First Insights into the Genetic Diversity of the Pinewood Nematode in Its Native Area Using New Polymorphic Microsatellite Loci

The pinewood nematode, Bursaphelenchus xylophilus, native to North America, is the causative agent of pine wilt disease and among the most important invasive forest pests in the East-Asian countries, such as Japan and China. Since 1999, it has been found in Europe in the Iberian Peninsula, where it also causes significant damage. In a previous study, 94 pairs of microsatellite primers have been identified in silico in the pinewood nematode genome. In the present study, specific PCR amplifications and polymorphism tests to validate these loci were performed and 17 microsatellite loci that were suitable for routine analysis of B. xylophilus genetic diversity were selected. The polymorphism of these markers was evaluated on nematodes from four field origins and one laboratory collection strain, all originate from the native area. The number of alleles and the expected heterozygosity varied between 2 and 11 and between 0.039 and 0.777, respectively. First insights into the population genetic structure of B. xylophilus were obtained using clustering and multivariate methods on the genotypes obtained from the field samples. The results showed that the pinewood nematode genetic diversity is spatially structured at the scale of the pine tree and probably at larger scales. The role of dispersal by the insect vector versus human activities in shaping this structure is discussed.     

Secretome Analysis of the Pine Wood Nematode Bursaphelenchus xylophilus Reveals the Tangled Roots of Parasitism and Its Potential for Molecular Mimicry

Since it was first introduced into Asia from North America in the early 20^th century, the pine wood nematode Bursaphelenchus xylophilus has caused the devastating forest disease called pine wilt. The emerging pathogen spread to parts of Europe and has since been found as the causal agent of pine wilt disease in Portugal and Spain. In 2011, the entire genome sequence of B. xylophilus was determined, and it allowed us to perform a more detailed analysis of B. xylophilus parasitism. Here, we identified 1,515 proteins secreted by B. xylophilus using a highly sensitive proteomics method combined with the available genomic sequence. The catalogue of secreted proteins contained proteins involved in nutrient uptake, migration, and evasion from host defenses. A comparative functional analysis of the secretome profiles among parasitic nematodes revealed a marked expansion of secreted peptidases and peptidase inhibitors in B. xylophilus via gene duplication and horizontal gene transfer from fungi and bacteria. Furthermore, we showed that B. xylophilus secreted the potential host mimicry proteins that closely resemble the host pine’s proteins. These proteins could have been acquired by host–parasite co-evolution and might mimic the host defense systems in susceptible pine trees during infection. This study contributes to an understanding of their unique parasitism and its tangled roots, and provides new perspectives on the evolution of plant parasitism among nematodes.     

Population genetics of Mioscirtus wagneri,a grasshopper showing a highly fragmented distribution

The genetic consequences of population fragmentation and isolation are major issues in conservation biology. In this study we analyse the genetic variability and structure of the Iberian populations of Mioscirtus wagneri, a specialized grasshopper exclusively inhabiting highly fragmented hypersaline low grounds. For this purpose we have used seven species‐specific microsatellite markers to type 478 individuals from 24 localities and obtain accurate estimates of their genetic variability. Genetic diversity was relatively low and we detected genetic signatures suggesting that certain populations of M. wagneri have probably passed through severe demographic bottlenecks. We have found that the populations of this grasshopper show a strong genetic structure even at small geographical scales, indicating that they mostly behave as isolated populations with low levels of gene flow among them. Thus, several populations can be regarded as independent and genetically differentiated units which require adequate conservation strategies to avoid eventual extinctions that in highly isolated localities are not likely to be compensated for with the arrival of immigrants from neighbouring populations. Overall, our results show that these populations probably represent the ‘fragments’ of a formerly more widespread population and highlight the importance of protecting Iberian hypersaline environments due to the high number of rare and endangered species they sustain.     

Population genetic structure in the Iberian cyprinid fish Iberochondrostoma lemmingii (Steindachner, 1866): disentangling species fragmentation and colonization processes

Interplay between the complex geography, hydrogeomorphological history, past climatic changes, and anthropogenic pressures is likely responsible for the current diversity and species' distribution of freshwater fishes in the Iberian Peninsula. To further disentangle the evolutionary processes promoting the diversification of endemic Iberian Cyprinids through time and space, we explored the patterns of genetic diversity of the Iberian arched‐mouth nase, Iberochondrostoma lemmingii (Steindachner, 1866), using molecular markers rendering at different timescales: the mitochondrial gene cytochrome b and seven microsatellite loci. Both markers showed significant differentiation of populations though the relative genetic distances among populations were different between markers. Mitochondrial DNA results indicate the isolation of hydrographic basins as the main driver of population differentiation, with Tejo as the centre of diversification. The results also support connections between Tejo, Guadiana, and Guadalquivir, with levels of divergence suggesting an earlier severance of Guadalquivir, whereas Guadiana and Tejo maintained connections until a more recent past. Establishment of more peripherial populations in small southern basins (Quarteira and Almargem) could have been ruled by founder events. However, the analysis of present‐day genetic configuration suggested by microsatellite data implies, for the first time, the involvement of other factors in the evolution of arched‐mouth Iberian nase populations. Relative low genetic distances between inter‐basin populations (Tejo and Guadiana) and the lack of concordance between differentiation and geography suggest a possible influence of human‐mediated translocations in the population genetic patterns of I. lemmingii. High intra‐basin differentiation levels were found within Tejo and Guadiana and may be associated with factors intrinsic to the species (e.g. low dispersal capability) or natural and/or artificial barriers to gene flow. The low vagility of the species appears to be an important factor influencing the evolutionary processes shaping the phylogeographical patterns of I. lemmingii, which could be relevant for the conservation of this threatened species. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 559–572.     

Signatures of demographic bottlenecks in European wolf populations

Monitoring the loss of genetic diversity in wild populations after a bottleneck event is a priority in conservation and management plans. Here, we used diverse molecular markers to search for signatures of demographic bottlenecks in two wolf populations; an isolated population from the Iberian Peninsula and a non-isolated population from European Russia. Autosomal, mtDNA and Y-chromosomal diversity and the effective population size (N_e) were significantly lower in the Iberian population. Neutrality tests using mtDNA sequences, such as R_2, Fu and Li’s F*, Tajima’s D and Fu’s F_s, were positively significant in the Iberian population, suggesting a population decline, but were not significant for the Russian population, likely due to its larger effective population size. However, three tests using autosomal data confirmed the occurrence of the genetic bottleneck in both populations. The M-ratio test was the only one providing significant results for both populations. Given the lack of consistency among the different tests, we recommend using multiple approaches to investigate possible past bottlenecks. The small effective population size (about 50) in the Iberian Peninsula compared to the presumed extant population size could indicate that the bottleneck was more powerful than initially suspected or an overestimation of the current population. The risks associated with small effective population sizes suggest that the genetic change in this population should be closely monitored in the future. On the other hand, the relatively small effective population size for Russian wolves (a few hundred individuals) could indicate some fragmentation, contrary to what is commonly assumed.     

Multilocus genetic analyses provide insight into speciation and hybridization in aquatic grasses,genus Ruppia

Aquatic plants of the genus Ruppia inhabit some of the most threatened habitats in the world, such as coastal lagoons and inland saline to brackish waters where their meadows play several key roles. The evolutionary history of this genus has been affected by the processes of hybridization, polyploidization, and vicariance, which have resulted in uncertainty regarding the number of species. In the present study, we apply microsatellite markers for the identification, genetic characterization, and detection of hybridization events among populations of putative Ruppia species found in the southern Iberian Peninsula, with the exception of a clearly distinct species, the diploid Ruppia maritima. Microsatellite markers group the populations into genetically distinct entities that are not coincident with geographical location and contain unique diagnostic alleles. These results support the interpretation of these entities as distinct species: designated here as (1) Ruppia drepanensis, (2) Ruppia cf. maritima, and (3) Ruppia cirrhosa. A fourth distinct genetic entity was identified as a putative hybrid between R. cf. maritima and R. cirrhosa because it contained a mixture of microsatellite alleles that are otherwise unique to these putative species. Hence, our analyses were able to discriminate among different genetic entities of Ruppia and, by adding multilocus nuclear markers, we confirm hybridization as an important process of speciation within the genus. In addition, careful taxonomic curation of the samples enabled us to determine the genotypic and genetic diversity and differentiation among populations of each putative Ruppia species. This will be important for identifying diversity hotspots and evaluating patterns of population genetic connectivity.     

Understanding resistant germplasm‐induced virulence variation through analysis of proteomics and suppression subtractive hybridization in a maize pathogen Curvularia lunata

Curvularia lunata is an important pathogen causing Curvularia leaf spot in maize. Significant pathogenic variation has been found in C. lunata. To better understand the mechanism of this phenomenon, we consecutively put the selective pressures of resistant maize population on C. lunata strain WS18 (low virulence) artificially. As a result, the virulence of this strain was significantly enhanced. Using 2DE, 12 up‐regulated and four down‐regulated proteins were identified in virulence‐increased strain compared to WS18. Our analysis revealed that melanin synthesis‐related proteins (Brn1, Brn2, and scytalone dehydratase) and stress tolerance‐related proteins (HSP 70) directly involved in the potential virulence growth as crucial markers or factors in C. lunata. To validate 2DE results and screen differential genes at mRNA level, we constructed a subtracted cDNA library (tester: virulence‐increased strain; driver: WS18). A total of 188 unigenes were obtained this way, of which 14 were indicators for the evolution of pathogen virulence. Brn1 and hsp genes exhibited similar expression patterns corresponding to proteins detected by 2DE. Overall, our results indicated that differential proteins or genes, being involved with melanin synthesis or tolerance response to stress, could be considered as hallmarks of virulence increase in C. lunata.     

Amplified fragment length polymorphism among Rhynchosporium secalis isolates collected from a single barley field in Syria

AFLP markers were used to measure the amount and distribution of genetic variation among Rhynchosporium secalis isolates on a microgeographical scale in Syria. Forty isolates hierarchically sampled from a single barley field were assayed for AFLP variation using primer combinations not previously tested in populations of the pathogen from Syria. In contrast to a previous study, which showed high clonality within field populations of R. secalis in Syria, the present study revealed a much higher level of genetic diversity, stressing the important roles that sampling strategies and the choice of primers/primer combinations play in the evaluation of genetic variation in R. secalis populations at a microgeographical scale. A high level of genetic variation was found to occur on a fine scale throughout the pathogen population examined, with 40 different haplotypes being identified among the 40 isolates sampled. Data were consistent with the hypothesis that the primary inoculum originated from a genetically diverse founding population, which may have consisted of ascospores of an as yet undescribed teleomorph and/or asexual spores of a highly mutable local population.     

Evolution of resistance against powdery mildew in winter wheat populations conducted under dynamic management. I – Is specific seedling resistance selected?

Dynamic management has been proposed as a complementary strategy to gene banks for the conservation of genetic resources. The evolution of frequencies of genes for specific resistance towards powdery mildew (caused by Blumeria graminis f. sp. tritici) in populations of a French network for dynamic management of bread wheat genetic resources was investigated after 10 years of multiplication without human selection. The objective was to determine whether specific resistance gene diversity was maintained in the populations and whether any changes could be attributed to selection due to pathogen pressure. Seven populations, originating from four of the network sites, were characterized and compared to the initial population for six specific resistance gene frequencies detected by nine Blumeria graminis f. sp. tritici isolates. Diversity decreased at the population level, but because of a strong differentiation between the populations, this diversity was maintained at the network level. The comparison of Fst parameters estimated on neutral markers (RFLP) and on resistance gene data revealed that in two of the populations specific resistance genes had been selected by pathogen pressure, whereas evolution in two other populations seemed to be the result of genetic drift. For the three last populations, conclusions were less clear, as one had probably experienced a strong bottleneck and the other two presented intermediate Fst values. A dynamic management network with sites contrasted for pathogen pressure, allowing genetic drift in some populations and selection in others, appeared, at least on the short term, to be a good tool for maintaining the diversity of genes for specific resistance to powdery mildew. Received: 15 December 1999 / Accepted: 30 December 1999     

Study of Resistance of Musa acuminata to Fusarium oxysporum using RAPD markers

Suckers collected from different populations of Musa acuminata ssp. malaccensis were found to be highly resistant to race 4 of Fusarium oxysporum f. sp. cubense (FOC) suggesting that local wild banana populations co-evolved with the pathogen. Seedlings from these wild banana plants segregated for resistance to the pathogen. The infected seedlings were characterized based on external and internal symptoms and the variable response to FOC was mainly due to the genetic factors. Using the technique of random amplified polymorphic DNA (RAPD), 96 major amplification products from 15 primers were identified. Only 10 out of 96 markers were monomorphic and shared among the seed progenies, whereas the remaining 86 were highly polymorphic. Three primers showed banding patterns specific to resistant or susceptible seedlings. These results showed the great potential of the wild Musa acuminata ssp. malaccensis as a source for banana improvement and also for the synthesis of segregating populations for linkage mapping, gene cloning and DNA markers related to FOC resistance.     

Asymmetric gene flow and the evolutionary maintenance of genetic diversity in small,peripheral Atlantic salmon populations

Small populations may be expected to harbour less genetic variation than large populations, but the relation between census size (N), effective population size (N _e), and genetic diversity is not well understood. We compared microsatellite variation in four small peripheral Atlantic salmon populations from the Iberian peninsula and three larger populations from Scotland to test whether genetic diversity was related to population size. We also examined the historical decline of one Iberian population over a 50-year period using archival scales in order to test whether a marked reduction in abundance was accompanied by a decrease in genetic diversity. Estimates of effective population size (N _e) calculated by three temporal methods were consistently low in Iberian populations, ranging from 12 to 31 individuals per generation considering migration, and from 38 to 175 individuals per generation if they were regarded as closed populations. Corresponding N _e/N ratios varied from 0.02 to 0.04 assuming migration (mean=0.03) and from 0.04 to 0.18 (mean=0.10) assuming closed populations. Population bottlenecks, inferred from the excess of heterozygosity in relation to allelic diversity, were detected in all four Iberian populations, particularly in those year classes derived from a smaller number of returning adults. However, despite their small size and declining status, Iberian populations continue to display relatively high levels of heterozygosity and allelic richness, similar to those found in larger Scottish populations. Furthermore, in the R. Asón no evidence was found for a historical loss of genetic diversity despite a marked decline in abundance during the last five decades. Thus, our results point to two familiar paradigms in salmonid conservation: (1)␣endangered populations can maintain relatively high levels of genetic variation despite their small size, and (2) marked population declines may not necessarily result in a significant loss of genetic diversity. Although there are several explanations for such results, microsatellite data and physical tagging suggest that high levels of dispersal and asymmetric gene flow have probably helped to maintain genetic diversity in these peripheral populations, and thus to avoid the negative consequences of inbreeding.     

Genetic structure of montane isolates of Pinus sylvestris L. in a Mediterranean refugial area

Aim This work investigates the population genetic effects of periodic altitudinal migrations and interstadial fragmentation episodes in long‐term Scots pine (Pinus sylvestris L.) populations at a regional scale. Location The study focuses on Scots pine populations in the northern Meseta and peripheral mountain chains, central and north‐western Iberian Peninsula. The ample macrofossil record in the area shows that this 60,000‐km² region represent a glacial refugium for Scots pine. The species occupied large areas on the Meseta plains during glacial cold stages, but it has periodically sheltered at high elevation in the surrounding mountain chains during warm episodes, conforming to a fragmented pattern similar to its present‐day distribution. Methods We perform a fine‐scale chloroplast microsatellite (cpSSR) survey to assess the genetic structure of 13 montane Scots pine isolates in the northern Meseta (total N = 322 individuals). Using a hierarchical analysis of molecular variance (amova ), we test the hypothesis of genetic isolation among disjunct mountain areas. We use a standard coalescence model to estimate genealogical relationship among populations, investigating the potential role of the regional relief as a factor influencing historic gene exchange among Scots pine populations. Results Population haplotypic diversity was high among Scots pine populations (H_e = 0.978), greater than values reported for other more thermophilic pine species in the Iberian Peninsula. The amova revealed low (but significant) differentiation among populations (Φ_ST = 0.031, P = 0.010), showed that the disjoint montane distribution could not account for the genetic divergence among areas (Φ_CT = 0.012, P = 0.253), and that there was non‐trivial subdivision among populations within the same mountain region (Φ_SC = 0.021, P = 0.012). The genealogical relationships among populations showed that Scots pine isolates growing on disjoint mountain blocks, but on slopes flowing to the same basin, were genetically closer than populations growing on different slopes of the same mountain chain, flowing to different basins. Main conclusions The observed genetic structure for Scots pine is consistent with its population history, inferred from the palaeobotanical record, with vertical migrations throughout climatic pulses and with the drainage basins and large long‐term population sizes connecting different mountain blocks during the cooler glacial periods. Overall, the results suggest that, despite periodic interstadial fragmentation episodes, Scots pine biology provides for the long‐term maintenance of high within‐population and low among‐population genetic diversity at neutral genetic markers.     

Patterns of nuclear and mitochondrial DNA variation in Iberian populations of <Emphasis Type="Italic">Emys orbicularis</Emphasis> (Emydidae): conservation implications

The European pond turtle (Emys orbicularis) is threatened and in decline in several regions of its natural range, due to habitat loss combined with population fragmentation. In this work, we have focused our efforts on studying the genetic diversity and structure of Iberian populations with a fine-scale sampling (254 turtles in 10 populations) and a representation from North Africa and Balearic island populations. Using both nuclear and mitochondrial markers (seven microsatellites, ∼1048 bp nDNA and ∼1500 bp mtDNA) we have carried out phylogenetic and demographic analyses. Our results show low values of genetic diversity at the mitochondrial level although our microsatellite dataset revealed relatively high levels of genetic variability with a latitudinal genetic trend decreasing from southern to northern populations. A moderate degree of genetic differentiation was estimated for Iberian populations (genetic distances, F _ST values and clusters in the Bayesian analysis). The results in this study combining mtDNA and nDNA, provide the most comprehensive population genetic data for E. orbicularis in the Iberian Peninsula. Our results suggest that Iberian populations within the Iberian–Moroccan lineage should be considered as a single subspecies with five management units, and emphasize the importance of habitat management rather than population reinforcement (i.e. captive breeding and reintroduction) in this long-lived species.     

2‐D DIGE reveals changes in wheat xylanase inhibitor protein families due to Fusarium graminearum ΔTri5 infection and grain development

Wheat contains three different classes of proteinaceous xylanase inhibitors (XIs), i.e. Triticum aestivum xylanase inhibitors (TAXIs) xylanase‐inhibiting proteins (XIPs), and thaumatin‐like xylanase inhibitors (TLXIs) which are believed to act as a defensive barrier against phytopathogenic attack. In the absence of relevant data in wheat kernels, we here examined the response of the different members of the XI protein population to infection with a ΔTri5 mutant of Fusarium graminearum, the wild type of which is one of the most important wheat ear pathogens, in early developing wheat grain. Wheat ears were inoculated at anthesis, analyzed using 2‐D DIGE and multivariate analysis at 5, 15, and 25 days post anthesis (DPA), and compared with control samples. Distinct abundance patterns could be distinguished for different XI forms in response to infection with F. graminearum ΔTri5. Some (iso)forms were up‐regulated, whereas others were down‐regulated. This pathogen‐specific regulation of proteins was mostly visible at five DPA and levelled off in the samples situated further from the inoculation point. Furthermore, it was shown that most identified TAXI‐ and XIP‐type XI (iso)forms significantly increased in abundance from the milky (15 DPA) to the soft dough stages (25 DPA) on a per kernel basis, although the extent of increase differed greatly. Non‐glycosylated XIP forms increased more strongly than their glycosylated counterparts.     

Genetic Diversity of Fusarium oxysporum f.sp. lentis Population Affecting Lentil in Syria

Lentil (Lens culinaris Medik.) is an important food legume crop in Syria. Fusarium wilt (Fusarium oxysporum f.sp. lentis – Fol) is a key yield‐limiting factor in the country. The genetic diversity of Fol population was studied using 96 isolates collected from different parts of the country using molecular markers. A total of 16 markers, random amplified polymorphic DNA, simple sequence repeats and inter‐simple sequence repeats were used and 218 polymorphic markers (scorable bands) were obtained. Cluster and structure analyses grouped the isolates into three major groups and subgroups indicating high genetic diversity in the pathogen populations. The molecular variance within the population accounted 87% of the total variation indicating high diversity within population than among geographic locations. The result of this study showed that no alleles were linked to specific province, and therefore, screening for the Fusarium wilt in one location using virulent isolates could be enough to save time and resources.     

Proteome of monoclonal antibody‐purified haustoria from Puccinia triticina Race‐1

Puccinia triticina causes leaf rust, a disease that causes annual yield losses in wheat. It is an obligate parasite that invades the host leaf and forms intracellular structures called haustoria, which obtain nutrients and suppress host immunity using secreted proteins called effectors. Since effector proteins act at the frontier between plant and pathogen and help determine the outcome of the interaction, it is critical to understand their functions. Here, we used a direct proteomics approach to identify effector candidates from P. triticina Race 1 haustoria isolated with a specific monoclonal antibody. Haustoria were >95% pure and free of host contaminants. Using high resolution MS we have identified 1192 haustoria proteins. These were quantified using normalized spectral counts and spanned a dynamic range of three orders of magnitude, with unknown proteins and metabolic enzymes as the most highly represented. The dataset contained 140 candidate effector proteins, based on the presence of a signal peptide and the absence of a known function for the protein. Some of these candidates were significantly enriched with cysteine, with up to 13 residues per protein and up to 6.8% cysteine in composition.