Identification and genetic mapping of highly polymorphic microsatellite loci from an EST database of the septoria tritici blotch pathogen Mycosphaerella graminicola

A database of 30,137 EST sequences from Mycosphaerella graminicola, the septoria tritici blotch fungus of wheat, was scanned with a custom software pipeline for di- and trinucleotide units repeated tandemly six or more times. The bioinformatics analysis identified 109 putative SSR loci, and for 99 of them, flanking primers were developed successfully and tested for amplification and polymorphism by PCR on five field isolates of diverse origin, including the parents of the standard M. graminicola mapping population. Seventy-seven of the 99 primer pairs generated an easily scored banding pattern and 51 were polymorphic, with up to four alleles per locus, among the isolates tested. Among these 51 loci, 23 were polymorphic between the parents of the mapping population. Twenty-one of these as well as two previously published microsatellite loci were positioned on the existing genetic linkage map of M. graminicola on 13 of the 24 linkage groups. Most (66%) of the primer pairs also amplified bands in the closely related barley pathogen Septoria passerinii, but only six were polymorphic among four isolates tested. A subset of the primer pairs also revealed polymorphisms when tested with DNA from the related banana black leaf streak (Black Sigatoka) pathogen, M. fijiensis. The EST database provided an excellent source of new, highly polymorphic microsatellite markers that can be multiplexed for high-throughput genetic analyses of M. graminicola and related species.     

The interaction among evolutionary forces in the pathogenic fungus Mycosphaerella graminicola

The population genetic dynamic of a species is driven by interactions among mutation, migration, drift, mating system, and selection, but it is rare to have sufficient empirical data to estimate values for all of these forces and to allow comparison of the relative magnitudes of these evolutionary forces. We combined data from a mark-release-recapture experiment, extensive population surveys, and computer simulations to evaluate interactions among these evolutionary forces in the pathogenic fungus Mycosphaerella graminicola. The results from these studies showed that, on average, the immigration rate was 0.027, the fraction of outcrossing individuals was 0.035, and the selection coefficient associated with immigrants was 0.106 each generation. We also estimated that effective population sizes for this fungus were larger than 24,000 and the mutation rate for the RFLP markers used in surveys and field experiments was approximately 4 x 10(-5). Computer simulations based on these estimates indicate that, on average, the global population of M. graminicola has reached equilibrium. Population genetic parameters including number of alleles, gene diversity, and population subdivision estimated from the computer simulations were surprisingly close to empirical estimates. Simulations also revealed that random drift is the major evolutionary force decreasing genetic variation in this fungus, followed by natural selection. The major force adding to genetic variation was mutation, followed by gene flow and sexual recombination. Gene flow played the leading role in decreasing population subdivision while natural selection was the major factor increasing population subdivision.     

The global genetic structure of the wheat pathogen Mycosphaerella graminicola is characterized by high nuclear diversity,low mitochondrial diversity,regular recombination,and gene flow

A total of 1673 Mycosphaerella graminicola strains were assayed for DNA fingerprints and restriction fragment length polymorphism (RFLP) markers in the nuclear and mitochondrial genomes. The isolates were collected from 17 wheat fields located in 11 countries on five continents over a six year period (1989-1995). Our results indicate that genetic diversity in the nuclear genome of this fungus was high for all but three of the field populations surveyed and that populations sampled from different continents had similar frequencies for the most common RFLP alleles. Hierarchical analysis revealed that more than 90% of global gene diversity was distributed within a wheat field, while approximately 5% of gene diversity was distributed among fields within regions and approximately 3% was distributed among regions on different continents. These findings suggest that gene flow has occurred on a global scale. On average, each leaf was colonized by a different nuclear genotype. In contrast, only seven mtDNA haplotypes were detected among the 1673 isolates and the two most common mtDNA haplotypes represented approximately 93% of the world population, consistent with a selective sweep. Analysis of multilocus associations indicated that all field populations were in gametic equilibrium, suggesting that sexual recombination is a regular occurrence globally.     

Sexual Reproduction Plays a Major Role in the Genetic Structure of Populations of the Fungus Mycosphaerella Graminicola

The relative contributions of sexual and asexual reproduction to the genetic structure of populations can be difficult to determine for fungi that use a mixture of both types of propagation. Nuclear RFLPs and DNA fingerprints were used to make indirect and direct measures of departures from random mating in a population of the plant pathogenic fungus Mycosphaerella graminicola during the course of an epidemic cycle. DNA fingerprints resolved 617 different genotypes among 673 isolates sampled from a single field over a 3-month period. Only 7% of the isolates represented asexual clones that were found more than once in the sample. The most common clone was found four times. Genotypic diversity averaged 85% of its maximum possible value during the course of the epidemic. Analyses of multilocus structure showed that allelic distributions among RFLP loci were independent. Pairwise comparisons between individual RFLP loci showed that the majority of alleles at these loci were in gametic equilibrium. Though this fungus has the capacity for a significant level of asexual reproduction, each analysis suggested that M. graminicola populations maintain a genetic structure more consistent with random-mating over the course of an epidemic cycle.     

Mating type idiomorphs from a French population of the wheat pathogen Mycosphaerella graminicola: widespread equal distribution and low but distinct levels of molecular polymorphism

Septoria tritici blotch caused by the heterothallic ascomycete Mycosphaerella graminicola is currently the most frequent and the most economically damaging disease on wheat worldwide. Five hundred and ten strains of this fungus were sampled from 16 geographical locations representing the major wheat producing areas in France. Multiplex PCR amplification, PCR-RFLP-SSCP screening and sequencing of parts of mating type encoding sequences were performed in order to assess the distribution and molecular polymorphism of the mating type idiomorphs. The two idiomorphs were scored at similar frequencies within all sampled locations. Both mating types were also identified at the leaf spatial scale, on 42% of leaves from which two or three strains were isolated. No correlation was found between distribution of mating types and either host cultivars from which the sampling was carried out or in vitro colony phenotypes observed during the culture of strains on potato dextrose agar (PDA) medium. PCR-RFLP-SSCP assay highlighted only one MAT1-1 strain exhibiting a profile distinct from all other MAT1-1 strains, whereas ten MAT1-2 strains (among which two and four with same profiles, respectively) showed profiles differing from the other MAT1-2 strains. Sequencing revealed that all polymorphisms corresponded to single nucleotide variations and all strains displaying the same single strand conformation polymorphism (SSCP) profiles showed identical nucleotide sequences, thereby confirming the high sensitivity of SSCP. Only two out of the disclosed nucleotide variations were nonsynonymous. This study strongly suggests a large potential for sexual reproduction in the French population of M. graminicola and reports a high conservation of mating type sequences in the fungus at both nucleotide and population levels, with a great difference in molecular variability between the two idiomorphs.     

Migration patterns among global populations of the pathogenic fungus Mycosphaerella graminicola

DNA sequences from five nuclear loci and data from three microsatellites were collected from 360 isolates representing 14 globally distributed populations of the plant pathogenic fungus Mycosphaerella graminicola. Haplotype networks were constructed for the five sequence loci and population subdivision was assessed using Hudson's permutation test. Migration estimates were calculated using six regional populations for both the sequence and microsatellite loci. While subdivision was detected among the six regional populations, significant gene flow was indicated among some of the populations. The European and Israeli populations contributed the majority of historical immigrants to the New World. Migration estimates for microsatellite loci were used to infer more recent migration events among specific New World populations. We conclude that gene flow was an important factor in determining the demographic history of Mycosphaerella graminicola.     

基于线粒体COⅠ基因序列的中国二点委夜蛾遗传多态性分析

二点委夜蛾Athetis lepigone是2005年首次在河北省发现危害夏玉米苗的新害虫, 2011年7月在河北、 河南、 山东、 山西、 安徽和江苏6省47市夏玉米苗期大面积暴发成灾, 严重威胁玉米生产。为了从种群水平探讨该虫暴发成灾的机制, 我们通过分析线粒体细胞色素氧化酶亚基Ⅰ(mtCOⅠ）基因序列来研究不同地区二点委夜蛾种群的进化关系。本研究采集了河北、 河南、 山东和山西等地的19个不同地理种群样本, 用同源序列比对的方法分析样本mtCOⅠ基因片段, 利用DnaSP 5.0软件和Arlequin 3.5软件对不同地理种群间的mtCOⅠ单倍型多样性分析和Tajima’s D中性检测, 建立了单倍型邻接（N-J）系统发育进化树和单倍型网络图。结果表明, 在203头个体的658 bp mtCOⅠ基因片段中, 得到17种单倍型和18个变异位点, 河北省的二点委夜蛾的单倍型多态性最丰富, 而河南、 山东和山西3省采集的二点委夜蛾样品其单倍型均有与河北种群单倍型一致的类型。二点委夜蛾不同地理种群间基因流水平较高, 种群间没有明显的遗传分化, 并且在较近的历史时期未经历明显的种群扩张。     

Genetic diversity measures of local European beef cattle breeds for conservation purposes

This study was undertaken to determine the genetic structure, evolutionary relationships, and the genetic diversity among 18 local cattle breeds from Spain, Portugal, and France using 16 microsatellites. Heterozygosities, estimates of Fst, genetic distances, multivariate and diversity analyses, and assignment tests were performed. Heterozygosities ranged from 0.54 in the Pirenaica breed to 0.72 in the Barrosã breed. Seven percent of the total genetic variability can be attributed to differences among breeds (mean F_st = 0.07; P < 0.01). Five different genetic distances were computed and compared with no correlation found to be significantly different from 0 between distances based on the effective size of the population and those which use the size of the alleles. The Weitzman recursive approach and a multivariate analysis were used to measure the contribution of the breeds diversity. The Weitzman approach suggests that the most important breeds to be preserved are those grouped into two clusters: the cluster formed by the Mirandesa and Alistana breeds and that of the Sayaguesa and Tudanca breeds. The hypothetical extinction of one of those clusters represents a 17% loss of diversity. A correspondence analysis not only distinguished four breed groups but also confirmed results of previous studies classifying the important breeds contributing to diversity. In addition, the variation between breeds was sufficiently high so as to allow individuals to be assigned to their breed of origin with a probability of 99% for simulated samples.     

Microsatellite markers provide evidence for sexual reproduction of Mycosphaerella graminicola in Saskatchewan.

This study examined the genetic structure of a Saskatchewan population of Mycosphaerella graminicola, cause of the foliar disease Septoria tritici blotch of wheat. Such knowledge is valuable for understanding the evolutionary potential of this pathogen and for developing control strategies based on host resistance. Nine pairs of single-locus microsatellite primers were used to analyze the genomic DNA of 90 isolates of M. graminicola that were collected using a hierarchical sampling procedure from different locations, leaves, and lesions within a wheat field near Saskatoon. Allelic series at eight different loci were detected. The number of alleles per locus ranged from one to five with an average of three alleles per locus. Genetic diversity values ranged from 0.04 to 0.67. Partitioning the total genetic variability into within- and among-location components revealed that 88% of the genetic variability occurred within locations, i.e., within areas of 1 m(2), but relatively little variability occurred among locations. Low variability among locations and a high degree of variability within locations would result if the primary source of inoculum was airborne ascospores, which would be dispersed uniformly within the field. This finding was confirmed by gametic disequilibrium analysis and suggests that the sexual reproduction of M. graminicola occurs in Saskatchewan.     

Multilocus ISSR Markers Reveal Two Major Genetic Groups in Spanish and South African Populations of the Grapevine Fungal Pathogen Cadophora luteo-olivacea

Cadophora luteo-olivacea is a lesser-known fungal trunk pathogen of grapevine which has been recently isolated from vines showing decline symptoms in grape growing regions worldwide. In this study, 80 C. luteo-olivacea isolates (65 from Spain and 15 from South Africa) were studied. Inter-simple-sequence repeat-polymerase chain reaction (ISSR-PCR) generated 55 polymorphic loci from four ISSR primers selected from an initial screen of 13 ISSR primers. The ISSR markers revealed 40 multilocus genotypes (MLGs) in the global population. Minimum spanning network analysis showed that the MLGs from South Africa clustered around the most frequent genotype, while the genotypes from Spain were distributed all across the network. Principal component analysis and dendrograms based on genetic distance and bootstrapping identified two highly differentiated genetic clusters in the Spanish and South African C. luteo-olivacea populations, with no intermediate genotypes between these clusters. Movement within the Spanish provinces may have occurred repeatedly given the frequent retrieval of the same genotype in distant locations. The results obtained in this study provide new insights into the population genetic structure of C. luteo-olivacea in Spain and highlights the need to produce healthy and quality planting material in grapevine nurseries to avoid the spread of this fungus throughout different grape growing regions.     

Phenotypic and genetic analysis of the Triticum monococcum-Mycosphaerella graminicola interaction

Here, the aim was to understand the cellular and genetic basis of the Triticum monococcum-Mycosphaerella graminicola interaction. Testing for 5 yr under UK field conditions revealed that all 24 T. monococcum accessions exposed to a high level of natural inocula were fully resistant to M. graminicola. When the accessions were individually inoculated in the glasshouse using an attached leaf seeding assay and nine previously characterized M. graminicola isolates, fungal sporulation was observed in only three of the 216 interactions examined. Microscopic analyses revealed that M. graminicola infection was arrested at four different stages post-stomatal entry. When the inoculated leaves were detached 30 d post inoculation and incubated at 100% humidity, abundant asexual sporulation occurred within 5 d in a further 61 interactions. An F(2) mapping population generated from a cross between T. monococcum accession MDR002 (susceptible) and MDR043 (resistant) was inoculated with the M. graminicola isolate IPO323. Both resistance and in planta fungal growth were found to be controlled by a single genetic locus designated as TmStb1 which was linked to the microsatellite locus Xbarc174 on chromosome 7A(m). Exploitation of T. monococcum may provide new sources of resistance to septoria tritici blotch disease.     

Finished genome of the fungal wheat pathogen Mycosphaerella graminicola reveals dispensome structure, chromosome plasticity, and stealth pathogenesis

The plant-pathogenic fungus Mycosphaerella graminicola (asexual stage: Septoria tritici) causes septoria tritici blotch, a disease that greatly reduces the yield and quality of wheat. This disease is economically important in most wheat-growing areas worldwide and threatens global food production. Control of the disease has been hampered by a limited understanding of the genetic and biochemical bases of pathogenicity, including mechanisms of infection and of resistance in the host. Unlike most other plant pathogens, M. graminicola has a long latent period during which it evades host defenses. Although this type of stealth pathogenicity occurs commonly in Mycosphaerella and other Dothideomycetes, the largest class of plant-pathogenic fungi, its genetic basis is not known. To address this problem, the genome of M. graminicola was sequenced completely. The finished genome contains 21 chromosomes, eight of which could be lost with no visible effect on the fungus and thus are dispensable. This eight-chromosome dispensome is dynamic in field and progeny isolates, is different from the core genome in gene and repeat content, and appears to have originated by ancient horizontal transfer from an unknown donor. Synteny plots of the M. graminicola chromosomes versus those of the only other sequenced Dothideomycete, Stagonospora nodorum, revealed conservation of gene content but not order or orientation, suggesting a high rate of intra-chromosomal rearrangement in one or both species. This observed "mesosynteny" is very different from synteny seen between other organisms. A surprising feature of the M. graminicola genome compared to other sequenced plant pathogens was that it contained very few genes for enzymes that break down plant cell walls, which was more similar to endophytes than to pathogens. The stealth pathogenesis of M. graminicola probably involves degradation of proteins rather than carbohydrates to evade host defenses during the biotrophic stage of infection and may have evolved from endophytic ancestors.     

Database of European chestnut cultivars and definition of a core collection using simple sequence repeats

We have evaluated 271 accessions corresponding to 118 European cultivars, 96 from Spain, 16 from Italy, four from France and two from Portugal with the following objectives: (1) to provide a European database based on reference simple sequence repeats (SSRs) and (2) to define a core collection. A set of 24 highly polymorphic SSRs were used for the genetic analysis. Two main clusters were identified using a model-based Bayesian procedure, which correspond to Spanish and Italian cultivar clusters, with the latter showing a higher genetic diversity. An additional genetic substructure was observed among five different groups of cultivars. A core collection with a minimum of 37 cultivars was selected. We provided a database including 132 European accessions with unique genotypes evaluated with 24 SSRs as a reference for distinction, registering and traceability. Finally, we found that a core collection based on 14% of the total accessions conserves all allelic diversity.     

Comparing the genetic structure of codling moth Cydia pomonella (L.) from Greece and France: long distance gene-flow in a sedentary pest species

Codling moth Cydia pomonella L. (Lepidoptera: Tortricidae) is the most important insect pest of apple production in Europe. Despite the economic importance of this pest, there is not information about the genetic structure of its population in Greece and the patterns of gene-flow which might affect the success of control programs. In this study, we analysed nine samples from apple, pear and walnut from various regions of mainland Greece using 11 microsatellite loci. Six samples from the aforementioned hosts from southern France were also examined for comparison. Bayesian clustering and genetic distance analyses separated the codling moth samples in two genetic clusters. The first cluster consisted mainly of the individuals from Greece, and the second of those from France, although admixture and miss-classified individuals were also observed. The low genetic differentiation among samples within each country was also revealed by F(ST) statistics (0.009 among Greek samples and 0.0150 among French samples compared to 0.050 global value among all samples and 0.032 the mean of the pair-wise values between the two countries). These F(ST) values suggest little structuring at large geographical scales in agreement with previous published studies. The host species and local factors (climatic conditions, topography, pest control programs) did not affect the genetic structure of codling moth populations within each country. The results are discussed in relation to human-made activities that promote gene-flow even at large geographic distances. Possible factors for the genetic differentiation between the two genetic clusters are also discussed.     

A combined amplified fragment length polymorphism and randomly amplified polymorphism DNA genetic kinkage map of Mycosphaerella graminicola,the septoria tritici leaf blotch pathogen of wheat

An F(1) mapping population of the septoria tritici blotch pathogen of wheat, Mycosphaerella graminicola, was generated by crossing the two Dutch field isolates IPO323 and IPO94269. AFLP and RAPD marker data sets were combined to produce a high-density genetic linkage map. The final map contained 223 AFLP and 57 RAPD markers, plus the biological traits mating type and avirulence, in 23 linkage groups spanning 1216 cM. Many AFLPs and some RAPD markers were clustered. When markers were reduced to 1 per cluster, 229 unique positions were mapped, with an average distance of 5.3 cM between markers. Because M. graminicola probably has 17 or 18 chromosomes, at least 5 of the 23 linkage groups probably will need to be combined with others once additional markers are added to the map. This was confirmed by pulsed-field gel analysis; probes derived from 2 of the smallest linkage groups hybridized to two of the largest chromosome-sized bands, revealing a discrepancy between physical and genetic distance. The utility of the map was demonstrated by identifying molecular markers tightly linked to two genes of biological interest, mating type and avirulence. Bulked segregant analysis was used to identify additional molecular markers closely linked to these traits. This is the first genetic linkage map for any species in the genus Mycosphaerella or the family Mycosphaerellaceae.     

Population structure and virulence analysis of <Emphasis Type="Italic">Alternaria carthami</Emphasis> isolates of India using ISSR and SSR markers

Alternaria leaf spot caused by Alternaria carthami is one of the most devastating diseases of safflower. Diversity among 95 isolates of A. carthami was determined using virulence assays, enzyme assays, dominant (ISSR) and co-dominant (SSR) markers. Collections and isolations were made from three major safflower producing states of India. The virulence assays categorised the population into four groups based on level of virulence. Estimation of activities of cell wall degrading enzymes (CWDE) yielded concurrent results to virulence assays with maximum CWDE activities in most virulent group. Eighteen ISSR primers were used and 23 polymorphic microsatellite markers were developed to assess the genetic diversity and determine the population structure of A. carthami. Analysis of ISSR profiles revealed high genetic diversity (Nei’s Genetic diversity index; h?=?0.36). Microsatellite markers produced a total of 56 alleles with an average of 2.43 alleles per microsatellite marker and Nei’s genetic diversity index as h?=?0.43. Unweighted Neighbor-joining and population structure analysis using both the marker systems differently arranged the isolates into three clusters. Distance analysis of the marker profiles provided no evidence for geographical clustering of isolates, indicating that isolates are randomly spread across India, signifying high potential of the fungus to adapt to diverse regions. Microsatellite markers clustered the isolates in consonance to the virulence groups in the dendrogram. This implies that the fungus has a high potential to adapt to resistant cultivars or fungicides. The information can aid in the breeding and deployment of A. carthami resistant varieties, and in early blight disease management in all safflower growing regions of the world.     

Detection of strobilurin-resistant isolates of Mycosphaerella graminicola in Morocco

Septoria tritici blotch caused by Mycosphaerella graminicola (anamorph: Septoria tritici) is nowadays one of the most frequently occurring diseases on both bread and durum wheat crops. Two hundred and thirty isolates of the fungus were sampled from six distinct wheat-producing regions of Morocco in order to investigate the resistance of M. graminicola to strobilurins in this country, where this fungicide class is increasingly used in wheat-pest management. A subset of 134 isolates was first collected in 2008 from Meknes-Tafilalet, Tadla-Azilal, Gharb and Chaouia. Furthermore, 96 additional isolates were sampled in 2010 from the fourth regions investigated in 2008 plus Fes-Boulmane and Doukkala-Abda. Sensitivity or resistance within the isolates were determined by screening the G143A cytochrome b substitution conferring resistance. We used a mismatch amplification mutation assay allowing the amplification of either G143 (sensitive) or A143 (resistant) allele. All the 2008 isolates were found to be sensitive since they carry the wild-type allele G143. However, 9 (9%) out of the 2010 isolates were found to contain the resistant allele A143 and therefore to be resistant. Four of them were from Gharb and five from Fes-Boulmane. This study highlighted for the first time the occurrence of strobilurin-resistant isolates of M. graminicola in Morocco. Further genetic investigations should determine if the resistant isolates emerged independently in Morocco or traveled by wind-migration from Europe.     

Spatial structuring of the population genetics of a European subterranean termite species

In population genetics studies, detecting and quantifying the distribution of genetic variation can help elucidate ecological and evolutionary processes. In social insects, the distribution of population‐level genetic variability is generally linked to colony‐level genetic structure. It is thus especially crucial to conduct complementary analyses on such organisms to examine how spatial and social constraints interact to shape patterns of intraspecific diversity. In this study, we sequenced the mitochondrial COII gene for 52 colonies of the subterranean termite Reticulitermes grassei (Isoptera: Rhinotermitidae), sampled from a population in southwestern France. Three haplotypes were detected, one of which was found exclusively in the southern part of the study area (near the Pyrenees). After genotyping 6 microsatellite loci for 512 individual termites, we detected a significant degree of isolation by distance among individuals over the entire range; however, the cline of genetic differentiation was not continuous, suggesting the existence of differentiated populations. A spatial principal component analysis based on allele frequency data revealed significant spatial autocorrelation among genotypes: the northern and southern groups were strongly differentiated. This finding was corroborated by clustering analyses; depending on the randomized data set, two or three clusters, exhibiting significant degrees of differentiation, were identified. An examination of colony breeding systems showed that colonies containing related neotenic reproductives were prevalent, suggesting that inbreeding may contribute to the high level of homozygosity observed and thus enhance genetic contrasts among colonies. We discuss the effect of evolutionary and environmental factors as well as reproductive and dispersal modes on population genetic structure.     

Evidence of population structuring following population genetic analyses of Fasciola hepatica from Argentina

Fasciola hepatica, the liver fluke, is a trematode parasite that causes disease of economic importance in livestock. As a zoonosis this parasite also poses a risk to human health in areas where it is endemic. Population genetic studies can reveal the mechanisms responsible for genetic structuring (non-panmixia) within parasite populations and provide valuable insights into population dynamics, which in turn enables theoretical predictions of evolutionary dynamics such as the evolution of drug resistance. Here we genotyped 320 F. hepatica collected from 14 definitive hosts from four provinces in Argentina. STRUCTURE analysis indicated three population clusters, and principal coordinate analysis confirmed this, showing population clustering across provinces. Similarly, pairwise F_ST values amongst all four provinces were significant, with standardised pairwise F_ST (F′_ST) ranging from 0.0754 to 0.6327. Therefore, population genetic structure was evident across these four provinces in Argentina. However, there was no evidence of deviation from Hardy–Weinberg equilibrium, so it appears that within these sub-populations there is largely random mating. We identified 263 unique genotypes, which gave a clonal diversity of 82%. Parasites with identical genotypes, clones, accounted for 26.6% of the parasites studied and were found in 12 of the 14 hosts studied, suggesting some clonemate transmission.