Genetic Variability of Hymenoscyphus pseudoalbidus on Ash Leaf Rachises in Leaf Litter of Forest Stands in Poland

Two hundred and thirty cultures of Hymenoscyphus pseudoalbidus were obtained from ascospores created in apothecia on the previous years' ash leaf rachises in the stand floor. Fruiting bodies of the pathogen were collected in four regions of Poland differing by geographical location, the altitude above sea level and climatic conditions. Isolates were identified based on the sequences of ribosomal DNA (ITS1‐5.8S‐ITS2) and the calmodulin gene. Only the presence of H. pseudoalbidus was identified in the decaying ash stands in Poland; morphologically similar, saprotrophic species of H. albidus was absent. Intrapopulation and interpopulation genetic variability of isolates was determined based on 84 RAMS markers obtained using four primers. Genetic variability of the fungus populations, measured by the Dice coefficient of genetic similarity and the Shannon coefficient of genetic diversity, decreased along with a decrease in the location of isolate collection area above sea level. A significant dependency was shown between intrapopulation genetic variability of isolates and altitude of regions above sea level. The Mantel test excluded existence of dependence between geographical and genetic distance among populations (r = ?0.038, P = 0.55). A significant correlation was found between the genetic distances of individuals within populations and locations above sea level. Based on PCA and geographical location of populations, it was shown that populations create four distinct groups. amova showed that a majority of total genetic variability (65.80%) constitutes intrapopulation variability. Variability between populations was high (28.7%), and individual regions had a smallest influence (5.5%) on the level of total variability.     

Genetic variability of Chalara fraxinea, dieback cause of European ash (Fraxinus excelsior L.)

A total of 159 colonies of Chalara fraxinea were isolated between 2005 and 2006 from dying trees of European ash (Fraxinus excelsior L.) aged between 3 and 10 years. They derived from five regions of Poland differing by geographic location and climatic conditions. On the basis of 90 RAMS markers, pathogen intra- and inter-population variability, as well as its dependency on geographic distance and climatic conditions in the regions of strain origin, was analysed. The applied measures of intrapopulation genetic variability (genetic distance, Nei’s unbiased diversity, Shannon’s Information Index and percentage of polymorphic loci) allowed for differentiation of two strain groups: the first deriving from lowlands and the second from uplands and mountainous areas. Strains in lowlands were characterised by smaller number of markers, smaller number of polymorphic loci and smaller intrapopulation genetic variability. Positive and statistically significant correlation was shown between variability of isolates and elevation of regions above sea level. Pair-wise genetic distances between groups of isolates (Nei’s unbiased genetic distance) from particular regions were not significantly correlated with the corresponding geographic distances. On the basis of AMOVA, it was shown that 85% of variability was within-region differences and 2% between-region differences, whereas differences between lowlands and uplands were 13%. Principal Components Analysis (PCA) for the investigated regions confirmed the results from Nei’s genetic distance matrix.     

Geographic distribution and genetics of killer phenotypes for the yeast Pichia kluyveri across the United States.

Representative strains (n = 61) of the yeast Pichia kluyveri from across the United States were studied for their ability to kill 71 other strains (representing 25 species) of yeast. This survey showed killing activity in 69% of the P. kluyveri strains tested. More extensive analysis of killer activity of 197 P. kluyveri strains against strains of five tester species showed comparable activity (67% of strains tested). This activity was shown to be equally variable within localities, within regions, and across the continent. The genetic basis of the variability was ascertained by tetrad analysis and is most likely due to alleles segregating at three epistatic loci. Evidence for the idea that killer toxins have a role in excluding other yeasts from particular habitats is discussed.     

Geographic distribution and genetics of killer phenotypes for the yeast Pichia kluyveri across the United States.

Representative strains (n = 61) of the yeast Pichia kluyveri from across the United States were studied for their ability to kill 71 other strains (representing 25 species) of yeast. This survey showed killing activity in 69% of the P. kluyveri strains tested. More extensive analysis of killer activity of 197 P. kluyveri strains against strains of five tester species showed comparable activity (67% of strains tested). This activity was shown to be equally variable within localities, within regions, and across the continent. The genetic basis of the variability was ascertained by tetrad analysis and is most likely due to alleles segregating at three epistatic loci. Evidence for the idea that killer toxins have a role in excluding other yeasts from particular habitats is discussed.     

Combined effects of bottlenecks and selfing in populations of Corella eumyota, a recently introduced sea squirt in the English Channel

The success of an exotic species depends notably on its capacity to initiate a new population from a few individuals, to survive genetic bottlenecks and to adapt locally. Species with multiple reproductive strategies (e.g. mixed-mating system with both self- and cross-fertilization) can be efficient colonizers. Herein we focus on Corella eumyota , an exotic ascidian that has rapidly invaded English Channel coasts in recent years. Interestingly, this brooding hermaphroditic ascidian is capable of self-fertilization in the laboratory. We developed 12 microsatellite markers from an enriched library of genomic DNA to investigate the level of inbreeding and selfing in two putatively native populations (South Africa, N  = 34, and New Zealand, N  = 28) and to examine if founder effects were possibly associated with its recent introduction in two French populations (Perros-Guirec, N  = 22 and Brest; N  = 25). Genetic polymorphism was very low in both native populations (i.e. less than 60% of the loci were polymorphic) and even lower in the introduced populations, one of which was monomorphic at all loci, suggesting a recent bottleneck. F _is and a new method based on multi-locus heterozygosity were used to provide estimates of inbreeding. A high selfing rate was inferred in the South Africa population with both methods ( s  = 0.90), whereas in the other native population (New Zealand) a lower but significant estimate of selfing rate ( s  = 0.29) was obtained with the multi-locus method. This variability of population selfing rate might be explained by a mixed-mating system, allowing C. eumyota to reproduce through inbreeding and outbreeding according to mating possibilities; this trait may have favoured the rapid establishment of new populations in Europe.     

Population genetics of the hazel hen <Emphasis Type="Italic">Bonasa bonasia</Emphasis> in Poland assessed with non-invasive samples

Despite a severe decrease in the number of hazel hens during the 20^th century, nowadays this grouse species is rather common in the forests of Northeastern and Southern Poland. We used mitochondrial control region and microsatellite markers to examine the genetic variability of Polish populations of hazel hens. We used non-invasively collected faeces to estimate genetic variability within populations, genetic differentiation among populations as well as genetic differentiation between two regions inhabited by two different subspecies of hazel hens. Our results confirm the usefulness of DNA from faeces to obtain reliable information on the population genetics of hazel hens. We found a rather high level of genetic variability in the Polish population. Genetic variability was higher in birds from continuous forests in the South of the country than in birds from fragmented habitats in the Northeast. Genetic differentiation was higher among subpopulations from Northeastern Poland. Additionally, both classes of molecular markers suggested the presence of two distinct genetic groups of birds, corresponding to previously described subspecies. We conclude that the genetic variability of the Polish hazel hen population has been influenced by habitat fragmentation and the history of the population during its post-glacial colonization of Poland from different glacial refugia.     

The genetic architecture of correlations among growth‐related traits and male age at maturation in rainbow trout

Heritabilities and genetic correlations among growth‐related traits of two cultured strains (Rainbow Springs and Spring Valley) of rainbow trout Oncorhynchus mykiss were estimated using restricted maximum likelihood methods with a three‐generation pedigree. Heritability was high (>0·50 ± 0·03) for body mass and condition factor but moderate (0·35 ± 0·04) for age at sexual maturity in males. Body mass and age at sexual maturation were phenotypically correlated in the families of one experimental strain, Rainbow Springs, and had a positive genetic correlation (0·26 ± 0·03) across families from both test strains (Rainbow Springs and Spring Valley). This indicates that faster growing individuals were more likely to mature at 2 years of age than slower growing individuals in the two hatchery strains investigated. Microsatellite markers of body mass quantitative tract loci (QTL) were reconfirmed as being located on linkage groups B, G, N, 5 and new markers on Oi were detected. Some QTL effects were restricted to specific sampling dates suggesting temporal expression of QTL. QTL for condition factor were limited to linkage group G in both strains. Three suggestive QTL for precocious maturation mapped to similar regions as those for body mass in the Rainbow Springs families while no associations were evident in the Spring Valley families. The results suggest that these regions may play a role in the basis for genetic and phenotypic correlations between body mass and precocious maturation in this species.     

Biochemical and DNA markers yield strikingly different results regarding variability and differentiation of roe deer (Capreolus capreolus, Artiodactyla: Cervidae) populations from northern Germany

Three mainland and two island roe deer ( Capreolus capreolus ) populations with a total sample size of 105 individuals from Schleswig–Holstein, northern Germany, were analysed with regard to genetic variability within and differentiation among populations as revealed by eight allozyme loci known to be polymorphic in roe deer, eight microsatellite loci and 404 bp of the mitochondrial control region. Surprisingly, the allozymes were completely monomorphic, but microsatellite and control region variability were high. Hypotheses as to demographic reasons for the variability patterns found, including bottlenecks, founder effects and translocations, are put forward. There were no statistically significant differences between the island and the mainland populations in terms of genetic variability as measured by expected heterozygosity, inbreeding coefficient and allelic richness. The correlations of the various variability indices were not statistically significant after Bonferroni correction. Nevertheless, there was a clear tendency for differentiation indices to yield concordant results for microsatellite and mitochondrial markers.     

Identification of Molecular Marker and Aggressiveness for Different Groups of <Emphasis Type="Italic">Bipolaris sorokiniana</Emphasis> Isolates Causing Spot Blotch Disease in Wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

One hundred fifty-five isolates of Bipolaris sorokiniana of wheat were studied for their morphopathological characterization. These isolates were grouped in five categories--black, brown/dull black, gray cottony growth, dull white/greenish black, and white--on the basis of their growth pattern. The frequency of the black suppressed type was maximum (45.63%), whereas the white isolate displayed lowest frequency (6.96%) in the natural population. Twenty RAPD (random amplified polymorphic DNA) primers were used to observe the variability among the identified groups of B. sorokininana. From each group, eight random isolates were investigated. A total of 143 bands were amplified, out of which 107 (74.83%) were polymorphic and 36 (25.17%) were monomorphic. On an average, the total numbers of bands generated per primer were 7.15, of which 5.35 and 1.80 were polymorphic and monomorphic, respectively. Dendrograms based on molecular polymorphism unveiled a considerable amount of diversity among the isolates. Specific DNA bands were identified for selected isolates. The distinct markers appeared to be potential enough to be employed as genetic fingerprints for future strain identification and classification. The study indicated that the RAPD primers provide an easy, rapid, and simple technique for the preliminary assessment of genetic diversity among the fungal isolates.     

Intraspecific variability among NADH dehydrogenase subunit 1 sequences of Taenia hydatigena.

This paper describes intraspecific variability of the partial sequences of the mitochondrial ND1 gene among isolates of Taenia hydatigena from pigs in Poland, Ukraine and Wales. The differences between studied isolates ranged from 0.4 to 5.5%, which exceeds the variability within the same fragment between the different genetic variants of Echinococcus multilocularis and is comparable with the variability between the most closely related strains (G5/G6/G7) of E. granulosus. The biggest difference (5.5%) was found between the geographically most distant Ukrainian and Welsh samples of T. hydatigena while the samples collected from the neighbouring locations in Poland, were most similar to each other.     

To what extent do microsatellite markers reflect genome‐wide genetic diversity in natural populations?

Microsatellite variability is widely used to infer levels of genetic diversity in natural populations. However, the ascertainment bias caused by typically selecting only the most polymorphic markers in the genome may lead to reduced sensitivity for judging genome-wide levels of genetic diversity. To test this potential limitation of microsatellite-based approaches, we assessed the degree of nucleotide diversity in noncoding regions of eight different carnivore populations, including inbred as well as outbred populations, by sequencing 10 introns (5.4–5.7 kb) in 20 individuals of each population (wolves, coyotes, wolverines and lynxes). Estimates of nucleotide diversity varied 30-fold (7.1 × 10⁻⁵ –2.1 × 10⁻³), with densities of one single nucleotide polymorphism every 112–5446 bp. Microsatellite genotyping (10–27 markers) of the same animals revealed mean multilocus heterozygosities of 0.54–0.78, a 1.4-fold difference among populations. There was a positive yet not perfect ( r ²  = 0.70) correlation between microsatellite marker heterozygosity and nucleotide diversity at the population level. For example, point estimates of nucleotide diversity varied in some cases with an order of magnitude despite very similar levels of microsatellite marker heterozygosity. Moreover, at the individual level, no significant correlation was found. Our results imply that variability at microsatellite marker sets typically used in population studies may not accurately reflect the underlying genomic diversity. This suggests that researchers should consider using resequencing-based approaches for assessing genetic diversity when accurate inference is critical, as in many conservation and management contexts.     

Phylogenetic relationship of street rabies virus strains and their antigenic reactivity with antibodies induced by vaccine strains. I. Analysis of phylogenetic relationship of street rabies virus strains isolated in Poland

The aims of these studies were: genetic characteristic of street rabies virus strains isolated from different animal species in Poland and determination of phylogenetic relationships to reference laboratory strains of the street rabies viruses belonging to genotype 1 and 5. The variability of rabies isolates and their phylogenetic relationship were studied by comparing the nucleotide sequence of the virus genome fragment. The Polish strains of genotype 1 belong to four phylogenetic groups (NE, CE, NEE, EE) corresponding to four variants: fox-racoon dog (F-RD); European fox 1 (F1); European fox 2 (F2) and European fox 3 (F3). On the Polish territories there are no rabies strains representing the variant dog-wolf and typical for arctic fox variant. The similarity of nucleotide and amino acid sequences of street rabies strains belonging to genotype 1 and laboratory strain CVS is very high. It is about 91% similarity at nucleotide level and 95% at amino acid level. Rabies strain CVS is similar to genotype 5 bat strains (EBL 1) only in about 69% and 74% at nucleotide and amino acid level, respectively. The genetic divergence of rabies strains circulating in Poland raised the need of permanent epidemiological and virological surveillance. The genotype and variant of isolated strains should be determined (using PCR and RLFP methods).     

Biochemical genetics of phosphohydrolase polymorphism in Culex pipiens complex.

1. Single 4th-instar larvae were used in the investigation of alkaline phosphohydrolase (APH) variability in Culex pipiens quinequefasciatus. The genetic basis of isozyme variability was determined from genetic crosses performed with isogenic and hybrid strains of mosquitoes. 2. Isozyme electromorphs presented four enzyme activity zones, three monomorphic and one polymorphic, correspondent with four APH gene loci (aph1, aph2, aph3 and aph4). 3. All isozymes migrated anodically, with aph4 isozymes migrating most rapidly. 4. Enzyme polymorphism was evident only at aph4 locus, with three allozymes present. 5. aph4 allozymes are conditioned by multiple, co-dominant alleles transmitted in a Mendelian manner. 6. Differential frequencies and selection for aph4 alleles and genotypes are suggested by data from genetic crosses.     

Microsatellite DNA analysis of northern pike (Esox lucius L.) populations: insights into the genetic structure and demographic history of a genetically depauperate species

The northern pike Esox lucius L. is a freshwater fish exhibiting pronounced population subdivision and low genetic variability. However, there is limited knowledge on phylogeographical patterns within the species, and it is not known whether the low genetic variability reflects primarily current low effective population sizes or historical bottlenecks. We analysed six microsatellite loci in ten populations from Europe and North America. Genetic variation was low, with the average number of alleles within populations ranging from 2.3 to 4.0 per locus. Genetic differentiation among populations was high (overall θ_ST = 0.51; overall ρ_ST = 0.50). Multidimensional scaling analysis of genetic distances between populations and spatial analysis of molecular variance suggested a single phylogeographical race within the sampled populations from northern Europe, whereas North American and southern European populations were highly distinct. A population from Ireland was monomorphic at all loci, presumably reflecting founder events associated with introduction of the species to the island in the sixteenth century. Bayesian analysis of demographic parameters showed differences in θ (a product of effective population size and mutation rate) among populations from large and small water bodies, but the relative differences in θ were smaller than expected, which could reflect population subdivision within the larger water bodies. Finally, the analyses showed drastic population declines on a time scale of several thousand years within European populations, which we ascribe to either glacial bottlenecks or postglacial founder events.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 91–101.     

The uses of AFLP for detecting DNA polymorphism, genotype identification and genetic diversity between yeasts isolated from Mexican agave-distilled beverages and from grape musts

AIMS: The objectives were to determine the variability and to compare the genetic diversity obtained using amplified fragment length polymorphism (AFLP) markers in analyses of wine, tequila, mezcal, sotol and raicilla yeasts. METHODS AND RESULTS: A molecular characterization of yeasts isolated from Mexican agave musts, has been performed by AFLP marker analysis, using reference wine strains from Italian and South African regions. CONCLUSIONS: A direct co-relation between genetic profile, origin and fermentation process of strains was found especially in strains isolated from agave must. In addition, unique molecular markers were obtained for all the strains using six combination primers, confirming the discriminatory power of AFLP markers. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of molecular characterization between yeasts isolated from different Mexican traditional agave-distilled beverages, which shows high genetic differences with respect to wine strains.     

Population isolation rather than ecological variation explains the genetic structure of endangered myrmecophilous butterfly Phengaris (=Maculinea) arion

Genetic variation of the globally threatened obligatorily myrmecophilous Large Blue butterfly Phengaris (Maculinea) arion (Lepidoptera) was studied, using six microsatellite markers, in a country where its decline is dramatic (Poland). Material was collected on 13 sites showing considerable ecological variation as far as biotope, larval food plant and host ants of the butterfly were concerned. Genetic variability, estimated in terms of number of alleles and heterozygosity, was the lowest in the most isolated populations. However on sites localized in areas where suitable biotopes were extensive and interconnected, P. arion still held relatively high genetic diversity. Pairwise F _ST values indicated small and moderate differentiation among samples (F _ST = 0.01–0.15), with the exceptions of two isolated localities (0.20). We did not find clear evidence of isolation by distance. The presence of four or five genetic clusters was indicated. Analysis of the membership of each individual to each cluster showed that the vast majority of individuals from three isolated populations were clustered in three separate genetic groups. The most distinct population was the one, which had been found to be specialized towards Myrmica lobicornis in previous studies. Individuals from the remaining populations could not be clustered in separate genetic groups, however some dominance of different clusters in geographical regions was observed. Some portion of the population’s genetic variability could be explained by geographical distribution, however the percentage of variation, explaining the differences between two main regions (S and NE Poland), was very low. We conclude that the main factor shaping the current genetic structure of P. arion in Poland is the recent isolation of populations related to habitat fragmentation but local ecological specializations may be also a potential factor. Therefore the necessity of activities aiming to halt the further reduction of genetic variability, as well as the monitoring of priority populations (e.g. those belonging to unique host races), should be emphasized in future action plans in Central Europe.     

Auxin production by plant associated bacteria: impact on endogenous IAA content and growth of Triticum aestivum L.

Aims:  The aim of this study was to investigate the potential of bacterial strains of Bacillus, Pseudomonas, Escherichia, Micrococcus and Staphylococcus genera associated with wild herbaceous flora to enhance endogenous indole-3-acetic acid (IAA) content and growth of Triticum aestivum var. Inqalab-91.
Methods and Results:  Gas chromatography and mass spectrometric (GC–MS) analysis revealed that bacterial strains produced 0·6–8·22 μg IAA ml⁻¹ in the presence of L-tryptophan. Plant microbe experiments showed a significant positive correlation between auxin production by bacterial strains and endogenous IAA content of T. aestivum for GC–MS ( r  = 0·618; P  = 0.05) and colorimetric analysis ( r  = 0·693; P  = 0.01). Similarly, highly significant positive correlation for shoot length ( r  = 0·627; P  = 0.01) and shoot fresh weight ( r  = 0·626; P  = 0.01) was observed with auxin production under axenic conditions. Bacterial inoculations also enhanced shoot length (up to 29·16%), number of tillers (up to 97·35%), spike length (up to 25·20%) and seed weight (up to 13·70%) at final harvest.
Conclusions:  Bacterial strains have the ability to increase the endogenous IAA content and growth of T. aestivum var. Inqalab-91.
Significance and Impact of the Study:  Microbial strains of wild herbaceous flora can be effectively used to enhance the growth and yield of agronomically important crops.     

High resolution genotyping of Bacillus anthracis outbreak strains using four highly mutable single nucleotide repeat markers

Aims: Bacillus anthracis is a genetically monomorphic bacterium with little diversity to be expected during an outbreak. This study used more rapidly evolving genetic markers on outbreak samples to ascertain genetic diversity. Methods and Results: Forty‐seven isolates from a B. anthracis outbreak during the summer of 2005 in South Dakota were analysed using single nucleotide polymorphisms (SNP) and multi‐locus VNTR analysis (MLVA). Results indicated that all of the outbreak strains belonged to a single clonal lineage. However, analysis of four single nucleotide repeat (SNR) markers resolved these isolates into six distinct genotypes providing insights into disease transmission. Conclusions: Strain determination of unknown B. anthracis samples can be ascertained by SNP and MLVA markers. However, comparison of many samples obtained during an outbreak will require markers with higher rates of mutation to ascertain genetic diversity. Significance and Impact of the Study: SNR4 analysis allowed discrimination of closely related B. anthracis isolates and epidemiological tracking of the outbreak. When used in conjunction with other genotyping schemes that allow broad genetic relationships to be determined, SNR markers are powerful tools for detailed tracking of natural B. anthracis outbreaks and could also prove useful in forensic investigations.     

Efficiency of ISSR and RAPD markers in genetic divergence analysis and conservation management of Justicia adhatoda L., a medicinal plant

Genetic variation within and among population is the basis for survival of the population both in short and long term. Thus, studying the plant genetic diversity is essential for any conservation program. Indigenous medicinal plants like Justicia adhatoda L. which are facing high rate of depletion from the wild population need immediate attention. DNA-based dominant molecular marker techniques, random amplification of polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) were used to unravel the genetic variability and relationships across thirty-two wild accessions of J. adhatoda L., a valuable medicinal shrub widespread throughout the tropical regions of Southeast Asia. Amplification of genomic DNA using 38 primers (18 RAPD and 20 ISSR) yielded 434 products, of which 404 products were polymorphic revealing 93.11 % polymorphism. The average polymorphic information content value obtained with RAPD and ISSR markers was 0.25 and 0.24, respectively. Marker index (RAPD = 3.94; ISSR = 3.53) and resolving power (RAPD = 4.24; ISSR = 3.94) indicate that the RAPD markers were relatively more efficient than the ISSR assay revealing the genetic diversity of J. adhatoda. The Shannon diversity index obtained with RAPD and ISSR markers was 0.40 and 0.38, respectively. The similarity coefficient ranged from 0.26 to 0.89, 0.33 to 0.93 and 0.31 to 0.90 with RAPD, ISSR and combined UPGMA dendrogram, respectively. PCA derived on the basis of pooled data of both the markers illustrated that the first three principal coordinate components accounted 79.27 % of the genetic similarity variance. The mantel test between two Jaccard’s similarity matrices gave r = 0.901, showing the fit correlation between ISSR- and RAPD-based similarities. Based on the results, ex-situ methods may be the most suitable and efficient measure for long-term conservation.     

Comparison of Lactococcus garvieae strains isolated in northern Italy from dairy products and fishes through molecular typing

Aims:  To investigate the genetic relatedness between Lactococcus garvieae strains isolated from fish and dairy samples collected in northern Italy, using random-amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR), Sau -PCR and amplified fragment length polymorphism (AFLP).
Methods and Results:  Eighty-one isolates from bovine and caprine dairy products ( n  = 53) and from diseased rainbow trouts and other fishes ( n  = 28) were examined. All methods showed a typeability of 100%, repeatability ranging from 84·4% to 97·5% and discriminatory powers from 0·798 to 0·986. Dairy and fish strains revealed a low genetic relatedness as they are often grouped into distinct clusters. RAPD analysis discriminated 52 genotypes when primer M13 was used, whereas with primer P5 only 27 genotypes were identified. When Sau -PCR was performed, 13 genotypes were detected while AFLP analysis allowed the differentiation of 32 genotypes.
Conclusion:  L. garvieae strains isolated from dairy samples are generally not related to those collected from fish lactococcosis outbreaks.
Significance and Impact of the Study:  L. garvieae strains exhibit a genetic diversity related to the specific animal host they colonize. RAPD M13 fingerprinting proved to be a molecular tool for comparing isolates, whereas Sau -PCR and AFLP analyses were useful techniques to investigate the distribution of L. garvieae populations in the environment.