Mitochondrial genome variation in domesticated sable (<Emphasis Type="Italic">Martes zibellina</Emphasis>)

The first comparison of mitochondrial variations in sables from captive and natural populations of the Urals, Central Siberia, Yakutia, Kamchatka, and Japan has been performed. The object of comparative analysis was a 427-bp 5′ fragment of the mitochondrial control region, including the D-loop. Two main haplogroups of the sable mitochondrial genome have been found, which provides new data for reconstruction of the spread of the sable over its current range. Asymmetry of the haplotype abundances in the captive populations of sables has been detected. The mitochondrial haplotypes characteristic of sable breeds have been identified. The possible role of the frequent mitochondrial haplotypes of the captive population in the sable adaptation to the conditions of captivity is discussed.     

A PCR-RFLP-based method to distinguish sable (<Emphasis Type="Italic">Martes zibellina</Emphasis>) and pine marten (<Emphasis Type="Italic">Martes martes</Emphasis>)

Species identification is an important issue in conservation and a particular focus for wildlife forensics. Molecular biological methods retain a unique power to differentiate between difficult samples that lack other identifiable characteristics. The pine marten (Martes martes) and sable (Martes zibellina) are closely related species with very similar pelage characteristics and are often difficult to distinguish from each other. The sable, however, in contrast to the pine marten, remains an endangered and protected animal in China with both hunting and fur trade strictly prohibited for this species. Here, we present a polymerase chain reaction-based restriction fragment length polymorphism method for distinguishing the two species. We sequenced a 638-bp fragment of cytochrome b gene in 39 sables, 68 pine martens, and 10 stone martens and identified all variable nucleotides. A new primer pair was subsequently designed to amplify a 316-bp fragment containing restriction sites of enzyme BseG I and BamH I that are different among martens. When the fragment was cut using BseG I, the resulting restriction pattern was identical in the sable and pine marten, but differed from all other martens. When cut using BamH I, the fragment generated two diagnostic fragments in the sable which could distinguish them from pine martens. This method was valid for all haplotypes of sable and pine marten thus far identified and has high potentially applicability for the identification of the two species.     

Microsatellite analysis of two captive populations of sable (<Emphasis Type="Italic">Martes zibellina</Emphasis> L.)

The high value of sable (Martes zibellina L.) fur and stable demand for it over the centuries have led to suboptimal hunting patterns and, as a result, considerable fluctuations in the sizes of natural populations of this species. To maintain the traditional export of sable fur, efforts towards commercial domestication of sable have been made in Russia. The first farm population of sable consisted of animal from eight natural populations was founded in 1929. After the problems related to breeding in captivity were solved, directional selection began. Eighty years of breeding have resulted in sable herds with homogeneous quantitative characters. Prospects for further breeding depend on the current level of genetic diversity in the captive populations of sables formed during the first stages of domestication. The sable populations of the Pushkinsky and Saltykovsky fur farms located in Moscow oblast, which were the objects of this study, are the progenitors of the existing captive populations. The first estimation of genetic variation of this species by means of a panel of micro-satellite markers was developed for this study. Two captive sable populations were analyzed using ten micro-satellite loci; a total of 75 alleles were found in both populations. Population-specific alleles were identified (6 and 13 in the Pushkinsky and Saltykovsky populations, respectively). The populations studied were found to be differentiated with respect to four microsatellite loci.     

Intraspecific structure of sable <Emphasis Type="Italic">Martes zibellina</Emphasis> L. Inferred from nucleotide variation of the mitochondrial DNA cytochrome <Emphasis Type="Italic">b</Emphasis> gene

A fragment of the mitochondrial DNA (mtDNA) cytochrome b gene was sequenced in sable from Magadan oblast, Khabarovsk krai, and Kamchatka. Using phylogenetic analysis, the presence of two clusters (A and BC), with the divergence value of 1.4%, was demonstrated. Analysis of the cytochrome b gene median networks indicated that split of the ancestral population took place in early Pleistocene (about one Myr ago), while expansion of its more young phylogenetic group A occurred in late Pleistocene, about 120000 years ago.     

Genetic diversity of MHC class II <Emphasis Type="Italic">DRB</Emphasis> alleles in the continental and Japanese populations of the sable <Emphasis Type="Italic">Martes zibellina</Emphasis> (Mustelidae,Carnivora, Mammalia)

The sable (Martes zibellina) is a medium-sized mustelid inhabiting forest environments in Siberia, northern China, the Korean Peninsula, and Hokkaido Island, Japan. To further understand the molecular evolution of the major histocompatibility complex (MHC), we sequenced part of exon 2 in MHC class II DRB genes, including codons encoding the antigen binding site, from 33 individuals from continental Eurasia and Japan. We identified 16 MHC class II DRB alleles (Mazi-DRBs), some of which were geographically restricted and others broadly distributed, and eight putative pseudogenes. A single-breakpoint recombination analysis detected a recombination site in the middle of exon 2. A mixed effects model of evolution analysis identified five amino acid sites presumably under positive selection. These sites were all located in the region 3′ to the recombination site, suggesting that positive selection and recombination could be committed to the diversity of the M. zibellina DRB gene. In a Bayesian phylogenetic tree, all Mazi-DRBs and the presumed pseudogenes grouped within a Mustelidae clade. The Mazi-DRBs showed trans-species polymorphism, with some alleles most closely related to alleles from other mustelid species. This result suggests that the sable DRBs have evolved under long-lasting balancing selection.     

Genetic structure of the sable <Emphasis Type="Italic">Martes zibellina</Emphasis> L. populations from Magadan oblast as inferred from mitochondrial DNA variation

Restriction polymorphism of the mtDNA cytochrome b gene was studied in nine sable Martes zibellina L. populations from three introduction foci of Khabarovsk and Kamchatka sables in Magadan oblast: Olya, Kolyma, and Omolon. For comparison, similar studies were performed with the populations of central Kamchatka and Khabarovsk krai. In total, 444 DNA specimens were examined. Three mtDNA haplotypes (A, B, and C) proved to occur at various frequencies in the populations under study. The sable population system displayed high differentiation (F _ST = 22.3%). The populations of the Olya focus were most similar genetically to the populations of Kamchatka; those of the Omolon focus were similar to the Khabarovsk populations, and those of the Kolyma focus occupied an intermediate place. The observed spatial heterogeneity of the sable populations of Magadan oblast was explained in terms of the formation of the introduction foci of Kamchatka and Khabarovsk sables, starting from the 1950s.     

A PCR-RFLP method on faecal samples to distinguish <Emphasis Type="Italic">Martes martes</Emphasis>, <Emphasis Type="Italic">Martes foina</Emphasis>, <Emphasis Type="Italic">Mustela putorius</Emphasis> and <Emphasis Type="Italic">Vulpes vulpes</Emphasis>

A non-invasive genetic approach has been recently employed in the population genetics of wild species, using faeces that could be easily collected, particularly of elusive or endangered species. However, faeces of pine (Martes martes) and beech marten (M. foina) can be morphologically similar and could be confused with those of polecat (Mustela putorius) and red fox (Vulpes vulpes). On this basis, a rapid, simple and inexpensive RFLP protocol using a cytochrome b (Cyt b) gene fragment of mtDNA, isolated from faecal samples, was performed to distinguish the above-mentioned species.     

The effect of artificial selection for coat color on fitness in a farm population of the sable (<Emphasis Type="Italic">Martes zibellina</Emphasis>)

The relationship between the response to artificial selection for darker coat color and fitness in a farm population of the sable (Martes zibellinaL.) from the Pushkinskoe Fur Farm (Moscow oblast) was studied. The selection was performed during 41 years. By the moment of the study, a response to the selection for this character had been obtained: the coat color in the selected population had become darker, and the proportion of black animals in it increased. In addition, sables with black heads, which were absent in the original population, had appeared. Artificial selection was accompanied by a decrease in the fitness of the selected population, which was expressed in decreased female reproductive capacity parameters (the fertility, maturation rate, and duration of the reproductive period). A selection technique consisting in the use of only highly fertile animals in the selection originally made it possible to restore the fitness parameters to the initial level almost without a decrease in the dark shade of the fur. However, further selection led to a drastic decrease in fitness that could not be precluded by any selection method used. The possible ways to overcome this unfavorable effect of artificial selection are discussed.     

Genetic Effects of Sable (<Emphasis Type="Italic">Martes zibellina</Emphasis> L.) Reintroduction in Western Siberia

In the middle of the 20th century, massive introductions of sables were performed to recover the area of this valuable fur species. In this work, genetic variation of a naturalized sable population from the Vakh River basin (Nizhnevartovskiy district, Khanty-Mansi Autonomous Okrug) was investigated. This population developed as a result of sable introduction from Cisbaikalia in 1952?1957. The naturalized sable population of the Vakh basin occupies an intermediate position between two autochthonous sable populations from the Ob River area and Cisbaikalia, as assessed by variation of five microsatellite loci. Apparently, the genetic structure of the modern sable population from the Vakh basin was formed by mixing the gene pools of the original Cisbaikalian population and the neighboring autochthonous populations from the Ob River area which recovered their numbers in a natural way. Data on genetic variation in the naturalized sable population agree with the results of previous morphological studies and can be employed for long-term monitoring of the outcome of sable introduction.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.     

Effect of coat color mutations on behavioral polymorphism in farm populations of American minks (<Emphasis Type="Italic">Mustela vison</Emphasis> Schreber, 1777) and sables (<Emphasis Type="Italic">Martes zibellina</Emphasis> Linnaeus, 1758)

Behavioral polymorphism estimated by the expression of the defensive reaction towards humans has been studied in farm-bred American minks and sables with different color types. Most animals (both minks and sables) from farm populations displayed passive defensive behavior towards humans in the standard hand catch test. Coat color genes have been found to have pleiotropic effects; they influence both the penetrance and expressivity of domestication behavior: in animals with aberrant color types (both sapphire minks and white-and-black sables), the proportion of animals with domestication behavior and the expressivity of this behavior are significantly higher (p < 0.01 and p < 0.001, respectively).     

Pedogamy in <Emphasis Type="Italic">Neidium</Emphasis> (<Emphasis Type="Italic">Bacillariophyceae</Emphasis>)

Single (unpaired) vegetative cells of freshwater pennate diatom Neidium cf. ampliatum differentiated into gametangia and produced a single zygote (auxospore) via a pedogamic process. The gametic nuclei fused after auxospore expansion had begun. The auxospore expanded in parallel to the apical axis of the gametangium.     

<Emphasis Type="Italic">In silico</Emphasis> and <Emphasis Type="Italic">in situ</Emphasis> characterization of the zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) <Emphasis Type="Italic">gnrh3</Emphasis> (sGnRH) gene

Background

Gonadotropin releasing hormone (GnRH) is responsible for stimulation of gonadotropic hormone (GtH) in the hypothalamus-pituitary-gonadal axis (HPG). The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio).

Results

We have characterized a zebrafish [Trp⁷, Leu⁸] or salmon (s) GnRH variant, gnrh 3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH), was shown capable of driving cell specific reporter gene expression in transgenic zebrafish.

Conclusions

The characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar) GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III.

     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Quinua</Emphasis> and Relatives (<Emphasis Type="Italic">Chenopodium</Emphasis> sect.<Emphasis Type="Italic">Chenopodium</Emphasis> subsect.<Emphasis Type="Italic">Celluloid</Emphasis>)

Traditionally viewed as an Andean grain crop,Chenopodium quinoa Willd. includes domesticated populations that are not Andean, and Andean populations that are not domesticated. Comparative analysis of leaf morphology and allozyme frequencies have demonstrated that Andean populations, both domesticated(quinua) and free-living(ajara), represent an exceptionally homogeneous unit that is well differentiated from allied domesticates of coastal Chile(quingua) and freeliving populations of the Argentine lowlands(C. hircinum). This pattern of relationships indicates that Andean populations represent a monophyletic crop/weed system that has possibly developed through cyclic differentiation (natural vs. human selection) and introgressive hybridization. Relative levels of variation suggest that this complex originated in the southern Andes, possibly from wild types allied withC. hircinum, with subsequent dispersal north to Colombia and south to the Chilean coast. Coastal populations were apparently isolated from post-dispersal differentiation and homogenization that occurred in the Andes. Other data point toward a center of origin in the northern Andes with secondary centers of genetic diversity subsequently developing in the southern Andes and the plains of Argentina. Comparative linkage of South American taxa, all tetraploid, with North American tetraploids of the subsection will eventually clarify this problem. While the possibility of a direct phyletic connection betweenC. quinoa and the Mexican domesticate(C. berlandieri subsp. nuttalliae,) cannot be excluded, available evidence indicates that the latter represents an autonomous lineage that is associated with the basal tetraploid, C. b. subsp.berlandieri, through var.sinuatum, whereas South American taxa show possible affinities to either var. zschackei or var.berlandieri. An extinct domesticate of eastern North America,C. b. subsp.jonesianum, represents either another instance of independent domestication, possibly from subsp. b. var.zschackei, or a northeastern outlier of subsp.nuttalliae.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.