Galina N. Chelomina

A spatial aspect on mitochondrial DNA genealogy in Apodemus peninsulae from East Asia.

Apodemus peninsulae is a field mouse that inhabits the broad-leafed forests of temperate Eurasia. We examined the mitochondrial cytochrome b gene in 57 individuals of A. peninsulae from northeastern Asia, including Siberia, Primorye, Magadan region, Sakhalin, Hokkaido, and the Korean Peninsula. The genealogy of the mitochondrial DNA (mtDNA) in A. peninsulae was shown to have substantial geographic affinity, suggesting geographic architecture of northeastern Asia, including the islands of Sakhalin and Hokkaido, played important roles on the cladogenesis. Taking into account the presence of region-specific anciently divergent mtDNA types, three parts of the regions of Primorye, Siberia, and the Korean Peninsula can be denoted as refugia for A. peninsulae during the substantial period of the Quaternary glacial ages. Among the geographic regions examined, Primorye is likely to be the most influential one, from which the mtDNA is thought to have migrated to the neighboring regions of Sakhalin, Hokkaido, the Magadan region, and Siberia during the evolution of this species.

Genetic variation and differentiation in striped field mouse Apodemus agrarius inferred from RAPD-PCR analysis

Genetic variation and differentiation of the trans-Palearctic species Apodemus agrarius (striped field mouse), whose range consists of two large isolates—European-Siberian and Far Eastern-Chinese, were examined using RAPD-PCR analysis. The material from the both parts of the range was examined (41 individual of A. agrarius from 18 localities of Russia, Ukraine, Moldova, and Kazakhstan); the Far Eastern-Chinese part was represented by samples from the Amur region, Khabarovsk krai, and Primorye (Russia). Differences in frequencies of polymorphic RAPD loci were found between the European-Siberian and the Far Eastern population groups of striped field mouse. No “fixed” differences between them in RAPD spectra were found, and none of the used statistical methods permitted to distinguish with absolute certainty animals from the two range parts. Thus, genetic isolation of the European-Siberian and the Far Eastern population groups of A. agrarius is not strict. These results support the hypothesis on recent dispersal of striped field mouse from East to West Palearctics (during the Holocene climatic optimum, 7000 to 4500 years ago) and subsequent disjunction of the species range (not earlier than 4000–4500 years ago). The Far Eastern population group is more polymorphic than the European-Siberian one, while genetic heterogeneity is more uniformly distributed within it. This is probably explained by both historical events that happened during the species dispersal in the past, and different environmental conditions for the species in different parts of its range. The Far Eastern population group inhabits the area close to the distribution center of A. agrarius. It is likely that this group preserved genetic variation of the formerly integral ancestral form, while some amount of genetic polymorphism could be lost during the species colonization of the Siberian and European areas. To date, the settlement density and population number in general are higher than within the European-Siberian isolate, which seems to account for closer interpopulation associations, intense genetic exchange, and “smoothing” of polymorphism within the Far Eastern population group of A. agrarius.

Genetic and Taxonomic Diversity of the House Mouse Mus musculus from the Asian Part of the Former Soviet Union

Genetic diversity of the house mouse Mus musculus from 12 local populations (n = 65) of the central and eastern parts of the former Soviet Union was examined using RAPD–PCR. About 400 loci were identified, encompassing approximately 500 kb of the mouse genome. Genetic diversity was assessed using NTSYS, POPGENE, TFPGA, and TREECON software programs. In general, the house mouse sample from the regions examined was characterized by moderate genetic variation: polymorphism P = 95.6%, P99 = 60.7%, P95 = 24.2%; heterozygosity H = 0.089; the mean observed number of alleles na = 1.97; effective number of alleles ne = 1.13; intrapopulation differentiation δS = 0.387; gene diversity h = 0.09. Individual local populations displayed different levels of genetic isolation: the genetic subdivision index Gst varied from 0.086 to 0.324 at gene flow Nm varying from 5.3 to 1.05, while the interpopulation genetic distance DN ranged from 0.059 to 0.186. Most of the genetic diversity of the total sample resided within the local populations: HS = 0.06, total gene diversity HT = 0.09. The exact test for differentiation, however, did not confirm the affiliation of all the mice examined to one population: χ2 = 1446, d.f. = 724, P = 0.000. Molecular markers specific to four subspecies (musculus, castaneus, gansuensis, and wagneri) were identified. Moreover, in some cases the populations and individual animals exhibited traits of different subspecies, suggesting their introgressive hybridization. It was demonstrated that the house mouse fauna on the territories investigated was characterized by the prevalence of musculus-specific markers, while gansuensis-specific markers ranked second. The castaneus-specific markers were highly frequent in the Far East, but almost absent in Central Asia, where wagneri-specific markers were detected. It was suggested that house mice from Turkmenistan could belong to one of the southern subspecies, which had not deeply penetrated into the Asian fauna of the former Soviet Union. In phenogenetic (UPGMA) and phylogenetic (NJ) reconstructions this form with the high bootstrap support was placed at the tree base, while the isolation of other clusters was not statistically significant. It is thus likely that the house mice from Turkmenistan are closest to the ancestral form of the genus Mus on the territory of the former Soviet Union.

Analysis of genetic variation and differentiation in the pygmy wood mouse Sylvaemus uralensis (Rodentia, Muridae) aided by the RAPD-PCR method

The present work ascertains distinct differentiation of the pygmy wood mouse into two groups of populations, which correspond to the European and Asian races, but cannot, however, be considered valid species due to the low values of genetic distances between them. Neither of the used statistical methods explicitly verifies genetic isolation of the East European and South European chromosomal forms, which are close to one another and together constitute the European race. However, these chromosomal forms/races of the pygmy wood mouse differentiates each from other by a character of polymorphism. It can be explained both by the history of their formation (the degree of proximity to the ancestral population complex, isolation, etc.) and by unequal current environmental conditions.

Genetic diversity and phylogenetic relationships of the Manchurian zokor Myospalax psilurus (Rodentia, Muridae) according to RAPD-PCR analysis

AbstractsThe population structuring and low genetic diversity of the Manchurian zokor Myospalax psilurus Milne-Edwards, 1874, an East Asian endemic included in the Red List of Russia, were demonstrated. Two separate geographical groups differing in the level of their genetic diversity were found on the territory of the Primorskii krai. The subpopulation located closest to the main area of this species was determined as ancestral. A subspecies differentiation of the Primorskii krai and Transbaikal M. psilurus populations was shown, as was the monophyletic origin of M. psilurus and its high divergence from M. aspalax. The animals from northern localities are recommended for reintroduction in nature under species recovery programs in Primorskii krai.

Variation in the number of nucleoli and incomplete homogenization of 18S ribosomal DNA sequences in leaf cells of the cultivated Oriental ginseng (Panax ginseng Meyer)

Background Wild ginseng, Panax ginseng Meyer, is an endangered species of medicinal plants. In the present study, we analyzed variations within the ribosomal DNA (rDNA) cluster to gain insight into the genetic diversity of the Oriental ginseng, P. ginseng, at artificial plant cultivation. Methods The roots of wild P. ginseng plants were sampled from a nonprotected natural population of the Russian Far East. The slides were prepared from leaf tissues using the squash technique for cytogenetic analysis. The 18S rDNA sequences were cloned and sequenced. The distribution of nucleotide diversity, recombination events, and interspecific phylogenies for the total 18S rDNA sequence data set was also examined. Results In mesophyll cells, mononucleolar nuclei were estimated to be dominant (75.7%), while the remaining nuclei contained two to four nucleoli. Among the analyzed 18S rDNA clones, 20% were identical to the 18S rDNA sequence of P. ginseng from Japan, and other clones differed in one to six substitutions. The nucleotide polymorphism was more expressed at the positions 440–640 bp, and distributed in variable regions, expansion segments, and conservative elements of core structure. The phylogenetic analysis confirmed conspecificity of ginseng plants cultivated in different regions, with two fixed mutations between P. ginseng and other species. Conclusion This study identified the evidences of the intragenomic nucleotide polymorphism in the 18S rDNA sequences of P. ginseng. These data suggest that, in cultivated plants, the observed genome instability may influence the synthesis of biologically active compounds, which are widely used in traditional medicine.

Increased genetic diversity of 18S rDNA in the genomes of F1 hybrids of sturgeons Acipenser schrenckii × A. baerii and A. schrenckii × Huso dauricus

Study on Acipenseriformes fishes demonstrated that different species of the Acipenser genus have various 18S rDNA copies, which is a trait much distinguishing this fish from other animals (including Polyodon spathula , a phylogenetic ancestor of all sturgeons) and suggesting incompleteness of concerted evolution in their genomes [2, 3]. Most researchers explain this phenomenon by polyploidy [3]. Other factors could also disturb the mechanisms of concerted evolution, e.g., a low rate of molecular evolution in sturgeons [3]. In plants, polyploidy is widespread, and intraspecific variation of rDNA sequences is in most cases related to multiplicity of nucleolus-organizing regions (NORs) and to their location in different chromosomes [4]. The amount of gene copies may depend on the genome size; in organisms with relatively small genomes, pseudogenes are known to be eliminated faster than mutations are accumulated [5]. Some authors suggest that hybridization is the most probable and most potent factor of rDNA evolution, along with mutations, homogenization, and selection [6].

Genetic variation of Microtus fortis pelliceus in mainland and island populations of the Russian Far East based on random amplified polymorphic DNA markers

ABSTRACT Random amplified polymorphic DNA (RAPD) markers were used to estimate the level and pattern of genetic diversity in Microtus fortis pelliceus populations from the mainland and islands of the Russian Far East. No markers were found both for individual populations and for the mainland or island voles as a whole. However, they appeared to be different in both their allele frequencies and microevolution mode based on correlation pleiad analysis. The island populations demonstrated a higher level of genetic differentiation among themselves than did mainland populations and each mainland population probably represents more genetic diversity than any population of islands. Overall, genetic variation between island and mainland M. f. pelliceus populations was low (Dst = 0.003), and can be explained by a low value of gene differentiation coefficient (Gst = 0.058). Nevertheless, exact test did not support the hypothesis that both united mainland and united island populations belong to the same genetic unit (p = 0.0025). RAPD data reliably distinguished voles of Matveeva Island, which are strongly differentiated by morphological traits, and this in turn did not exclude the island speciation event. Unlike UPGMA and NJ trees for individuals, population UPGMA strongly separated island voles from the mainland conspecifics, and MS reconctructions identified the Amur population as an ancestor.

Quantitative sexing (Q-sexing) technique for animal sex-determination based on X chromosome-linked loci: Empirical evidence from the Siberian tiger

Here we report a mammal sexing procedure based on the detection of quantitative differences between females and males in the X-linked loci (quantitative sexing, Q-sexing). This novel technique was validated using samples from Siberian tigers ( Panthera tigris altaica ) whose sexes were known. The Q-sexing technique relies on the fact that amplifications proceeding exclusively from the two X chromosomes in a female mammal should reach the threshold cycle (CT) in a real-time quantitative real time polymerase chain reaction (qPCR) assay sooner than amplifications from the single male X chromosome. Nevertheless, given that the amplification efficiency may vary between samples, results have to be calibrated to a marker that does not vary in copy number between the sexes (for example, an autosomal-linked locus). For this purpose we used quantitative real time polymerase chain reaction (RT qPCR) assays to quantify the amount of three specific Siberian tiger microsatellite markers (X-/Y- and autosomal-linked loci) in individual samples in order to determine the sex of an animal. A difference of one CT between the X and the autosome-linked loci was detected in males, but no such difference was present in female samples. The Q-sexing technique unambiguously separates female from male Siberian tigers. The future of RT qPCR is bright as technology is becoming ever more rapid, cost-effective, easier to use and capable of processing higher throughputs. Thus, we expect that our novel technique for animal sexing will have a wide applicability, although further studies are still needed to adapt it to other animal species using specific primers. Key words : Polymerase chain reaction (PCR), quantitative real time polymerase chain reaction (qPCR), quantitative sexing, Siberian tiger.

Genetic diversity and phylogenetic relations of salmon trematode Nanophyetus japonensis

Nanophyetiasis is the severe zoonotic disease caused by parasitic worms from the genus Nanophyetus. Humans and carnivorous animals become infected when they ingest raw fish containing metacercariae, especially Pacific salmonids. Nanophyetiasis is detected in limited geographical areas which include the coastal regions of the North Pacific: the United States of America, Russian Federation and Japan. Despite the epidemiological significance, Nanophyetus species have not been well studied genetically. In this research, we for the first time explored genetic diversity of Nanophyetus japonensis from Japan in comparison with those of related species, N. salmincola from North America and N. schikhobalowi from the Russian Far East, based on sequence variation in the nuclear ribosomal gene family (18S, ITS1-5.8S-ITS2 and 28S) and mitochondrial nad1 gene, encoding subunit I of the respiratory chain NADH dehydrogenase. The results confirmed the independent species status for the compared flukes, demonstrated a greater genetic similarity of Asian species between themselves than each of them with the North American one, suggesting that N. japonensis and N. schikhobalowi are close sister species, and also revealed discrepancy between the levels of morphological and genetic differentiation.