007. E-mail address: [email protected] (L. Orba´n). 0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 ( 0 2 ) 0 0 5 7 1 - 9
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1. Introduction Common carp (Cyprinus carpio L.) belongs to Cyprinidae, the largest family among freshwater teleosts (Nelson, 1994), for which the world’s annual total catch in 1999 was estimated above 15.6 million metric tons, compared to the 2.3 million tons of salmonids (FAO, 2001). Classical taxonomic analysis divides the currently existing common carp forms into three categories: (1) European (Cyprinus carpio carpio), (2) Far Eastern (C. carpio haematopterus) and (3) SouthEast Asian (C. carpio viridiviolaceus) (Kirpitchnikov, 1999). Common carp varieties (e.g. races, landraces, strains, breeds and stocks) ‘‘developed through a combination of forces including geographic isolation, adaptation accumulation of mutations and natural as well as human selection pressures’’ (Hulata, 1995). The Middle and Eastern European region is home for many common carp varieties. However, their origin and relatedness are not well described. Despite the commercial importance of the species, genetic data on common carp stocks are relatively scarce. Analysis of protein polymorphisms was performed on some populations (Csizmadia et al., 1995; Kohlmann and Kersten, 1999). However, reports have only been published recently on common carp genotypes with random amplified polymorphic DNA (RAPD) (Dong and Zhou, 1998) and microsatellite markers (Crooijmans et al., 1997; Aliah et al., 1999; Tanck et al., 2000, 2001; Desvignes et al., 2001; David et al., 2001; Lehoczky et al., 2002). The present work deals with the genetic analysis of two famous common carp varieties of Hungary: the entire Attala and Dinnyes broodstocks and their comparison with over 100 individuals collected from various other sources.
2. Materials and methods 2.1. Stocks, sample collection and DNA isolation Samples from the Dinnyes broodstock (196 individuals) were collected during the April – May period of 1996, whereas the Attala broodstock (80 individuals) was sampled 1 year later. All individuals of both broodstocks were sampled and labeled by inserting an electronic passive integrated transponder (PIT) tag (Destron Fearing, South, St. Paul, MN, USA). One hundred and four samples were also obtained between 1996 and 1998 from the following sources located in Hungary: Boszormeny (50 individuals), Tata (35), Bikal (6), Szajol (5) and wild (8; four from Tisza river and four from Danube river). The 96 samples from the first four sources (all domesticated stocks) were merged into one group (‘‘others’’) in order to obtain similar sample size with that of the stocks, whereas the remaining eight was labeled as the ‘‘wild’’ group. Fin clips were cut from brooders anaesthetized with Quinaldin (Reanal T-2051, Hungary). They were then placed into 1 ml SET buffer (10 mM Tris/HCl, pH 8.0, containing 50 mM EDTA, 200 mM NaCl and 0.5% SDS). Tissue samples were digested with 0.5 Ag/Al Proteinase K at 55 jC overnight, the resulting solution was centrifuged, the supernatant precipitated in ethanol and dissolved in 1 TE buffer. From some of the samples, genomic DNA was isolated by the phenol – chloroform procedure (Sambrook et
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al., 1989). The quality and concentration of DNA from both sources were assessed by agarose gel electrophoresis then samples were stored at 4 jC until use. 2.2. RAPD assay RAPD reactions were performed as described in Bercsenyi et al. (1998), but proteinasetreated and ethanol-precipitated fin clip sample was used as a template. The following primers were tested in the preliminary assay: OPF, OPM, OPO and OPQ series, with 20 primers each, plus three OPE primers (Operon, Alameda, CA, USA). RAPD reactions were run in a PCR1 Thermocycler (Perkin Elmer, Palo Alto, CA, USA). RAPD products (20 Al load) were separated on 2% agarose gels, containing 0.5 Ag/ml ethidium bromide. Band patterns were photographed under UV light. 2.3. Microsatellite analysis Primers were synthesized to the flanking regions of eight microsatellites: MFW4, MFW7, MFW9, MFW13, MFW17, MFW20, MFW26 and MFW31, according to Crooijmans et al. (1997). For preliminary testing of microsatellite markers, mastermixes contained (in 10 Al): 1 PCR buffer (Promega, Madison, WI, USA), 200 AM dNTP mix, 200 nM for both primers, 2 mM MgCl2, 40 ng template and 0.25 U Taq polymerase (Promega). PCR cycles were: 5Vpreamplification denaturation at 95 jC, then 20U at 94 jC, 30U at 63 jC and 20U at 72 jC for 10 cycles, followed by 20U at 94 jC, 30U at 53 jC and 20U 72 jC for 25 cycles. As a final step, products were fully elongated for 7V at 72 jC. Amplifications were performed on a PE 9700 thermocycler (Perkin Elmer) with ramp speed set identical to that of PE 9600. For parallel genotyping, the eight selected markers were grouped into four pairs (MFW7/9, MFW4/31, MFW13/20 and MFW17/26) on the basis of their allele range and their ability to be co-amplified in the same PCR reaction. For the duplex PCR, the same reaction mixture was used as above with the following primer concentrations: 200 nM of MFW7 with 400 nM of MFW9 for the first duplex, while 200– 200 nM of both MFW4 and MFW31 for the second one. For separation on denaturing polyacrylamide gels, 8-Al sequencing stop solution was added to the PCR product following amplification and it was denatured at 96 jC. Eight microliters of this solution was loaded onto 5%T, 2%CBis PA gels containing 6 M urea. Gels were cast in a 38 50-cm Sequi-Gen GT System (BioRad, Hercules, CA, USA) using a comb with 49 ‘‘sharkteeth wells’’. For separation of single microsatellite markers, a gel with uniform 0.4-mm thickness, while for duplexes, a 0.4– 1.2-mm wedge gel was used. Since the allele ranges of any two duplexes were at least partially overlapping in order to separate the alleles of four microsatellites on the same gel, the second pair had to be loaded with delay. Two duplex pairs (MFW7/9 + MFW4/31 and MFW13/20 + MFW17/ 26) were tested. In the first case, the products of the MFW4/31 amplification were loaded when bromophenol blue dye from MFW7/9 sample migrated to a 3-cm distance from the wells, while in the second case, MFW17/26 products were loaded when the dye from MFW13/20 reached 4.5 cm. Such a ‘‘delayed double duplex’’ setup allowed us to separate
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all alleles from the four markers with a few exceptions. In case of a suspected overlap of allele range, both markers in question were reamplified and separated in individual lanes. Following electrophoresis, gel cassettes were disassembled and a custom-made plastic frame was clamped to the glass plate holding the gel. Detection of microsatellite alleles was achieved by silver staining in this special container according to Varga et al. (1997). To determine the size of alleles obtained by the silver staining methods, labeled PCR products of 20 individuals were later analyzed on the ABI 377 sequencer as described previously (Yue et al., 2000). One of the two primers from each pair was labeled with either 6FAM or HEX dyes (Genset, Paris, France). PCR conditions for each locus were the same as described above. The length of the fluorescently labeled PCR products was determined against the size standards (Tamra-GS-500) using Genescan and Genotyper software (Applied Biosystems, Foster City, CA, USA). The 20 individuals were selected to cover most of the alleles of the three sample groups; for those not present, the allele length was estimated by comparison to their closest known neighbors on the scale. 2.4. Analysis of data RAPD patterns were visually analyzed and scored from photographs. For the analysis and comparison of the patterns, a set of distinct, well-separated bands were selected (see Fig. 1). The genotypes were determined by recording the presence (1) or absence (0) of these bands only, neglecting other (weak and unresolved groups of) bands. Two assumptions were made for the analysis of the RAPD data: (1) markers from different loci did not migrate to the same position on the gel, (2) each band was assumed to represent the dominant genotype at the locus, whereas lack of the same band in another individual was assumed to correspond to the alternative homozygous recessive genotype in the Hardy– Weinberg equilibrium (Lynch and Milligan, 1994). Genetic similarity (GS) between individuals i and j was estimated according to the formula given by Nei and Li (1979): GSij ¼ 2Nij =ðNi þ Nj Þ where Nij is the number of bands common in individuals i and j, and Ni and Nj are the total number of bands in individuals i and j, respectively, with regard to all assay units. Thus, GS reflects the proportion of bands shared between two individuals and ranges from 0 (no common bands) to 1 (all bands identical). Genetic dissimilarity (GD) was calculated as: GD ¼ 1 GS The expected heterozygosity (He) and percentage of the polymorphic loci were calculated by using the software called Tools for Population Genetic Analysis (TFPGA; Miller, 1997). Microsatellite data were scored visually from silver-stained, dried gels. Allele lengths were assigned on the basis of the results obtained with labeled primers on a subset of samples. In the majority (>60%) of the individuals tested, the primers for marker MFW31 amplified extra bands (ca. 240 – 255 bp) confirming the observation of Crooijmans et al. (1997). These bands were not scored. The expected (He) and observed heterozygosity
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(Ho), number of alleles, fixation index (Fis), allele frequencies and Hardy –Weinberg equilibrium were analyzed using software Genetic Data Analysis (GDA; Lewis and Zaykin, 2000). The definition of ‘‘private alleles’’ labels alleles, which are only present in one stock and absent from all others tested. The pairwise similarity between individuals was calculated by using software Microsat (Minch, 1996). The dendrograms from the GD values determined by RAPD and from the genetic dissimilarity data determined by microsatellite analysis were constructed based on the NJ method by using software NTSYSpc (Exeter Software, Setauket, NY, USA). We tested both stocks, whether they went through a bottleneck during their history by using the software Bottleneck 1.2.02 (Cornuet and Luikart, 1996; Piry et al., 1999). The software tests for the departure from mutation drift equilibrium based on heterozygosity excess or deficiency. Out of the three options provided, we used stepwise mutation model (SMM) and the two-phased model (TPM). By the using the same software, the allele frequency distribution of the microsatellite loci was also examined for model shift (Luikart et al., 1998), which would indicate a recent genetic bottleneck, if any.
3. Results 3.1. Preliminary analysis for selection of markers Six genomic DNA samples were selected randomly from among the individuals of the Dinnyes stock. These templates were then amplified with a total of 81 10 mer random primers (see Section 2.2 for details). Three primers (OPE-12, OPM-14 and OPQ-09) generating polymorphic patterns, which were clear and easy-to-interpret (for representative band patterns, see Fig. 1A) were selected for further analysis. Out of more than 30 bands amplified by the three primers, 17 stabile and characteristic bands (i.e. those which were reasonably strong, easy to score and reproducible) were selected for genotyping of all individuals from both stocks. For preliminary analysis of the microsatellites, samples were chosen randomly from the following sources: Attala (12), Dinnyes (8), Boszormeny (8), Bikal (4), Szajol (4), wild-Danube (4) and wild-Tisza (4). The eight markers were split into two groups (four markers each) and tested in the ‘‘delayed double duplex’’ system (see Section 2.3 for details). The marker quadruplet containing MFW4/31 and MFW7/9 showed more stabile bands and better separation of alleles than the other did (MFW13/20 + MFW17/ 26). Thus, the MFW4/31+ MFW7/9 ‘‘delayed double duplex’’ was selected for further use. 3.2. Analysis of the Attala and Dinnyes broodstocks by RAPD markers Seventeen bands were selected from the three RAPD patterns for genotyping of the broodstocks. Nine of the markers were polymorphic, while seven failed to show sufficient polymorphism (>5%) in both stocks (for complete list of markers and their polymorphisms, see Fig. 1B.). The remaining marker (M14-950) was missing from all individuals of the Attala stock and present in one quarter of the other population.
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Fig. 1. RAPD analysis of the Attala and Dinnyes common carp stocks. Panel A: representative RAPD patterns obtained with the three selected primers (OPM-14, OPE-12 and OPQ-09). Each subpanel contains a gel picture with a molecular weight marker (lambda/PstI) on the left followed by patterns amplified from four individual samples. On the right of each picture, a sketch is shown indicating those bands with their approximate size, which were scored from the RAPD pattern. Panel B: frequencies of the 17 bands amplified by the three primers from the two broodstocks. Bands are identified by the primer and their estimated length in basepairs.
The expected heterozygosity was lower in the Attala stock (0.16) and somewhat higher (0.18) in the Dinnyes stock. Four of the polymorphic markers (M14-1400, E12-1900, Q91300, Q9-950) showed very similar frequency in the two broodstocks, while the percentage values for the others were different, especially for M14-950 and M14-850. When a dendrogram was generated from the RAPD genotypes, individuals from the two stocks were not separated into distinct groups; instead, they were mixed throughout the tree (data not shown). 3.3. Analysis of the two broodstocks and samples from other stocks by microsatellite markers All DNA samples from the two stocks (80 and 196 individuals) and those from the other sources (104 individuals) were tested for the four selected microsatellite markers (MFW4/31 + MFW7/9) by using the ‘‘delayed double duplex’’ method. At the four loci, a total of 47 alleles were detected across the four groups: Attala, Dinnyes, ‘‘others’’ and ‘‘wild’’. Allele frequencies at each locus were basically similar in their distribution, except for the wild carps (Fig. 2). Equal number of private alleles (three each) were found in the Dinnyes stock, the ‘‘others’’ and the wild carps, whereas in the Attala stock only one private allele was detected at the four loci. The frequency of private alleles was very low, ranging from 0.003 to 0.027 in the three large groups, but about two magnitudes higher (0.125 – 0.25) in case of the eight wild individuals, as expected (Fig. 2). The average expected heterozygosity was 0.83 in the Attala stock and 0.81 in the Dinnyes stock, while the observed heterozygosity was 0.69 for both (Table 1). No
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Fig. 2. Allele frequencies for the four microsatellite loci tested on the four sample groups. Number of samples tested per group: Attala = 80, Dinnyes = 196, others = 96 and wild = 8.
statistically significant (df = 3, P>0.05) difference has been detected between the heterozygosity values observed in the two populations. The inbreeding coefficient was also similar in the two stocks (Attala: 0.17, Dinnyes: 0.15). When the four markers were used to test for deviations from the Hardy – Weinberg equilibrium, only MFW7 gave a probability bigger than 0.05 ( P = 0.245) in the Attala stock (data not shown), indicating that both populations are in disequilibrium for most loci tested. When tested for signs of bottlenecks, neither of the two broodstocks displayed significant heterozygosity excess ( P>0.05) under either the SMM or the TPM models (Table 2). The allele frequency distribution showed an L-shape in both broodstock (data not shown).
Table 1 Genetic analysis of common carp stocks by microsatellites Marker
All samples
Attala stock
Dinnyes stock
Allele No. of No. of Private He size range alleles alleles alleles MFW4 132 – 152 MFW31 284 – 308 MFW7 186 – 276 MFW9 84 – 136 Mean
8 11 15 13 12
8 9 11 8 9
0 1 0 0
0.86 0.80 0.87 0.82 0.83
Ho
Fis
No. of Private He alleles alleles
0.78 0.43 0.90 0.68 0.69
0.1 7 0.47 9 0.03 13 0.17 9 0.17 9.5
0 0 2 1
0.78 0.78 0.87 0.81 0.81
Ho
Fis
0.71 0.42 0.89 0.73 0.69
0.09 0.46 0.02 0.1 0.15
He: expected heterozygosity. Ho: observed heterozygosity. Fis: fixation index. ‘‘All samples’’ labels all of the 380 individuals genotyped, including the groups labeled ‘‘others’’ and ‘‘wild’’.
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Table 2 Wilcoxon test for heterozygosity excess at four microsatellite loci in two common carp broodstocks Population
SMM
TPM
He/Hd
P
He/Hd
P
Attala (n = 80) Dinnyes (n = 192)
3/1 3/1
0.32 0.88
4/0 4/0
0.06 0.06
SMM: stepwise mutation model. TPM: two-phase model of mutation. He: no. of loci showing heterozygosity excess. Hd: no. of loci showing heterozygosity deficiency. P: statistical possibility under the Wilcoxon test that the population exhibits overall heterozygosity excess over all loci given the model.
The dendrogram generated from the microsatellite data showed a similar picture to the RAPD dendrogram. However, the individuals from the two stocks were even more evenly mixed in the former tree, than in the latter (data not shown).
4. Discussion Despite the importance of common carp as a foodfish worldwide, our knowledge on the genetic background of natural or farmed populations is generally not very extensive. Isoenzyme analysis of common carp samples indicated that the genetic diversity between stocks originating from the same geographic region is small (Csizmadia et al., 1995; Kohlmann and Kersten, 1999). Desvignes et al. (2001) observed that microsatellites were more efficient in detecting subtle differences between closely related French and Czech stocks than isoenzymes. Our publication deals with DNA marker-based genetic analysis of full broodstocks of two fish farms. The Attala stock was established at around 1920 from several mirror carp stocks of the Southern Hungarian region. The broodstock population was maintained by ‘‘closed breeding’’ and continuous replenishment from its own offspring with phenotypic selection. The Attala stock has successfully survived the carp spring viremia epidemic of the 1950s, which resulted in the loss of several other famous varieties of the region. At the Dinnyes Fish Farm breeding of common carp was started in 1961. The original brooders (80 females and 20 males) were obtained from the Derekegyhazi ponds of the Hortobagy Hatchery (Eastern Hungary). Until 1984, brooders were crossed on several occasion with individuals from one of the following varieties: Attala, Nasice (Croatia) and Szarvas (Hungary). Since that time the Dinnyes stock was maintained by ‘‘closed breeding’’ similar to that followed in Attala. On the basis of the RAPD and microsatellite data neither of the two broodstocks tested shows a large number of identical/similar genotypes, which would be typical for a highly uniform population. The present genetic status of the broodstocks is appropriate for the breeding practices followed at the two farms (see above). Both stocks are in disequilibrium for all except one loci (MFW7 for Attala). However, this is not surprising taking the selection procedures and the repeated crosses with other strains (for the Dinnyes stock) into account. No signs of possible bottlenecks were found in either of the two populations, a potential indicator for good breeding practices at both farms. Data derived from the dendrograms would now allow the farmers to perform more targeted breeding programs.
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Our results obtained by the two different DNA markers support the prediction of Kirpitchnikov (1999) on the genetic similarity of stocks originating from the same geographic region. Band frequencies (RAPDs) and allele frequencies (microsatellites) were very similar and dendrograms generated from the two data sets failed to show clear grouping of samples according to their origin (stocks). Further studies should be performed on farmed and natural stocks as well as ‘‘live strain banks’’ (e.g. Szarvas and Vodnany) of the Middle European region. Results of a large-scale analysis with the same set of 25 – 30 microsatellite markers as recommended for all domestic animals (Barker et al., 1998) would help to further clarify relationships of strains. At the beginning of our studies microsatellites from common carp were not available, thus, RAPD assay was selected for its simplicity and speed. Later, as microsatellite markers from common carp have been described (Crooijmans et al., 1997), the samples were retested. As expected, microsatellites proved to be more useful for analysis of genetic diversity and for isolation of private alleles. Multiplexed microsatellites were first used for analysis of fish stocks by O’Reilly et al. (1996). The optimization of multiplexes is a tedious work, but it pays off later, when larger number of individuals is analyzed. A possible disadvantage for such fine-tuned systems might appear when new stocks with different allele ranges are included in the testing, which might result in overlaps between alleles of two different markers. Silver staining of products generated by unlabeled primers is safer, than isotope labeling and it seems to be an ideal tool for those labs, which do not have access to automated sequencers. One of the four microsatellite markers (MFW7) selected by us was also used by Desvignes et al. (2001) for the analysis of French and Czech common carp populations. The size range of MFW7 in both Hungarian broodstocks appears to be considerably wider than that of the three French and four Czech sample groups. This difference might result from the vastly different sample sizes, but it might also indicate lower genetic diversity in the French and Czech populations. While the size ranges of the three French sample groups are partially overlapping with those obtained from the two Hungarian broodstocks, surprisingly, there is no such overlap with the Czech samples, which were crossbred lines from Czech, Hungarian, German and Russian strains (Flajshans et al., 1999). In a parallel study, 12 microsatellite loci have been analyzed on individuals from four domestic (Szarvas, Biharugra, Tata, Kis-Balaton) and two wild (Tisza, Danube) common carp strains from Hungary (Lehoczky et al., 2002). Although two out of the twelve microsatellites (MFW7 and 9) are the same as in our study, it would be hard to make direct comparisons only on the basis of published values. On the other hand, the results of Lehoczky et al. (2002) strengthen our observations at several points, including (i) similar He and Ho values, (ii) significant, but rather limited, differences between the genetic make-up of the farmed stocks and (iii) substantial differences between domestic and wild stocks with higher number of unique alleles in the latter group. With a haploid chromosome set of 52 and a relatively high genome size (C value: 1.7 pg; Hinegardner and Rosen, 1972), common carp belongs to those cyprinids, which are regarded as ‘‘tetraploids’’. According to Ohno et al. (1967), these species were formed by the hybridization of two closely related ancestors of the cyprinid species. Analysis of duplicated loci puts the hybridization event to about 16 million years ago (Larhammar and Risinger, 1994). Study on 35 microsatellite markers (Aliah et al., 1999; Crooijmans et al.,
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1997) in common carp identified six flanking primer pairs, which produced an additional set of products, indicating the presence of duplicate loci. Our data confirm this phenomenon for marker MFW31. Samples tested in our experiment included those of Hungarian wild common carps collected from the Danube and Tisza rivers (four individuals for both). The phenotype of wild carps with its elongated, torpedo-like and fully scaled body is distinct from most domesticated varieties, which show arched vertebral columns and scattered scales (Kirpitchnikov, 1999). We found three private alleles in the eight wild individuals as opposed to the total of seven identified in the three larger groups (Attala, Dinnyes and others). This finding is in agreement with that of Tanck et al. (2000), who genotyped individuals from Dutch wild and domesticated common carp populations by microsatellite markers and identified a number of unique alleles in both groups. The different allele pool might be the result of selection on domesticated stocks, however it might also indicate that wild carps might belong to a taxonomic category different from that of domesticated common carps. Future studies on wild carps could help us to determine not only their taxonomic status, but the genetic diversity of their populations and their relatedness to domesticated stocks, as well.
Acknowledgements The authors are indebted to the staff of the Attala, Dinnyes and Boszormeny fish farms for their help provided during the sample collections. Special thanks to Professors J. Dohy and J. Komen as well as Drs. L. Varga and A. Zsolnai for critical reading of the manuscript. Financial support was provided by the Hungarian Academy of Sciences (AKP 96/2-449 3,1/54) and the Hungarian Ministry of Agriculture (K + F, K-158/7/97). Work performed in Singapore was initiated at the Institute of Molecular Agrobiology with financial support from the Agency for Science, Technology and Research (A*STAR).
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Desvignes, J.F., Laroche, J., Durand, J.D., Bouvet, Y., 2001. Genetic variability in reared stocks of common carp (Cyprinus carpio L.) based on allozymes and microsatellites. Aquaculture 194, 291 – 301. Dong, Z., Zhou, E., 1998. Application of the random amplified polymorphic DNA technique in a study of heterosis in common carp, Cyprinus carpio L. Aquac. Res. 29, 595 – 600. FAO, 2001. FAOSTAT Database Results. http://apps.fao.org/. Flajshans, M., Linhart, O., Slechtova, V., Slechta, V., 1999. Genetic resources of commercially important fish species in the Czech Republic: present state and future strategy. Aquaculture 173, 471 – 483. Hinegardner, R., Rosen, D.E., 1972. Cellular DNA content and the evolution of teleostean fishes. Am. Nat. 106, 621 – 644. Hulata, G., 1995. A review of genetic improvement of the common carp (Cyprinus carpio L.) and other cyprinids by crossbreeding, hybridization and selection. Aquaculture 129, 143 – 155. Kirpitchnikov, V.S., 1999. Genetics and Breeding of Common Carp. INRA, Paris, France. Kohlmann, K., Kersten, P., 1999. Genetic variability of German and foreign common carp (Cyprinus carpio L.) populations. Aquaculture 173, 435 – 445. Larhammar, D., Risinger, C., 1994. Molecular genetic aspects of tetraploidy in the common carp, Cyprinus carpio L. Mol. Phylogenet. Evol. 3, 59 – 68. Lehoczky, I., Magyary, I., Hancz, Cs., 2002. Study of the genetic variability of six domestic common carp strains using microsatellite DNA markers. Allatenyesztes 51, 8 – 19 (in Hungarian). Lewis, P.O., Zaykin, D., 2000. Genetic Data Analysis: Computer Program for the Analysis of Allelic Data: http://lewis.eeb.uconn.edu/lewishome/software html/. Luikart, G., Allendorf, F.W., Cornuet, J.M., Sherwin, W.B., 1998. Distortion of allele frequency distributions provides a test for recent population bottlenecks. J. Heredity 89, 238 – 247. Lynch, M., Milligan, B.G., 1994. Analysis of population genetic structure with RAPD markers. Mol. Ecol. 3, 91 – 99. Miller, M.P., 1997. Tools for Population Genetic Analysis. TFPGA. Computer software distributed by the author. Minch, E., 1996. Microsat, The Microsatellite Distance Program: http://human.stanford.edu/microsat/microsat. html. Nei, M., Li, W.-H., 1979. Mathematical modelling for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. U. S. A. 76, 5269 – 5273. Nelson, J., 1994. Fishes of the World, 3rd ed. Wiley, New York, NY, USA. Ohno, S., Muramoto, J., Christian, L., Atkin, N., 1967. Diploid – tetraploid relationship among old-world members of the family Cyprinidae. Chromosoma 23, 1 – 9. O’Reilly, P.T., Hamilton, L.C., McConnell, S.K., Wright, J.M., 1996. Rapid analysis of genetic variation in Atlantic salmon (Salmo salar) by PCR multiplexing of dinucleotide and tetranucleotide microsatellites. Can. J. Fish. Aquat. Sci. 53, 2292 – 2298. Piry, S., Luikart, G., Cornuet, J.M., 1999. BOTTLENECK: a computer program for detecting recent reductions in the effective population size using allele frequency data. J. Heredity 90, 502 – 503. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning. CSH Laboratory Press, Cold Spring Harbor, NY, USA. Tanck, M.W.T., Baars, H.C.A., Kohlmann, K., Van der Poel, J.J., Komen, J., 2000. Genetic characterization of wild Dutch common carp (Cyprinus carpio L.). Aquac. Res. 31, 779 – 783. Tanck, M.W.T., Palstra, A.P., van de Weerd, M., Leffering, C.P., van der Poel, J.J., Bovenhuis, H., Komen, J., 2001. Segregation of microsatellite alleles and residual heterozygosity at single loci in homozygous androgenetic common carp (Cyprinus carpio L.). Genome 44, 743 – 751. Varga, L., Szabo, G., Darvasi, A., Muller, G., Sass, M., Soller, M., 1997. Inheritance and mapping of Compact (Cmpt), a new mutation causing hypermuscularity in mice. Genetics 147, 755 – 764. Yue, G.H., Chen, F., Orban, L., 2000. Rapid isolation and characterization of microsatellites from the genome of Asian arowana (Scleropages formosus, Osteoglossidae, Pisces). Mol. Ecol. 9, 1007 – 1009.
Genetic analysis of two common carp broodstocks by RAPD and microsatellite markers Richa´rd Ba´rtfai a,b, Sa´ndor Egedi b, Gen Hua Yue a, Bala´zs Kova´cs b, Be´la Urba´nyi c, Gizella Tama´s d, La´szlo´ Horva´th c, La´szlo´ Orba´n a,b,* a
Reproductive Genomics Group, Temasek Life Sciences Laboratory, 1 Research Link, The NUS, Singapore 117 604, Singapore b Laboratory of Aquatic Molecular Biology, Agricultural Biotechnology Center, POB 411, Go¨do¨llo , H-2101 Hungary c Department of Fish Culture, Faculty of Agricultural and Environmental Sciences, Szent Istva´n University, Pa´ter K. u.1, Go¨do¨llo , H-2103 Hungary d Dinnye´s Fish Farm, Dinnye´s H-2485 Hungary
Received 29 November 2001; received in revised form 28 April 2002; accepted 12 September 2002
Abstract The whole broodstock of two Hungarian common carp farms—80 and 196 individuals—was analyzed by using random amplified polymorphic DNA (RAPD) assay and microsatellite analysis. Ten polymorphic RAPD markers and four microsatellites were selected to genotype both of the stocks. As expected, microsatellite analysis revealed more detailed information on genetic diversities than RAPD assay. Results obtained with both types of DNA markers showed lack of major differences between the genetic structure of the two stocks: heterozygosity values and allele frequencies were very similar. Dendrograms created from both sets of data did not show grouping of individuals according to stocks. Genotypes from the two stocks were also compared to those from a limited number of samples collected from other hatcheries and two rivers. Allele frequencies in the groups were similar, with the exception of wild carps. An interesting observation was that three private microsatellite alleles were found in the eight wild carp individuals, compared to the seven detected in the rest of the samples tested (372 individuals). D 2003 Elsevier Science B.V. All rights reserved. Keywords: Cyprinus carpio; Cyprinid; Genotyping; Fish breeding; Landrace
* Corresponding author. Reproductive Genomics Group, Temasek Life Sciences Laboratory, 1 Research Link, The NUS, Singapore 117 604, Singapore. Tel.: +65-6872-7413; fax: +65-6872-7007. E-mail address: [email protected] (L. Orba´n). 0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 ( 0 2 ) 0 0 5 7 1 - 9
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1. Introduction Common carp (Cyprinus carpio L.) belongs to Cyprinidae, the largest family among freshwater teleosts (Nelson, 1994), for which the world’s annual total catch in 1999 was estimated above 15.6 million metric tons, compared to the 2.3 million tons of salmonids (FAO, 2001). Classical taxonomic analysis divides the currently existing common carp forms into three categories: (1) European (Cyprinus carpio carpio), (2) Far Eastern (C. carpio haematopterus) and (3) SouthEast Asian (C. carpio viridiviolaceus) (Kirpitchnikov, 1999). Common carp varieties (e.g. races, landraces, strains, breeds and stocks) ‘‘developed through a combination of forces including geographic isolation, adaptation accumulation of mutations and natural as well as human selection pressures’’ (Hulata, 1995). The Middle and Eastern European region is home for many common carp varieties. However, their origin and relatedness are not well described. Despite the commercial importance of the species, genetic data on common carp stocks are relatively scarce. Analysis of protein polymorphisms was performed on some populations (Csizmadia et al., 1995; Kohlmann and Kersten, 1999). However, reports have only been published recently on common carp genotypes with random amplified polymorphic DNA (RAPD) (Dong and Zhou, 1998) and microsatellite markers (Crooijmans et al., 1997; Aliah et al., 1999; Tanck et al., 2000, 2001; Desvignes et al., 2001; David et al., 2001; Lehoczky et al., 2002). The present work deals with the genetic analysis of two famous common carp varieties of Hungary: the entire Attala and Dinnyes broodstocks and their comparison with over 100 individuals collected from various other sources.
2. Materials and methods 2.1. Stocks, sample collection and DNA isolation Samples from the Dinnyes broodstock (196 individuals) were collected during the April – May period of 1996, whereas the Attala broodstock (80 individuals) was sampled 1 year later. All individuals of both broodstocks were sampled and labeled by inserting an electronic passive integrated transponder (PIT) tag (Destron Fearing, South, St. Paul, MN, USA). One hundred and four samples were also obtained between 1996 and 1998 from the following sources located in Hungary: Boszormeny (50 individuals), Tata (35), Bikal (6), Szajol (5) and wild (8; four from Tisza river and four from Danube river). The 96 samples from the first four sources (all domesticated stocks) were merged into one group (‘‘others’’) in order to obtain similar sample size with that of the stocks, whereas the remaining eight was labeled as the ‘‘wild’’ group. Fin clips were cut from brooders anaesthetized with Quinaldin (Reanal T-2051, Hungary). They were then placed into 1 ml SET buffer (10 mM Tris/HCl, pH 8.0, containing 50 mM EDTA, 200 mM NaCl and 0.5% SDS). Tissue samples were digested with 0.5 Ag/Al Proteinase K at 55 jC overnight, the resulting solution was centrifuged, the supernatant precipitated in ethanol and dissolved in 1 TE buffer. From some of the samples, genomic DNA was isolated by the phenol – chloroform procedure (Sambrook et
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al., 1989). The quality and concentration of DNA from both sources were assessed by agarose gel electrophoresis then samples were stored at 4 jC until use. 2.2. RAPD assay RAPD reactions were performed as described in Bercsenyi et al. (1998), but proteinasetreated and ethanol-precipitated fin clip sample was used as a template. The following primers were tested in the preliminary assay: OPF, OPM, OPO and OPQ series, with 20 primers each, plus three OPE primers (Operon, Alameda, CA, USA). RAPD reactions were run in a PCR1 Thermocycler (Perkin Elmer, Palo Alto, CA, USA). RAPD products (20 Al load) were separated on 2% agarose gels, containing 0.5 Ag/ml ethidium bromide. Band patterns were photographed under UV light. 2.3. Microsatellite analysis Primers were synthesized to the flanking regions of eight microsatellites: MFW4, MFW7, MFW9, MFW13, MFW17, MFW20, MFW26 and MFW31, according to Crooijmans et al. (1997). For preliminary testing of microsatellite markers, mastermixes contained (in 10 Al): 1 PCR buffer (Promega, Madison, WI, USA), 200 AM dNTP mix, 200 nM for both primers, 2 mM MgCl2, 40 ng template and 0.25 U Taq polymerase (Promega). PCR cycles were: 5Vpreamplification denaturation at 95 jC, then 20U at 94 jC, 30U at 63 jC and 20U at 72 jC for 10 cycles, followed by 20U at 94 jC, 30U at 53 jC and 20U 72 jC for 25 cycles. As a final step, products were fully elongated for 7V at 72 jC. Amplifications were performed on a PE 9700 thermocycler (Perkin Elmer) with ramp speed set identical to that of PE 9600. For parallel genotyping, the eight selected markers were grouped into four pairs (MFW7/9, MFW4/31, MFW13/20 and MFW17/26) on the basis of their allele range and their ability to be co-amplified in the same PCR reaction. For the duplex PCR, the same reaction mixture was used as above with the following primer concentrations: 200 nM of MFW7 with 400 nM of MFW9 for the first duplex, while 200– 200 nM of both MFW4 and MFW31 for the second one. For separation on denaturing polyacrylamide gels, 8-Al sequencing stop solution was added to the PCR product following amplification and it was denatured at 96 jC. Eight microliters of this solution was loaded onto 5%T, 2%CBis PA gels containing 6 M urea. Gels were cast in a 38 50-cm Sequi-Gen GT System (BioRad, Hercules, CA, USA) using a comb with 49 ‘‘sharkteeth wells’’. For separation of single microsatellite markers, a gel with uniform 0.4-mm thickness, while for duplexes, a 0.4– 1.2-mm wedge gel was used. Since the allele ranges of any two duplexes were at least partially overlapping in order to separate the alleles of four microsatellites on the same gel, the second pair had to be loaded with delay. Two duplex pairs (MFW7/9 + MFW4/31 and MFW13/20 + MFW17/ 26) were tested. In the first case, the products of the MFW4/31 amplification were loaded when bromophenol blue dye from MFW7/9 sample migrated to a 3-cm distance from the wells, while in the second case, MFW17/26 products were loaded when the dye from MFW13/20 reached 4.5 cm. Such a ‘‘delayed double duplex’’ setup allowed us to separate
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all alleles from the four markers with a few exceptions. In case of a suspected overlap of allele range, both markers in question were reamplified and separated in individual lanes. Following electrophoresis, gel cassettes were disassembled and a custom-made plastic frame was clamped to the glass plate holding the gel. Detection of microsatellite alleles was achieved by silver staining in this special container according to Varga et al. (1997). To determine the size of alleles obtained by the silver staining methods, labeled PCR products of 20 individuals were later analyzed on the ABI 377 sequencer as described previously (Yue et al., 2000). One of the two primers from each pair was labeled with either 6FAM or HEX dyes (Genset, Paris, France). PCR conditions for each locus were the same as described above. The length of the fluorescently labeled PCR products was determined against the size standards (Tamra-GS-500) using Genescan and Genotyper software (Applied Biosystems, Foster City, CA, USA). The 20 individuals were selected to cover most of the alleles of the three sample groups; for those not present, the allele length was estimated by comparison to their closest known neighbors on the scale. 2.4. Analysis of data RAPD patterns were visually analyzed and scored from photographs. For the analysis and comparison of the patterns, a set of distinct, well-separated bands were selected (see Fig. 1). The genotypes were determined by recording the presence (1) or absence (0) of these bands only, neglecting other (weak and unresolved groups of) bands. Two assumptions were made for the analysis of the RAPD data: (1) markers from different loci did not migrate to the same position on the gel, (2) each band was assumed to represent the dominant genotype at the locus, whereas lack of the same band in another individual was assumed to correspond to the alternative homozygous recessive genotype in the Hardy– Weinberg equilibrium (Lynch and Milligan, 1994). Genetic similarity (GS) between individuals i and j was estimated according to the formula given by Nei and Li (1979): GSij ¼ 2Nij =ðNi þ Nj Þ where Nij is the number of bands common in individuals i and j, and Ni and Nj are the total number of bands in individuals i and j, respectively, with regard to all assay units. Thus, GS reflects the proportion of bands shared between two individuals and ranges from 0 (no common bands) to 1 (all bands identical). Genetic dissimilarity (GD) was calculated as: GD ¼ 1 GS The expected heterozygosity (He) and percentage of the polymorphic loci were calculated by using the software called Tools for Population Genetic Analysis (TFPGA; Miller, 1997). Microsatellite data were scored visually from silver-stained, dried gels. Allele lengths were assigned on the basis of the results obtained with labeled primers on a subset of samples. In the majority (>60%) of the individuals tested, the primers for marker MFW31 amplified extra bands (ca. 240 – 255 bp) confirming the observation of Crooijmans et al. (1997). These bands were not scored. The expected (He) and observed heterozygosity
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(Ho), number of alleles, fixation index (Fis), allele frequencies and Hardy –Weinberg equilibrium were analyzed using software Genetic Data Analysis (GDA; Lewis and Zaykin, 2000). The definition of ‘‘private alleles’’ labels alleles, which are only present in one stock and absent from all others tested. The pairwise similarity between individuals was calculated by using software Microsat (Minch, 1996). The dendrograms from the GD values determined by RAPD and from the genetic dissimilarity data determined by microsatellite analysis were constructed based on the NJ method by using software NTSYSpc (Exeter Software, Setauket, NY, USA). We tested both stocks, whether they went through a bottleneck during their history by using the software Bottleneck 1.2.02 (Cornuet and Luikart, 1996; Piry et al., 1999). The software tests for the departure from mutation drift equilibrium based on heterozygosity excess or deficiency. Out of the three options provided, we used stepwise mutation model (SMM) and the two-phased model (TPM). By the using the same software, the allele frequency distribution of the microsatellite loci was also examined for model shift (Luikart et al., 1998), which would indicate a recent genetic bottleneck, if any.
3. Results 3.1. Preliminary analysis for selection of markers Six genomic DNA samples were selected randomly from among the individuals of the Dinnyes stock. These templates were then amplified with a total of 81 10 mer random primers (see Section 2.2 for details). Three primers (OPE-12, OPM-14 and OPQ-09) generating polymorphic patterns, which were clear and easy-to-interpret (for representative band patterns, see Fig. 1A) were selected for further analysis. Out of more than 30 bands amplified by the three primers, 17 stabile and characteristic bands (i.e. those which were reasonably strong, easy to score and reproducible) were selected for genotyping of all individuals from both stocks. For preliminary analysis of the microsatellites, samples were chosen randomly from the following sources: Attala (12), Dinnyes (8), Boszormeny (8), Bikal (4), Szajol (4), wild-Danube (4) and wild-Tisza (4). The eight markers were split into two groups (four markers each) and tested in the ‘‘delayed double duplex’’ system (see Section 2.3 for details). The marker quadruplet containing MFW4/31 and MFW7/9 showed more stabile bands and better separation of alleles than the other did (MFW13/20 + MFW17/ 26). Thus, the MFW4/31+ MFW7/9 ‘‘delayed double duplex’’ was selected for further use. 3.2. Analysis of the Attala and Dinnyes broodstocks by RAPD markers Seventeen bands were selected from the three RAPD patterns for genotyping of the broodstocks. Nine of the markers were polymorphic, while seven failed to show sufficient polymorphism (>5%) in both stocks (for complete list of markers and their polymorphisms, see Fig. 1B.). The remaining marker (M14-950) was missing from all individuals of the Attala stock and present in one quarter of the other population.
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Fig. 1. RAPD analysis of the Attala and Dinnyes common carp stocks. Panel A: representative RAPD patterns obtained with the three selected primers (OPM-14, OPE-12 and OPQ-09). Each subpanel contains a gel picture with a molecular weight marker (lambda/PstI) on the left followed by patterns amplified from four individual samples. On the right of each picture, a sketch is shown indicating those bands with their approximate size, which were scored from the RAPD pattern. Panel B: frequencies of the 17 bands amplified by the three primers from the two broodstocks. Bands are identified by the primer and their estimated length in basepairs.
The expected heterozygosity was lower in the Attala stock (0.16) and somewhat higher (0.18) in the Dinnyes stock. Four of the polymorphic markers (M14-1400, E12-1900, Q91300, Q9-950) showed very similar frequency in the two broodstocks, while the percentage values for the others were different, especially for M14-950 and M14-850. When a dendrogram was generated from the RAPD genotypes, individuals from the two stocks were not separated into distinct groups; instead, they were mixed throughout the tree (data not shown). 3.3. Analysis of the two broodstocks and samples from other stocks by microsatellite markers All DNA samples from the two stocks (80 and 196 individuals) and those from the other sources (104 individuals) were tested for the four selected microsatellite markers (MFW4/31 + MFW7/9) by using the ‘‘delayed double duplex’’ method. At the four loci, a total of 47 alleles were detected across the four groups: Attala, Dinnyes, ‘‘others’’ and ‘‘wild’’. Allele frequencies at each locus were basically similar in their distribution, except for the wild carps (Fig. 2). Equal number of private alleles (three each) were found in the Dinnyes stock, the ‘‘others’’ and the wild carps, whereas in the Attala stock only one private allele was detected at the four loci. The frequency of private alleles was very low, ranging from 0.003 to 0.027 in the three large groups, but about two magnitudes higher (0.125 – 0.25) in case of the eight wild individuals, as expected (Fig. 2). The average expected heterozygosity was 0.83 in the Attala stock and 0.81 in the Dinnyes stock, while the observed heterozygosity was 0.69 for both (Table 1). No
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Fig. 2. Allele frequencies for the four microsatellite loci tested on the four sample groups. Number of samples tested per group: Attala = 80, Dinnyes = 196, others = 96 and wild = 8.
statistically significant (df = 3, P>0.05) difference has been detected between the heterozygosity values observed in the two populations. The inbreeding coefficient was also similar in the two stocks (Attala: 0.17, Dinnyes: 0.15). When the four markers were used to test for deviations from the Hardy – Weinberg equilibrium, only MFW7 gave a probability bigger than 0.05 ( P = 0.245) in the Attala stock (data not shown), indicating that both populations are in disequilibrium for most loci tested. When tested for signs of bottlenecks, neither of the two broodstocks displayed significant heterozygosity excess ( P>0.05) under either the SMM or the TPM models (Table 2). The allele frequency distribution showed an L-shape in both broodstock (data not shown).
Table 1 Genetic analysis of common carp stocks by microsatellites Marker
All samples
Attala stock
Dinnyes stock
Allele No. of No. of Private He size range alleles alleles alleles MFW4 132 – 152 MFW31 284 – 308 MFW7 186 – 276 MFW9 84 – 136 Mean
8 11 15 13 12
8 9 11 8 9
0 1 0 0
0.86 0.80 0.87 0.82 0.83
Ho
Fis
No. of Private He alleles alleles
0.78 0.43 0.90 0.68 0.69
0.1 7 0.47 9 0.03 13 0.17 9 0.17 9.5
0 0 2 1
0.78 0.78 0.87 0.81 0.81
Ho
Fis
0.71 0.42 0.89 0.73 0.69
0.09 0.46 0.02 0.1 0.15
He: expected heterozygosity. Ho: observed heterozygosity. Fis: fixation index. ‘‘All samples’’ labels all of the 380 individuals genotyped, including the groups labeled ‘‘others’’ and ‘‘wild’’.
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Table 2 Wilcoxon test for heterozygosity excess at four microsatellite loci in two common carp broodstocks Population
SMM
TPM
He/Hd
P
He/Hd
P
Attala (n = 80) Dinnyes (n = 192)
3/1 3/1
0.32 0.88
4/0 4/0
0.06 0.06
SMM: stepwise mutation model. TPM: two-phase model of mutation. He: no. of loci showing heterozygosity excess. Hd: no. of loci showing heterozygosity deficiency. P: statistical possibility under the Wilcoxon test that the population exhibits overall heterozygosity excess over all loci given the model.
The dendrogram generated from the microsatellite data showed a similar picture to the RAPD dendrogram. However, the individuals from the two stocks were even more evenly mixed in the former tree, than in the latter (data not shown).
4. Discussion Despite the importance of common carp as a foodfish worldwide, our knowledge on the genetic background of natural or farmed populations is generally not very extensive. Isoenzyme analysis of common carp samples indicated that the genetic diversity between stocks originating from the same geographic region is small (Csizmadia et al., 1995; Kohlmann and Kersten, 1999). Desvignes et al. (2001) observed that microsatellites were more efficient in detecting subtle differences between closely related French and Czech stocks than isoenzymes. Our publication deals with DNA marker-based genetic analysis of full broodstocks of two fish farms. The Attala stock was established at around 1920 from several mirror carp stocks of the Southern Hungarian region. The broodstock population was maintained by ‘‘closed breeding’’ and continuous replenishment from its own offspring with phenotypic selection. The Attala stock has successfully survived the carp spring viremia epidemic of the 1950s, which resulted in the loss of several other famous varieties of the region. At the Dinnyes Fish Farm breeding of common carp was started in 1961. The original brooders (80 females and 20 males) were obtained from the Derekegyhazi ponds of the Hortobagy Hatchery (Eastern Hungary). Until 1984, brooders were crossed on several occasion with individuals from one of the following varieties: Attala, Nasice (Croatia) and Szarvas (Hungary). Since that time the Dinnyes stock was maintained by ‘‘closed breeding’’ similar to that followed in Attala. On the basis of the RAPD and microsatellite data neither of the two broodstocks tested shows a large number of identical/similar genotypes, which would be typical for a highly uniform population. The present genetic status of the broodstocks is appropriate for the breeding practices followed at the two farms (see above). Both stocks are in disequilibrium for all except one loci (MFW7 for Attala). However, this is not surprising taking the selection procedures and the repeated crosses with other strains (for the Dinnyes stock) into account. No signs of possible bottlenecks were found in either of the two populations, a potential indicator for good breeding practices at both farms. Data derived from the dendrograms would now allow the farmers to perform more targeted breeding programs.
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Our results obtained by the two different DNA markers support the prediction of Kirpitchnikov (1999) on the genetic similarity of stocks originating from the same geographic region. Band frequencies (RAPDs) and allele frequencies (microsatellites) were very similar and dendrograms generated from the two data sets failed to show clear grouping of samples according to their origin (stocks). Further studies should be performed on farmed and natural stocks as well as ‘‘live strain banks’’ (e.g. Szarvas and Vodnany) of the Middle European region. Results of a large-scale analysis with the same set of 25 – 30 microsatellite markers as recommended for all domestic animals (Barker et al., 1998) would help to further clarify relationships of strains. At the beginning of our studies microsatellites from common carp were not available, thus, RAPD assay was selected for its simplicity and speed. Later, as microsatellite markers from common carp have been described (Crooijmans et al., 1997), the samples were retested. As expected, microsatellites proved to be more useful for analysis of genetic diversity and for isolation of private alleles. Multiplexed microsatellites were first used for analysis of fish stocks by O’Reilly et al. (1996). The optimization of multiplexes is a tedious work, but it pays off later, when larger number of individuals is analyzed. A possible disadvantage for such fine-tuned systems might appear when new stocks with different allele ranges are included in the testing, which might result in overlaps between alleles of two different markers. Silver staining of products generated by unlabeled primers is safer, than isotope labeling and it seems to be an ideal tool for those labs, which do not have access to automated sequencers. One of the four microsatellite markers (MFW7) selected by us was also used by Desvignes et al. (2001) for the analysis of French and Czech common carp populations. The size range of MFW7 in both Hungarian broodstocks appears to be considerably wider than that of the three French and four Czech sample groups. This difference might result from the vastly different sample sizes, but it might also indicate lower genetic diversity in the French and Czech populations. While the size ranges of the three French sample groups are partially overlapping with those obtained from the two Hungarian broodstocks, surprisingly, there is no such overlap with the Czech samples, which were crossbred lines from Czech, Hungarian, German and Russian strains (Flajshans et al., 1999). In a parallel study, 12 microsatellite loci have been analyzed on individuals from four domestic (Szarvas, Biharugra, Tata, Kis-Balaton) and two wild (Tisza, Danube) common carp strains from Hungary (Lehoczky et al., 2002). Although two out of the twelve microsatellites (MFW7 and 9) are the same as in our study, it would be hard to make direct comparisons only on the basis of published values. On the other hand, the results of Lehoczky et al. (2002) strengthen our observations at several points, including (i) similar He and Ho values, (ii) significant, but rather limited, differences between the genetic make-up of the farmed stocks and (iii) substantial differences between domestic and wild stocks with higher number of unique alleles in the latter group. With a haploid chromosome set of 52 and a relatively high genome size (C value: 1.7 pg; Hinegardner and Rosen, 1972), common carp belongs to those cyprinids, which are regarded as ‘‘tetraploids’’. According to Ohno et al. (1967), these species were formed by the hybridization of two closely related ancestors of the cyprinid species. Analysis of duplicated loci puts the hybridization event to about 16 million years ago (Larhammar and Risinger, 1994). Study on 35 microsatellite markers (Aliah et al., 1999; Crooijmans et al.,
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1997) in common carp identified six flanking primer pairs, which produced an additional set of products, indicating the presence of duplicate loci. Our data confirm this phenomenon for marker MFW31. Samples tested in our experiment included those of Hungarian wild common carps collected from the Danube and Tisza rivers (four individuals for both). The phenotype of wild carps with its elongated, torpedo-like and fully scaled body is distinct from most domesticated varieties, which show arched vertebral columns and scattered scales (Kirpitchnikov, 1999). We found three private alleles in the eight wild individuals as opposed to the total of seven identified in the three larger groups (Attala, Dinnyes and others). This finding is in agreement with that of Tanck et al. (2000), who genotyped individuals from Dutch wild and domesticated common carp populations by microsatellite markers and identified a number of unique alleles in both groups. The different allele pool might be the result of selection on domesticated stocks, however it might also indicate that wild carps might belong to a taxonomic category different from that of domesticated common carps. Future studies on wild carps could help us to determine not only their taxonomic status, but the genetic diversity of their populations and their relatedness to domesticated stocks, as well.
Acknowledgements The authors are indebted to the staff of the Attala, Dinnyes and Boszormeny fish farms for their help provided during the sample collections. Special thanks to Professors J. Dohy and J. Komen as well as Drs. L. Varga and A. Zsolnai for critical reading of the manuscript. Financial support was provided by the Hungarian Academy of Sciences (AKP 96/2-449 3,1/54) and the Hungarian Ministry of Agriculture (K + F, K-158/7/97). Work performed in Singapore was initiated at the Institute of Molecular Agrobiology with financial support from the Agency for Science, Technology and Research (A*STAR).
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