Population genetic structure of wild boar <Emphasis Type="Italic">Sus scrofa</Emphasis> in Bulgaria as revealed by microsatellite analysis

Over the last two decades wild boar Sus scrofa Linnaeus, 1758 became the most intensively managed game species in Bulgaria. In order to delineate the population genetic structure, which is essential for sustainable wildlife management, we screened 10 porcine microsatellites across 289 wild boar samples originating from all relevant bioregions of the country. Results based on F _ST values, Bayesian clustering methods and a multi-dimensional scaling analysis can be summarised as follows: (1) two main genetic groups were revealed for the Bulgarian data set: the first one included individuals collected from the Balkan Range Mountain and the northern part of the country and the second one comprised individuals from the Rhodope, Osogovo, Iskur Range and Rila mountains in southern and south-western Bulgaria; (2) all Bulgarian wild boar populations showed a higher level of genetic diversity compared to four populations from Germany which were included for outgroup comparison, and (3) wild boar sampled from a game enclosure were found to be genetically divergent from the other Bulgarian populations, indicating human impact on population genetic structure most likely resulted from fencing and former translocation actions. The evolutionary background leading to the two defined management units as well as conservation and management strategies are discussed.     

Reconstruction of the Transmission History of RNA Virus Outbreaks Using Full Genome Sequences: Foot-and-Mouth Disease Virus in Bulgaria in 2011

Improvements to sequencing protocols and the development of computational phylogenetics have opened up opportunities to study the rapid evolution of RNA viruses in real time. In practical terms, these results can be combined with field data in order to reconstruct spatiotemporal scenarios that describe the origin and transmission pathways of viruses during an epidemic. In the case of notifiable diseases, such as foot-and-mouth disease (FMD), these analyses provide important insights into the epidemiology of field outbreaks that can support disease control programmes. This study reconstructs the origin and transmission history of the FMD outbreaks which occurred during 2011 in Burgas Province, Bulgaria, a country that had been previously FMD-free-without-vaccination since 1996. Nineteen full genome sequences (FGS) of FMD virus (FMDV) were generated and analysed, including eight representative viruses from all of the virus-positive outbreaks of the disease in the country and 11 closely-related contemporary viruses from countries in the region where FMD is endemic (Turkey and Israel). All Bulgarian sequences shared a single putative common ancestor which was closely related to the index case identified in wild boar. The closest relative from outside of Bulgaria was a FMDV collected during 2010 in Bursa (Anatolia, Turkey). Within Bulgaria, two discrete genetic clusters were detected that corresponded to two episodes of outbreaks that occurred during January and March-April 2011. The number of nucleotide substitutions that were present between, and within, these separate clusters provided evidence that undetected FMDV infection had occurred. These conclusions are supported by laboratory data that subsequently identified three additional FMDV-infected livestock premises by serosurveillance, as well as a number of antibody positive wild boar on both sides of the border with Turkish Thrace. This study highlights how FGS analysis can be used as an effective on-the-spot tool to support and help direct epidemiological investigations of field outbreaks.     

Allelic constitution of the hemoglobin beta chain in wild populations of the house mouse,Mus musculus

We studied the allelic frequency of the hemoglobin beta chain (Hbb) of wild mice, Mus musculus, collected from 46 localities, mostly in Asia and surrounding areas. The wild populations in the northern part of China, Korea, and the central part of Japan exhibited an almost monomorphic distribution of Hbbp. In the southern part of Asia, the frequency of Hbbp decreased and Hbbd was predominant. Although Hbbs and Hbbd are generally found in Europe, the Hbbp allele was present in Southeastern Europe (Bulgaria). In the light of these results, the Hbbp allele might have originated in mice of northern Asia.     

Assessment of genetic structure of Greek brown hare (Lepus europaeus) populations based on variation in random amplified polymorphic DNA (RAPD)

The RAPD method was used to assess the genetic differentiation of brown hare (Lepus europaeus) populations from Central Greece. Greek wild populations were compared with samples from Austria, Poland, Germany, France, and Bulgaria, as well as with reared/released hares to investigate the impact of the releases on the native populations' genetic structure. The absence of diagnostic bands distinguishing between L. europaeus populations confirmed the high level of gene flow between brown hare populations over long geographic distances reported by other authors. Phylogenetic trees, derived from genetic distances estimated by RAPD band frequencies, suggested one major partitioning event of nuclear DNA lineages found in the samples. The reared individuals clustered with the Austrian, Polish, German, and French populations, whereas the Greek populations clustered apart with the Bulgarian population. Within Greece the distribution of the six wild populations did not follow any geographical trend, since their genetic divergence did not seem to correlate to geographic distances. However, RAPD profiles of some reared and wild specimens were different from the common RAPD pattern observed in the vast majority of sampled hares, probably reflecting an admixture of genetically differentiated individuals. The RAPD analysis indicates that releases might have begun to affect Greek population structure and reinforces the view that appropriate management is needed, adjusted to the local populations' biology and ecology.     

Signatures of de‐domestication in autochthonous pig breeds and of domestication in wild boar populations from MC1R and NR6A1 allele distribution

Autochthonous pig breeds are usually reared in extensive or semi‐extensive production systems that might facilitate contact with wild boars and, thus, reciprocal genetic exchanges. In this study, we analysed variants in the melanocortin 1 receptor (MC1R) gene (which cause different coat colour phenotypes) and in the nuclear receptor subfamily 6 group A member 1 (NR6A1) gene (associated with increased vertebral number) in 712 pigs of 12 local pig breeds raised in Italy (Apulo‐Calabrese, Casertana, Cinta Senese, Mora Romagnola, Nero Siciliano and Sarda) and south‐eastern European countries (Kr?kopolje from Slovenia, Black Slavonian and Turopolje from Croatia, Mangalitsa and Moravka from Serbia and East Balkan Swine from Bulgaria) and compared the data with the genetic variability at these loci investigated in 229 wild boars from populations spread in the same macro‐geographic areas. None of the autochthonous pig breeds or wild boar populations were fixed for one allele at both loci. Domestic and wild‐type alleles at these two genes were present in both domestic and wild populations. Findings of the distribution of MC1R alleles might be useful for tracing back the complex genetic history of autochthonous breeds. Altogether, these results indirectly demonstrate that bidirectional introgression of wild and domestic alleles is derived and affected by the human and naturally driven evolutionary forces that are shaping the Sus scrofa genome: autochthonous breeds are experiencing a sort of ‘de‐domestication’ process, and wild resources are challenged by a ‘domestication’ drift. Both need to be further investigated and managed.     

温度对假单胞rsmA突变株M-18R合成Plt和PCA的区别性影响

次生代谢物阻遏蛋白(Repressor of secondary metabolite,Rsm)A是一种全局性调控因子,与mRNA的RBS结合,转录后水平上抑制基因翻译。运用同源重组技术,构建了假单胞茵(Pseudomonas sp．)M-18的rsmA突变菌株M-18R。在37℃、28℃恒温和短期升温(37℃、4h培养,转28℃继续培养)条件下,比较野生株M-18和突变株M-18R生物合成藤黄绿菌素(Plt)和吩嗪-1-羧酸(PCA)的量。在37℃条件下,M-18和M-18R合成这两种抗生物质的能力几乎受到完全抑制。在28℃条件下,M-18R合成P11的量约为野生型M-18的10倍,达到270μg／mL,但是合成PCA的量仅为野生型的50％。经短期升温培养,M-18的Plt合成量明显下降,PCA产量降低不显;相反,M-18R合成Plt的量达到400μg／mL,但PCA产量的变化仍不明显。推测,M-18菌株细胞内存在着某种与RsmA相关联的温度敏感因子,在RsmA缺失条件下,作为专一性激活剂促进Plt的生物合成,但是,并不参与对PCA合成的调控。     

Genetic diversity and origin of weedy rice (Oryza sativa f. spontanea) populations found in North-eastern China revealed by simple sequence repeat (SSR) markers

BACKGROUND AND AIMS: Weedy rice (Oryza sativa f. spontanea) is one of the most notorious weeds occurring in rice-planting areas worldwide. The objectives of this study are to determine the genetic diversity and differentiation of weedy rice populations from Liaoning Province in North-eastern China and to explore the possible origin of these weedy populations by comparing their genetic relationships with rice varieties (O. sativa) and wild rice (O. rufipogon) from different sources. METHODS: Simple sequence repeat (SSR) markers were used to estimate the genetic diversity of 30 weedy rice populations from Liaoning, each containing about 30 individuals, selected rice varieties and wild O. rufipogon. Genetic differentiation and the relationships of weedy rice populations were analysed using cluster analysis (UPGMA) and principle component analysis (PCA). KEY RESULTS: The overall genetic diversity of weedy rice populations from Liaoning was relatively high (H(e) = 0.313, I = 0.572), with about 35 % of the genetic variation found among regions. The Liaoning weedy rice populations were closely related to rice varieties from Liaoning and japonica varieties from other regions but distantly related to indica rice varieties and wild O. rufipogon. CONCLUSIONS: Weedy rice populations from Liaoning are considerably variable genetically and most probably originated from Liaoning rice varieties by mutation and intervarietal hybrids. Recent changes in farming practices and cultivation methods along with less weed management may have promoted the re-emergence and divergence of weedy rice in North-eastern China.     

Genetic variation of the East Balkan Swine (Sus scrofa) in Bulgaria,revealed by mitochondrial DNA and Y chromosomal DNA

East Balkan Swine (EBS) Sus scrofa is the only aboriginal domesticated pig breed in Bulgaria and is distributed on the western coast of the Black Sea in Bulgaria. To reveal the breed's genetic characteristics, we analysed mitochondrial DNA (mtDNA) and Y chromosomal DNA sequences of EBS in Bulgaria. Nucleotide diversity (π_n) of the mtDNA control region, including two newly found haplotypes, in 54 EBS was higher (0.014 ± 0.007) compared with that of European (0.005 ± 0.003) and Asian (0.006 ± 0.003) domestic pigs and wild boar. The median‐joining network based on the mtDNA control region showed that the EBS and wild boar in Bulgaria comprised mainly two major mtDNA clades, European clade E1 (61.3%) and Asian clade A (38.7%). The coexistence of two mtDNA clades in EBS in Bulgaria may be the relict of historical pig translocation. Among the Bulgarian EBS colonies, the geographical differences in distribution of two mtDNA clades (E1 and A) could be attributed to the source pig populations and/or historical crossbreeding with imported pigs. In addition, analysis of the Y chromosomal DNA sequences for the EBS revealed that all of the EBS had haplotype HY1, which is dominant in European domestic pigs.     

Cyclic Oscillations in Melanin Composition within Hairs of Baboons

The wild‐type agouti‐banding pattern for hair is well characterized in lower mammals such as mice. The switch between eumelanin and pheomelanin in bands in the hair results from the interaction of α‐melanocyte stimulating hormone and agouti signal protein through the melanocortin 1 receptor on melanocytes. However, such banding patterns have not been described to date in higher mammals. We now report such ‘agouti’‐banding patterns that occur in several subspecies of baboons, and characterize those hairs using chemical and immunohistochemical methods. Hair and skin samples were obtained from the dorsa of adult male baboons of different subspecies (Papio cynocephalus hamadryas (PCH) and Papio cynocephalus anubis (PCA)). The hairs were excised with scissors into the gray and the white bands of the PCH subspecies and into the black and the yellow bands of the PCA subspecies, and were analyzed for total melanin, eumelanin, and pheomelanin by spectrophotometric and chemical methods. Hairs in the PCA subspecies oscillate between a eumelanic band (with high melanin content) and a pheomelanic band, while hairs in the PCH subspecies oscillate between a eumelanic band (with low melanin content) and a non‐pigmented band. Those chemical data are consistent with the histological appearance of the hair bulbs stained by the Fontana‐Masson technique. The difference in the melanin content between PCH and PCA subspecies is most likely related to tyrosinase levels, as suggested by the presence of unpigmented muzzle in the PCH subspecies compared with the black muzzle in the PCA subspecies.     

Geographic population structure and sequence divergence in the mitochondrial DNA control region of the Japanese wild boar (Sus scrofa leucomystax), with reference to those of domestic pigs

Mitochondrial DNA (mtDNA) control regions from 40 Japanese wild boars were examined by direct sequencing after amplification by PCR. From the DNA sequences obtained, we found eight haplotypes, whose differences arose via transitions. The geographical distribution of these different haplotypes indicated that wild boar populations inhabited limited areas and that there was some restricted gene flow between local populations. Eight mtDNA haplotypes from Eastern and Western domestic pigs and the Ryukyu wild boar were also analyzed as references to those from Japanese wild boars. The cluster analyses of the control-region sequences showed that those from Japanese wild boards belong to the Asian type as do those from Eastern domestic pigs and the Ryukyu wild boar, which differed from the European type (Western domestic pigs).     

假单胞菌M18调控基因ppbR的克隆及功能研究

假单胞菌(Pseudomonas sp.)M18是促进植物生长的根际细菌,能产生吩嗪-1-羧酸(PCA)和藤黄绿菌素(Plt)两种不同的抗生素.根据生物信息学分析,铜绿假单胞菌PA2572基因编码蛋白可能是一个双元调控系统的应答调节子.本研究从假单胞菌M18基因组中扩增出PA2572同源基因片段ppbR,利用体外定点插入突变和同源重组技术构建了M18的ppbR突变株M18P.研究结果表明,突变株M18P在泳动能力和群集运动能力上有显著的下降.突变株合成PCA的能力比野生型有显著的下降,在发酵液中PCA积累量仅为野生型的50%.在KMB培养基中,突变株Plt的积累量和野生型没有显著的差异.     

假单胞菌M18调控基因ppbR的克隆及功能研究

假单胞菌 (Pseudomonas sp.) M18 是促进植物生长的根际细菌, 能产生吩嗪-1-羧酸 (PCA) 和藤黄绿菌素 (Plt) 两种不同的抗生素。根据生物信息学分析, 铜绿假单胞菌PA2572基因编码蛋白可能是一个双元调控系统的应答调节子。本研究从假单胞菌M18基因组中扩增出PA2572同源基因片段ppbR, 利用体外定点插入突变和同源重组技术构建了M18 的ppbR突变株M18P。研究结果表明, 突变株M18P在泳动能力和群集运动能力上有显著的下降。突变株合成PCA 的能力比野生型有显著的下降, 在发酵液中PCA积累量仅为野生型的50%。在KMB培养基中, 突变株Plt的积累量和野生型没有显著的差异。     

假单胞菌gacA插入突变对藤黄绿脓菌素和吩嗪-1-羧酸合成代谢的差异性调控

假单胞菌株M18分泌藤黄绿脓菌素 (Pyoluteorin ,Plt )和吩嗪 1 羧酸 (Phenazine 1 carboxylicacid ,PCA)并抑制多种植物病菌的生长。从M18中克隆双基因调控系统gacS gacA的组成基因gacA ,并构建了该基因抗性插入突变株M18G。在KMB培养基中 ,M18G合成Plt的能力受到完全抑制 ,而PCA的积累约比野生型提高 31倍左右。Plt合成基因簇突变株M18T和在M18G基础上构建的PCA合成基因簇突变株M18GA的Plt和PCA合成的动力学变化表明 ,在M18G菌株中 ,Plt合成的抑制并不引起PCA的过量积累 ,PCA的过量积累也不引起Plt合成的抑制。由此推测 ,gacA在基因表达的水平上全局性地执行着调控功能     

Effect of Ammonia on Brain Serotonin Metabolism in Relation to Function in the Portacaval Shunted Rat

Four weeks following portacaval anastomosis (PCA) in the rat, severe liver atrophy, sustained hyperammonemia, and increased plasma and brain tryptophan are observed. Administration of ammonium acetate (NH4Ac) to rats with PCA precipitates severe signs of hepatic encephalopathy (HE) (loss of righting reflex progressing to loss of consciousness and ultimately deep coma). To evaluate the relationship between the deterioration of neurological status in HE and serotonin (5-HT) metabolism, the levels of 5-HT, its precursor 5-hydroxytryptophan, and its major metabolite 5-hydroxy-indole-3-acetic acid (5-HIAA) were measured by HPLC with ion-pairing and electrochemical detection in three well-defined areas of the cerebral cortex: anterior cingulate, piriform and entorhinal, and frontoparietal; as well as in the caudate-putamen, the raphe nuclei, and the locus ceruleus in rats with PCA at different stages of HE, before and after injection of NH4Ac, as well as in sham-operated controls. The results demonstrate increased 5-HIAA/5-HT ratios after PCA and NH4Ac loading, suggesting increased 5-HT turnover in the brains of these animals. However, these changes do not appear to be related to the precipitation of coma as no significant difference in 5-HT turnover was observed between precoma and coma stages of HE. Increased 5-HT turnover in brain of shunted rats may be related to early symptoms of HE such as altered sleep patterns and disorders of motor coordination.     

A Survey of Tospoviruses in Bulgaria

A survey of tospoviruses present in Bulgaria was conducted during three vegetation periods. A total of 258 different virus isolates were obtained from tobacco, tomato, weeds and ornamentals in the open fields and from glasshouses. Virus isolates were identified by inoculation to test plants and serology as Tomato spotted wilt virus (TSWV). No indications were obtained that other Tospovirus species were present in Bulgaria. In addition the transmission ability and efficiency of several Bulgarian populations of Thrips tabaci (Lind.) and Frankliniella occidentalis (Perg.) was determined. The experimental results indicated that Bulgarian TSWV isolates are transmitted with high efficiency by F. occidentalis and that only arrhenotokous and not thelytokouse populations of T. tabaci could transmit TSWV.     

Occurrence of eggs and oocysts of intestinal parasites of pheasant (<Emphasis Type="BoldItalic">Phasianus colchicus</Emphasis>) in droppings collected in differently managed protected areas of Tuscany (Italy)

The releasing of farm-reared pheasant (Phasianus colchicus) is a very common practice in order to sustain high hunting pressures. However farm-reared birds may be carriers of parasites and diseases for the natural populations. We compared the parasite egg and oocyst prevalence and abundance of excretions found in faecal droppings collected in 13 different protected areas of Tuscany: seven areas where farm-reared pheasants are released every year to increase the reproduction and dispersion of the wild population (restocking areas) and in six areas where the production of pheasants is guaranteed only by the wild population (wild areas). Eimeria spp. oocysts were found in 33 of 129 (25.6%) samples collected in wild areas and in 59 of 119 (51.3%) of samples collected in restocking areas. Nematode eggs were found in 21 of 129 (16.3%) samples collected in wild areas and in 59 of 119 (49.6%) of samples collected in restocking areas. Significant differences were found for Capillaria spp. (31.9% of restocking areas vs. 9.3% of wild areas) and Syngamus spp. (10.1% of restocking areas vs. 3.1% of wild areas) but not for Heterakis or Ascaridia spp. (7.6% of restocking areas vs. 3.9% of wild areas). Parasitic excretion abundance was higher in the droppings collected in restocking areas compared to those collected in wild areas, but differences were significant only for Eimeria and Capillaria spp. In order to reduce the risk of spreading parasites and diseases, we suggest to interpose a strip (larger than the home range of the pheasants) where hunting is not forbidden between the restocking areas and the wild areas, and pheasant releases should not be allowed at least within a “pheasant home range distance” from the wild areas.     

Sampling scale can cause bias in positive assortative mating estimates: evidence from two intertidal snails

Assortative mating in the wild is commonly estimated by correlating between traits in mating pairs (e.g. the size of males and females). Unfortunately, such an approach may suffer from considerable sampling bias when the distribution of different expressions of a trait in the wild is nonrandom (e.g. when segregation of different size classes of individuals occurs in different microhabitats or areas). Consequently, any observed trait correlation in the wild can be an artefact of pooling heterogeneous samples of mating pairs from different microhabitats or areas rather than true nonrandom matings. This bias in estimating trait correlations as a result of sampling scale is termed the scale‐of‐choice effect (SCE). In the present study, we use two intertidal littorinid species from Hong Kong to show how the SCE can bias size‐assortative mating estimates from mating pairs captured in the wild, empirically demonstrating the influence of this effect on measures of positive assortative mating. This finding cautions that studies overlooking the SCE may have misinterpreted the magnitude and the cause of assortative mating, and we provide a new analytical approach for protecting against this potential bias in future studies.     

Haplotype diversity of Bulgarian red deer (Cervus elaphus L.)

To estimate the hyplotype diversity of red deer (Cervus elaphus L.) from the Balkan Peninsula, a fragment of cytochrome b (427 bp) was analyzed. Six haplotypes were revealed in the investigated red deer from Bulgaria (16 individuals). In all, 32 sequences were included in the comparative analyses (15 from Genbank and 17 of our samples from different locations of the Cervus elaphus area). As an outgroup we used a C. e. xanthopygus sequence. Four Bulgarian red deer haplotypes were closely related to other European haplotypes, where two other haplotypes formed a separate branch, and also encompassed two samples from Sardinia and Tunisia. Thus, our results indicated the existence at least two different genetic lines of red deer in Bulgaria.     

铜绿假单胞菌M18 rsmY突变株的构建及其对PCA和Plt的区别性调控

【目的】在假单胞菌中,小RNA(sRNA)参与初级和次级代谢产物、多种毒素因子以及菌群传感系统的调控,通过在植物根际促生铜绿假单胞菌M18中研究RsmY对吩嗪-1-羧酸(PCA)和藤黄绿菌素(Plt)两种抗生素的调控作用,深入了解假单胞菌中次级代谢的途径并为构建高产抗生素工程菌株提供了一定的理论基础。【方法】运用同源重组技术,构建了铜绿假单胞菌M18株的rsmY突变菌株M18RY,通过基因过表达、lacZ报告基因融合分析实验,进一步验证了RsmY对抗生素合成基因的调控作用。【结果】比较野生型M18和突变株M18RY中PCA和Plt在同一培养条件下的生物合成量,突变菌株M18RY中PCA的产量显著增加,为野生型菌株的5倍左右,而Plt的产量降为野生型的1/8。LacZ报告基因融合分析进一步证明了RsmY对PCA的负调控作用主要是通过phz2基因簇来实现的。【结论】结果表明,rsmY基因区别性调控PCA和Plt的生物合成。