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

RAPD-PCR molecular analysis of the threatened Cabrera’s vole populations in the Iberian Peninsula

Optimal management and conservation programs of the threatened Cabrera’s vole require investigating potential molecular genetic markers in the genomic background, if the few remaining fragile populations are to be conserved. A collection of 30 Cabrera’s vole representing four populations in Spain and Portugal was characterized by 134 RAPD-PCR markers. Molecular analyses did not detect low level of the genetic diversity or population bottlenecks in all studied populations, in discordance with the expectation of low level of viability of the Cabrera’s vole. The results described Cabrera’s vole populations as a single genetic unit with slightly restricted gene flow. Phylogenetic reconstruction suggested genetic differentiation between Northern and Southern Cabrera’s vole populations, with the basal branches formed by the southern populations, which may be an evidence of the southern origin of Iberian vole ancestral population. To our knowledge, this is the first study on the genetic diversity of Microtus cabrerae, which may have further application for the conservation programs of this threatened species of Iberian vole.Keywords: Microtus cabrerae, RAPD-PCR, Spain, Portugal, gene flow, genetic diversity, bottleneck, conservationAfrican Journal of Biotechnology Vol. 12(26), pp. 4065-407

Genotoxicity of Paragonimus heterotremus Infection in a Rat Model of Simultaneous Pulmonary and Hepatic Paragonimiasis

Parasites cause numerous health issues in humans, eventually leading to significant social and economic damage; however, the mechanisms of parasite-mediated pathogenesis are not well understood. Nevertheless, it is clearly evidenced that cancerogenic fluke-induced chronic inflammations and cancer are closely associated with oxidative stress. (1) Methods: The Paragonimus heterotremus infection’s genotoxic potential was assessed in a rat model of simultaneous pulmonary and hepatic paragonimiasis by the alkaline version of single-cell gel electrophoresis (comet assay). Statistical analysis of comet parameters was based on the non-parametric Mann–Whitney U test. (2) Results: A clear and statistically significant increase in DNA damage was detected in the helminth-exposed group versus the control rats and the tissue areas adjacent to the parasite capsule versus remote ones; however, differences in DNA damage patterns between different tissues were not statistically significant. Infection resulted in up to 40% cells with DNA damage and an increased genetic damage index. (3) Conclusions: The data obtained contribute to understanding the pathogenesis mechanisms of paragonimiasis, suggesting oxidative stress as the most likely reason for DNA breaks; these findings allow us to consider P. heterotremus as a potentially cancerogenic species, and they are important for the monitoring and treatment of paragonimiasis

Comparative phylogeography of four Apodemus species (Mammalia: Rodentia) in the Asian Far East: evidence of Quaternary climatic changes in their genetic structure

The phylogeography of four Apodemus species (Apodemus agrarius, Apodemus peninsulae, Apodemus latronum, and Apodemus draco) was studied in the Far East of Asia, based on sequences of the mitochondrial DNA cytochrome b gene. The results obtained show the existence of many different genetic lineages within the studied Apodemus species, suggesting the isolation and differentiation of populations in multiple refuge areas. Higher genetic diversities in some regions such as Yunnan, Sichuan (China), and eastern Russia suggest these areas are potential refuges for these species. The existence of such complex genetic structures could be linked to the presence of many biogeographic barriers (Himalaya Mountains, Tien-shan Mountains, Altai Mountains, Tibetan Plateau, Gobi desert, Yunnan Guizhou Plateau, Dzungaria basin, and others) in these regions, which were probably reinforced during the Quaternary climate changes. These barriers also played an important role concerning the low dispersal abilities of the two studied Apodemus species adapted to forest habitats (A. latronum and A. draco) with respect to colonizing regions other than China. © 2010 The Linnean Society of London

A biogeographic view of Apodemus in Asia and Europe inferred from nuclear and mitochondrial gene sequences

Sequences of the mitochondrial cyt b gene and nuclear IRBP, RAGI, 17, and vWF genes were used to assess the evolutionary history of major lineages of Apodemus, in particular to better understand dispersal between Asia and Europe. Our data show eight extant lineages of Late Tertiary origin: Apodemus agrarius, A. semotus, A. peninsulae, A. speciosus, A. argenteus, A. gurkha, A. mystacinus, and A. sylvaticus. Monophyly of two European lineages (A. mystacinus and A. sylvaticus) and four Asian lineages (A. agrarius, A. semotus, A. peninsulae, and A. speciosus) was confirmed with high bootstrap support. Together with literature data, the available molecular data depict three crucial evolutionary events: (1) initial wide dispersal and subsequent radiation around 6 million years ago, (2) region-specific radiations in Europe and southern China around 2 million years ago, and (3) westward dispersal of A. agrarius to Europe in the Late Quaternary

Phylogeny of the genus Apodemus with a special emphasis on the subgenus Sylvaemus using the nuclear IRBP gene and two mitochondrial markers: cytochrome b and 12S rRNA.

Phylogenetic relationships among 17 extant species of Murinae, with special reference to the genus Apodemus, were investigated using sequence data from the nuclear protein-coding gene IRBP (15 species) and the two mitochondrial genes cytochrome b and 12S rRNA (17 species). The analysis of the three genes does not resolve the relationships between Mus, Apodemus, and Rattus but separates Micromys from these three genera. The analysis of the two mitochondrial regions supported an association between Apodemus and Tokudaia and indicated that these two genera are more closely related to Mus than to Rattus or Micromys. Within Apodemus, the mitochondrial data sets indicated that 8 of the 9 species analyzed can be sorted into two main groups: an Apodemus group, with A. agrarius, semotus, and peninsulae, and a Sylvaemus group, with uralensis, flavicollis, alpicola, sylvaticus, and hermonensis. The position of Apodemus mystacinus is ambiguous and might be either included in Sylvaemus or considered a distinct subgenus, Karstomys, more closely related to Sylvaemus than to Apodemus. Estimation of the divergence time for these taxa suggests a separation between 7 and 8 My ago for the three groups (mystacinus and the two subgenera Apodemus and Sylvaemus). Within each subgenus, divergence times are between 5.4 and 6 My for Apodemus and between 2.2 and 3.5 My for Sylvaemus and mystacinus

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