Polymorphism of the cytochrome oxidase b gene (<Emphasis Type="Italic">cyt b</Emphasis>) in Russian populations of common carp and wild common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.)

Polymorphism of the mitochondrial cyt b gene was examined in 35 individuals of common carp and wild common carp (Cyprinus carpio L.). The fish examined represented two natural populations from Khabarovsk krai (Ac and Am), Volga wild common carp, Don wild common carp, and two common carp breeds, Ropsha (strains BB and MM) common carp and Hungarian common carp. The highest level of nucleotide (π) and haplotype (h) diversity was detected in two strains of Ropsha common carp (MM, π = 0.67%, h = 0.7; and BB, π = 0.21%, h = 0.9) and in one population (Am) of Amur wild common carp (π = 0.26%; h = 0.6). The second population of Amur wild common carp (Ac) and Hungarian common carp were characterized by lower variation estimates (π = 0.035%, h = 0.4; and π = 0.09%, h = 0.7, respectively). Genetic homogeneity was demonstrated for the populations of Volga and Don wild common carp (π = 0, h = 0). In the sample of the cyt b sequences examined, three lineages were identified. Lineages I and II united all haplotypes of the Am Amur wild common carp along with two haplotypes of Ropsha common carp, strain MM. The third lineage (III) was formed by the haplotypes of three individuals of Ropsha common carp strain MM, all representatives of Ropsha common carp strain BB, Hungarian common carp, Ac Amur wild common carp, and Don and Volga wild common carps. Statistically significant amino acid differences were observed only for the sequences, corresponding to haplotypes of lineage III, and the sum of sequences of lineages I and II. Effectiveness of different types of markers to differentiate the two subspecies of European and Amur wild common carp (C. c. carpio and C. c. haematopterus) is discussed, as well as the issues of the origin and dispersal of Russian common carp and wild common carp breeds.     

Individual and population variation in cercariae of bird schistosomes of the <Emphasis Type="Italic">Trichobilharzia ocellata</Emphasis> species group as revealed with the polymerase chain reaction

The polymerase chain reaction with arbitrary (RAPD-PCR) or specific primers was used to study the population variation and to identify the species in cercariae of schistosomes of the Trichobilharzia ocellata species group (Trematoda, Schistosomatidae). In total, 28 cercariae were obtained from two spontaneously invaded mollusks Lymnaea stagnalis (LS) and L. ovata (LO), which were collected in different ponds of Moscow. RAPD-PCR was carried out with two arbitrary primers, OPA9 and OPB11, which each detected different levels of individual and among-group variation and revealed considerable genetic differentiation of cercariae from different host mollusks. To check whether the cercariae of the two samples belong to one species, sequencing was performed with a region corresponding to intergenic transcribed spacer 2 (ITS2), which was earlier proposed for cercaria identification in three European species of bird schistosomes of the genus Trichobilharzia (T. franki, T. regenti, and T. szidati). The ITS2 sequences of two LO cercariae were identical, each consisted of 319 bp, and showed 100% homology to the T. franki ITS2 sequence. The ITS2 sequences of two LS cercariae were identical, each consisted of 323 bp, and showed 99.4% homology to the T. szidati counterpart. The causes of genetic variation in cercariae and prospects of using RAPD markers to study different stages of the life cycle in trematodes are discussed.Translated from Genetika, Vol. 41, No. 1, 2005, pp. 17–22.Original Russian Text Copyright © 2005 by Semyenova, Chrisanfova, Filippova, Beer, Voronin, Ryskov.     

Polymorphism of the ND1 and CO1 Mitochondrial Genes in Populations of Liver Fluke Fasciola hepatica

Polymorphism of fragments of the ND1 and CO1 mitochondrial genes was for the first time found in four liver fluke Fasciola hepatica samples from Ukraine, Belarus, Moscow region, and Mordovia. The ND1 and CO1fragments were respectively 292 and 433 bp in size, with polymorphic sites amounting to 2.7 and 0.9% of the total sequence. Seven haplotypes were found in the four samples; two haplotypes (A and B) were most common (29.1 and 45.8%, respectively) in the pooled sample. The haplotype frequency distribution differed among the four populations. Haplotype B prevailed in the Mordovian and Moscow region samples. In addition, these samples had a higher number of unique haplotypes (A2, A3, B2). The results testify to genetic differences of the four geographically distant populations of F. hepatica.     

Genetic differentiation in eastern European and western Asian populations of the liver fluke, Fasciola hepatica, as revealed by mitochondrial nad1 and cox1 genes

Partial sequences of mitochondrial genes nad1 (316 bp) and cox1 (429 bp) were analyzed to estimate the variability of the liver fluke samples collected in 20 localities in Russia, Belarus, Ukraine, Bulgaria, Armenia, Azerbaijan, Georgia, Turkey, Turkmenistan, and China. The sequences had 4.1% (nad1) and 2.3% (cox1) of variable sites, and 13 and 10 haplotypes were identified among nad1 and cox1 genes, respectively. Spatial analysis of genetic and nucleotide diversity indicated little or no structuring of genetic variation between hosts or regions. The analysis of distribution of both separate and combined (nad1 + cox1) haplotypes revealed the existence of 2 well-defined lineages with 2 main haplotypes and a number of shared divergent haplotypes. Our study showed that the first lineage included the main N1-C1 haplotype, which was found in Australia, China, Georgia, Turkey, Armenia, Azerbaijan, and in all European populations (from Russia, Belarus, Ukraine, Bulgaria). The second lineage was found in all European populations and in populations from Armenia and Azerbaijan. It was suggested that one of the lineages (I) has an Asian origin. The possible source of mtDNA variability and associations between lineage divergence of parasite and its definitive hosts (cattle and sheep) are discussed.     

RAPD Fingerprinting of Common Bream Abramis brama L., Roach Rutilus rutilus L., and Their F1 Hybrids (A. brama × R. rutilus and R. rutilus × A. brama)

The polymerase chain reaction with arbitrary primers (RAPD–PCR) was used to study and to evaluate the genetic variation in the hybrid progeny of two Cyprinidae species, common bream Abramis brama L. and roach Rutilus rutilusL. Genetic polymorphism was studied in 20 fishes (young of the current year) obtained in four individual crosses: R. rutilus × R. rutilus (RR), A. brama × A. brama (AA), R. rutilus × A. brama (RA), and A. brama × R. rutilus (AR). Amplification spectra obtained with eight primers contained 288 fragments, 97.6% of which proved to be polymorphic. The proportion of polymorphic fragments was 75.0% in the RR progeny, 58.1% in the AA progeny, 84.9% in the AR progeny, and 77.8% in the RA progeny. Classification analysis in the space of principal components was performed with the first four components, which together accounted for 64% of the total variance of the character under study. The individual contributions of components I, II, III, and IV were 26.8, 16.8, 11.5, and 8.9%, respectively. Fishes of the two pure species and the hybrid progeny (direct and reverse hybrids together) were clearly differentiated in the space of principal components I and II. The best differentiation of the four samples (RR, AA, RA, and AR) was observed in the space of principal components II and IV. Possible causes of high genetic variation in interspecific hybrids are discussed.     

Polymorphism and structural features of two noncoding regions of the liver fluke <Emphasis Type="Italic">Fasciola hepatica</Emphasis> (Plathelminthes: Trematoda) mitochondrial genome

Structural characteristics and polymorphism of the long (LNR) and short (SNR) mitochondrial noncoding regions were studied in the liver fluke Fasciola hepatica. Flukes were sampled from several populations of Russia and Belarus. LNR amplification yielded a set of nine fragments, neighboring ones differing in length by one tandem repeat (85 bp), published for Australian flukes. The LNR amplification products of different lengths were cloned and sequenced. A comparison of the LNR sequences of Australian and Belarussian flukes revealed three nucleotide substitutions and one point heteroplasmy in the first positions of the imperfect repeat and four adjacent perfect repeats. The positions of the three mutations coincided in the perfect and imperfect repeats. The frequency of mutations was 4.0–4.7 %, while the frequency of heteroplasmic sites varied from 0.1 to 1.2%. It was shown that the mutations and the heteroplasmy of one site could change the structure and stability of the putative secondary structures of the perfect and imperfect repeats. SNR amplification in F. hepatica from several populations yielded fragments that differed from the published SNR sequence of Australian F. hepatica by one transversion (T → G in position 21). Both noncoding regions had several conserved and potential regulatory sequences. The possible causes of heteroplasmy and a concerted origin of substitutions in different repeats are discussed.     

Polymorphism of microsatellite markers in Russian common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.) breeds

Using five microsatellite loci, genotyping and genetic diversity estimates were obtained for nine samples representing seven common carp breeds most widespread in Russia. For comparison, the samples of Amur wild common carp (Cyprinus carpio haematopterus) and a sample of European Hungarian carp were used. In the samples examined (n = 148) a total of 78 alleles were revealed. The highest mean allele number per locus (7.3) was identified in Amur wild common carp, while the lowest number was found in Cherepets carps (4.0). In different breeds, the observed heterozygosities varied from 0.819 (Altai carp) to 0.651 (Cherepets scaly carp). Three out of five microsatellite loci (MFW-24, MFW-28, and MFW-19) revealed a high level of population differentiation. In the dendrogram of genetic differences, all breeds clustered into two groups. One of these groups was composed of the two strains of Ropsha carp, Stavropol carp, Amur wild common carp, and the two samples of Cherepets carp. The second cluster included Altai carp (Priobskii and Chumysh populations), two Angelinskii carp breeds (mirror and scaly), and Hungarian carp. The pairs of breeds/populations/strains, having common origin, were differentiated. Specifically, these were two populations of Altai carp, two strains of Ropsha carp, as well as the breeds of Angelinskii and Cherepets carps. The reasons for genetic differentiation of Russian common carp breeds, as well as the concordance of the evolutionary histories of these breeds, some of which originated from the European breeds, while the others contain substantial admixture of the Amur wild common carp, are discussed.     

Cloning and characterization of RAPD markers of parasitic nematodesTrichinella spiralis andT. pseudospiralis

A RAPD analysis was carried out in parasitic nematodesTrichinella spiralis andT. pseudospiralis, which are among the most common and hazardous agents in human and animal helminthoses. Several RAPD fragments were cloned and some of them sequenced. The fragments were shown to correspond to unique or low-repetitive sequences and to contain ORFs, microsatellites, and regions homologous to various pro- and eukaryotic sequences. Several fragments can be used to distinguish betweenT. spiralis andT. pseudospiralis.     

Genetic variability and differentiation of three Russian populations of potato cyst nematode <Emphasis Type="Italic">Globodera rostochiensis</Emphasis> as revealed by nuclear markers

Genetic variability of yellow potato cyst nematode G. rostochiensis from three Russian populations (Karelia, Vladimir oblast, and Moscow oblast) was investigated using two types of nuclear markers. Using RAPD markers identified with the help of six random primers (P-29, OPA-10, OPT-14, OPA-11, OPB-11, and OPH-20), it was possible to distinguish Karelian population from the group consisting of the populations from two adjacent regions (Moscow oblast and Vladimir oblast). Based on the combined matrix, containing 294 RAPD fragments, dendrogram of genetic differences was constructed, and the indices of genetic divergence and partition (P, H, and G _st), as well as the gene flow indices N _m between the nematode samples examined, were calculated. The dendrogram structure, genetic diversity indices, and variations of genetic distances between single individuals in each population from Karelia and Central Russia pointed to genetic isolation and higher genetic diversity of the nematodes from Karelia.Based on polymorphism of rDNA first intergenic spacer ITS1, attribution of all populations examined to the species G. rostochiensis was proved. Small variations of the ITS1 sequence in different geographic populations of nematodes from different regions of the species world range did not allow isolation of separate groups within the species. Possible factors (including interregional transportations of seed potato) affecting nematode population structure in Russia are discussed.     

Genetic Analysis and Estimation of Genetic Diversity in East-European Breeds of Windhounds (Canis familiaris L.) Based on the Data of Genomic Studies Using RAPD Markers

The method of polymerase chain reaction with a set of arbitrary primers (RAPD–PCR) was used to describe genetic variation and to estimate genetic diversity in East-European windhounds, Russian Borzoi and Russian Chortai. For comparison, windhounds of two West-European breeds (Whippet and Greyhound) and single dogs of other breed types (shepherd, terriers, mastiffs, and bird dogs) were examined. For all dog groups, their closest related species, the wolf Canis lupus, was used as an outgroup. Variation of RAPD markers was studied at several hierarchic levels: intra- and interfamily (for individual families of Russian Psovyi and Chortai windhounds), intra- and interbreed (for ten dog breeds), and interspecific (C. familiaris–C. lupus). In total, 57 dogs and 4 wolves were studied. Using RAPD–PCR with three primers, 93 DNA fragments with a length of 150–1500 bp were detected in several Windhound families with known filiation. These fragments were found to be inherited as dominant markers and to be applicable for estimation of genetic differences between parents and their offspring and for comparison of individuals and families with different level of inbreeding. A high level of intra- and interbreed diversity was found in Russian Borzoi and Russian Chortai. In these dogs, genetic similarity indices varied in a range of 72.2 to 93.4% (parents–offspring) and 68.0 to 94.5 (sibs). Based on the patterns of RAPD markers obtained using six primers, a dendrogram of genetic similarity between the wolf and different dog breeds was constructed, and indices of intragroup diversity were calculated. All studied breeds grouped into two clusters, windhounds (Borzoi-like dogs) and other dog breeds. Russian windhounds represent a very heterogeneous group, in which the Russian Borzoi is closer to Greyhound than the Russian Chortai. All studied wolves constituted a separate cluster. Significant differences were found between the wolf and dogs by the number of RAPD markers (92.8 and 86.1, respectively) and by the indices of genetic diversity (54.3 and 64.8%, respectively). The reason for the high intraspecific variation of dogs (including Russian windhounds) and the prospects of using the studied group of markers for genetic analysis and differentiation in C. familiaris are discussed.