The silver crucian carp, Carassius langsdorfii has three reproductive characteristics: gynogenesis, polyploidy (triploid or tetraploid), and genetic homogeneity within a family. In natural water, the silver crucian carp populations consist of multiple clonal lines. In the present study, three microsatellite DNA loci were used to distinguish several clonal lines of the silver crucian carp sampled from natural water. Progeny and the maternal fish had the same genotype in the three loci. In 237 fish collected from the wild, nine alleles were observed in GF1*, sixteen alleles in GF17*, and nine alleles in GF29*. Ten genotypes were observed in GF1*, seventeen in GF17* and eight in GF29*. The proportion of heterozygotes was very high in each locus (1.000). Sixteen clonal lines were distinguished by the combined genotypes of three microsatellite loci. Two subtypes were also detected within the clonal line KOC-011.
The silver crucian carp, Carassius langsdorfii (Matsubara and Ochiai, 1965) has three reproductive characteristics: gynogenesis, polyploidy (triploid or tetraploid) (Kobayasi, 1971; Kobayasi and Ochi, 1972), and clonal nature (Dong and Taniguchi, 1996; Dong et al., 1997; Umino et al., 1996). Sperms of other species initiate development of their eggs (Kobayasi, 1971; Kobayasi and Ochi, 1972). However, the introduced sperm nucleus does not transform into a male pronucleus and makes no contribution to the zygotic genome (Nakakuki et al., 1984; Yamashita et al., 1990, 1993). A cytological study concluded that a population in the Kanto district appeared to be comprised of both triploid and tetraploid types (Kobayasi et al., 1970). Thus it was suggested that the silver crucian carp is unisexual and produces unreduced polyploid eggs (Kobayasi, 1971).
Taniguchi and Sakata (1977) found that the silver crucian carp of western Japan has a specific band of muscle protein electrophoresis, which has been verified to be a creatine kinase in another study (Dong and Taniguchi, 1996). Taniguchi and Sakata (1977) also found that the silver crucian carp is independent from the gene pool of wild diploid crucian carp species. Murakami and Fujitani (1997) found that the repetitive DNA (Cal3nDr) are specific to polyploid silver crucian carp, and they suggested that this region is useful in the study of the genetic background of this species such as the origin of polyploidy and gynogenetic reproduction.
In terms of genetic homogeneity, several authors have observed that the progeny in this species are usually genetically identical to the maternal fish (Dong and Taniguchi, 1996; Dong et al., 1997; Umino et al., 1996). The wild silver crucian carp consist of several clonal lines (Dong et al., 1996; Umino et al., 1997; Ohara et al., 1998). These lines have been distinguished using tissue graft, isozymes, multilocus minisatellite DNA fingerprinting and mtDNA RFLP (Murayama et al., 1984; Dong et al., 1996; Umino et al., 1997; Ohara et al., 1998). Of these methods, tissue grafts, isozymes and mtDNA RFLP are time-consuming or lack the sensitivity to identify clonal lines. By contrast, multilocus minisatellite DNA fingerprinting is more sensitive method for the identification of clonal lines. This method, however, has limited application in population genetics because it is difficult to identify the zygotic condition (Takagi et al., 1997). Recently, a number of easily and highly variable genetic markers, known as microsatellite DNA, has been developed (Wright and Bentzen, 1994). Microsatellites are highly polymorphic, particularly in fish (Brocker et al., 1994).
In the present study, we used microsatellite DNA to examine the clonal nature of the artificially propagated silver crucian carp, and examined the zygotic condition of the loci of several clonal lines in this species collected from natural water in Kochi, Japan. As for the scientific name, we tentatively used C. langsdorfii for the triploid silver crucian carp while several other scientific names have been given to the silver crucian carp (Ginbuna), such as, C. auratus langsdorfii (Hosoya, 1993; Nakamura, 1969), C. gibelio langsdorfi (Miyadi et al., 1976) and C. langsdorfii (Matsubara and Ochiai, 1965).
MATERIALS AND METHODS
Sampling and DNA extraction
One silver crucian carp, C. langsdorfii, caught from the Monobe River in Kochi Prefecture, Japan, was used as the maternal fish. Sperm from several goldfishes were used to activate egg development without the effect of genetic contribution to the offspring (Kobayasi, 1971; Dong and Taniguchi, 1996). The fry were reared until 150 days after hatching, and 40 fish were used for the analysis.
Samples of silver crucian carp from natural waters were collected from two locations, the Niyodo and Monobe Rivers in Kochi Prefecture, Japan. The triploidy of these samples was determined by analyzing the red blood cell size using the methods detailed in Sezaki et al. (1977). In this study, we used the samples of triploid silver crucian carp that had previously been identified as 17 clones (designated as KOC-001 to 015 and 017) by minisatellite DNA fingerprinting in a previous report (Ohara et al., 1998). The samples of KOC-016 were not used due to denaturation of DNA. Total DNA was extracted from the blood of each fish by standard SDS-phenol/chloroform procedures and stored at 4°C prior to PCR analysis (Takagi et al., 1997).
Three microsatellite loci, GF1*, GF17*, and GF29*, developed by Zheng et al. (1995), were used in this study. Nomenclature of loci and alleles follow Shaklee et al. (1990). Radiolabeling and chemiluminescence methods were used to visualize microsatellite DNA. The radiolabeling method was performed using the methods reported by Takagi et al. (1997). The reverse primer was 5′ end-labeled with [γ32P]ATP. The PCR was programmed for 7 cycles of 1 min at 94°C, 30 sec at 53°C, 30 sec at 72°C, 33 cycles of 30 sec at 90°C, 30 sec at 53°C, and 30 sec at 72°C, respectively. Following amplification, PCR products were mixed with denaturing stop dye, heated at 95°C for 15 min, and electrophoresed on 6% polyacrylamide gel. Alleles were sized according to an M13 sequence ladder.
The chemiluminescence detection method was performed according to the methods given in Perez-Enriquez et al. (1998). The reverse primer was 5′ end-labeled with biotin. The PCR and electrophoresis conditions were the same as in the radiolabeling method. After electrophoresis, DNA was transferred to a nylon membrane by blotting, then the membrane was dried and UV crosslinked. DNA on the membrane was detected by using the Phototope™-Star Detection Kit (New England Biolabs). A sequence ladder obtained from the pUC19 plasmid was used as a size marker and was prepared with the CircumVent™ Phototope™ Kit (New England Biolabs).
Estimation of genetic distance
We estimated the genetic distance (D) among the clonal lines based on the band sharing index (BSI) at the three microsatellite loci in order to determine the genetic relationship among the clonal lines (Wetton et al., 1987; Gillbert et al., 1990). The BSI between clonal lines was calculated with the formula BSI=2Nab / Na+Nb, where Na and Nb are the number of bands (alleles) present in each clone, and Nab is the number of bands shared. The genetic distance (D) calculated with the formula D=1–BSI. The value varies from zero, when the two individuals are identical, to 1, when there are no bands in common. A dendrogram was drawn by the UPGMA method (Sneath and Sokal, 1973) based on D values, using a program in the PHYLIP Ver 3.5 software package (Felsenstein, 1994).
Microsatellite DNA patterns of artificial propagated off-spring
Microsatellite DNA markers of 40 offspring from one maternal fish were examined. Since both the offsprings and the maternal fish had the same genotype at the three polymorphic loci, they are of the same clonal line. The genotypes of the clonal line were determined as *307/(*307 or *311) / *311 in GF1*, *185 / *192, / *193 in GF17*, and *188 / (*188 or *194) / *194 in GF29*. This clonal line was identified as the clone KOC-001.
Microsatellite DNA polymorphism of wild crucian carp
Microsatellite polymorphism is shown in Fig. 1 as examples of the radiolabeling and chemiluminescence methods. A total of 237 silver crucian carp from natural water were analyzed. The number of alleles per locus, amplified product size range, proportion of heterozygotes, allelic combination and the number of genotypes for the three loci are summarized in Table 1. In the two bands types, the one allele was recognized to be duplicated to the one of two bands. The assumed genotypes of the silver crucian carp are given in Table 2. In GF1*, *303 and *311 were predominant and appeared in 13 and 10 clonal lines, respectively. In GF17*, *192 and *216 appeared in seven clonal lines. In GF29*, *188 and *194 appeared in 11 and 13 clonal lines, respectively.
Numbers of alleles, genotypes and fragment size as well as proportion of heterozygotic individuals and allelic combinations of 17 clonal lines of silver crucian carp, Carassius langsdorfii
Genotypes of three microsatellite DNA loci in 17 clonal lines of silver crucian carp, Carassius langsdorfii
The observed number of alleles and genotypes were quite large in the three loci comparing with isozyme analysis (Simizu et al., 1993; Don et al., 1996). The proportion of heterozygotes in the wild population was very high over the three microsatellite loci comparing with gold fish (Zheng et al., 1995). The observed number of genotypes was quite small although a large number of alleles was observed. In the results of allelic combinations, the ratio of triallelic types were high in GF17* (0.734). In GF1* and GF29*, the ratio of diallelic types were relatively high with values of 0.747 and 0.626, respectively, compared with isozyme analysis (Simizu et al., 1993; Dong et al., 1996). Yet, monoallelic type was not observed in this population.
The 16 clonal lines, which were identified by minisatellite DNA fingerprinting, are clearly distinguished based on the genotype in GF17*. Based on the analysis of each locus, all individuals within each clonal lines except KOC-011 had the same genotype. However, among the 66 individuals of KOC-011, one fish had two alleles (*182 and *194) while the others had three alleles (*182, *192, and *194) in GF17*. This fish had the same with the others in genotype of GF1* and GF29*. Thus KOC-011 was subdivided into two clonal lines: one with three alleles (*182, *192, and *194), and the other with two alleles (*182 and *194), which were designated KOC-011-1 and KOC-011-2, respectively.
The average genetic distance (D) between clonal lines was calculated as 0.637±0.191. The BSI within clonal lines, except for KOC-011, was equal to 1.000, indicating genetic uniformity. The dendrogram drawn by the UPGMA method based on D values is given in Fig. 2. KOC-011-1 and KOC-011-2 had a very close genetic relationship compared to other clones.
The analysis of microsatellite DNA loci can be used as a fingerprinting method to distinguish clonal lines. In the loci, information of the zygotic condition of each locus can also be obtained. The maximum allele number of an individual at each microsatellite locus was three as observed in this study. This suggests that the silver crucian carp C. langsdorfii is a true triploid. Since the genotypes can be recorded by each locus, in the future, microsatellite DNA loci will be applied to establish a registration system of clonal lines of silver crucian carp.
The proportion of heterozygotes was very high over the three microsatellite loci. Similar results were observed in the electrophoretic analysis (Simizu et al., 1993; Dong et al., 1996; Ohara et al., 1998). The fixed multilocus heterozygosity observed in this study is a characteristic of unisexual-hybrid vertebrates (Vrijenhoek, 1990). The unisexual vertebrates were generally originated from hybridization between congeneric species (Dawley, 1989). The silver crucian carp may be originated from hybridization (Simizu et al., 1993). One explanation for the high proportion of heterozygotes is that during the oogenesis of silver crucian carp, the first meiotic division does not occur, but rather the oocyte undergoes a homotypic nuclear division as in somatic cells, which are called apomixis (Kobayasi, 1976; Yamasita et al., 1993; Arai, 1997). Therefore, they can maintain an almost heterozygous state for each generation within a clonal population. The multilocus heterozygous condition may lead to an increase of fitness in the clonal line of silver crucian carp.
In this study, we detected two subtypes within the clone KOC-011 collected from natural water. Kojima et al. (1984) observed the partial pairing of homologue-like chromosomes at the zygote stage in silver crucian carp. Zhang et al. (1992) observed the putative recombinant in offspring from a female of the silver crucian carp based on the electrophoretic pattern of isozyme. Two different processes can be considered to explain the origin of the subtype: 1) Synapsis and recombination occurred between homologous chromosomes within the microsatellite locus existed. 2) The microsatellite DNA mutated to a new allele (size changed or null allele). Information on this mechanism is important to understand how genetic diversity is maintained within silver crucian carp populations. Further studies are needed to determine the developing processes of these subtypes.
In this study, we used three microsatellite loci, yet, it is not enough to fully discuss the genetic relationship of the silver crucian carp. It is necessary to investigate many other microsatellite loci. Nonetheless, we found that the microsatellite loci used in this study are very valuable due to their application not only to identify clonal lines of this species, but also to study the origin of the silver crucian carp.
We thank Dr. Chris Norman, lecturer of Chiba University for critical reading of the English manuscript.