Multilocus PCR Assays Elucidate Vegetative Incompatibility Gene Profiles of Cryphonectria parasitica in the United States

Chestnut blight is a devastating disease of Castanea spp. Mycoviruses that reduce virulence (hypovirulence) of the causative agent, Cryphonectria parasitica, can be used to manage chestnut blight. However, vegetative incompatibility (vic) barriers that restrict anastomosis-mediated virus transmission hamper hypovirulence efficacy. In order to effectively determine the vegetative incompatibility genetic structure of C. parasitica field populations, we have designed PCR primer sets that selectively amplify and distinguish alleles for each of the six known diallelic C. parasitica vic genetic loci. PCR assay results were validated using a panel of 64 European tester strains with genetically determined vic genotypes. Analysis of 116 C. parasitica isolates collected from five locations in the eastern United States revealed 39 unique vic genotypes and generally good agreement between PCR and tester strain coculturing assays in terms of vic diversity and genotyping. However, incongruences were observed for isolates from multiple locations and suggested that the coculturing assay can overestimate diversity at the six known vic loci. The availability of molecular tools for rapid and precise vic genotyping significantly improves the ability to predict and evaluate the efficacy of hypovirulence and related management strategies.     

Vegetative Incompatibility Loci with Dedicated Roles in Allorecognition Restrict Mycovirus Transmission in Chestnut Blight Fungus

Vegetative incompatibility (vic), a form of nonself allorecognition, operates widely in filamentous fungi and restricts transmission of virulence-attenuating hypoviruses in the chestnut blight fungus Cryphonectria parasitica. We report here the use of a polymorphism-based comparative genomics approach to complete the molecular identification of the genetically defined C. parasitica vic loci with the identification of vic1 and vic3. The vic1 locus in the C. parasitica reference strain EP155 consists of a polymorphic HET-domain-containing 771-aa ORF designated vic1a-2, which shares 91% identity with the corresponding vic1a-1 allele, and a small (172 aa) idiomorphic DUF1909-domain-containing ORF designated vic1b-2 that is absent at the vic1-1 locus. Gene disruption of either vic1a-2 or vic1b-2 in strain EP155 eliminated restrictions on virus transmission when paired with a vic1 heteroallelic strain; however, only disruption of vic1a-2 abolished the incompatible programmed cell death (PCD) reaction. The vic3 locus of strain EP155 contains two polymorphic ORFs of 599 aa (vic3a-1) and 102 aa (vic3b-1) that shared 46 and 85% aa identity with the corresponding vic3a-2 and vic3b-2 alleles, respectively. Disruption of either vic3a-1 or vic3b-1 resulted in increased virus transmission. However, elimination of PCD required disruption of both vic3a and vic3b. Additional allelic heterogeneity included a sequence inversion and a 8.5-kb insertion containing a LTR retrotransposon sequence and an adjacent HET-domain gene at the vic1 locus and a 7.7-kb sequence deletion associated with a nonfunctional, pseudo vic locus. Combined gene disruption studies formally confirmed restriction of mycovirus transmission by five C. parasitica vic loci and suggested dedicated roles in allorecognition. The relevance of these results to the acquisition and maintenance of vic genes and the potential for manipulation of vic alleles for enhanced mycovirus transmission are discussed.     

Genetic Analyses of ENDOTHIA PARASITICA: Linkage Data for Four Single Genes and Three Vegetative Compatibility Types

The loci cre, met and ts segregate independently in Endothia parasitica. The phenotype brown (br) seems to be determined by an allele at or very near the cre locus. The vegetative compatibility types (v-c) 5 and 39 are determined by different alleles at a locus that is not linked to cre, met or ts. Analysis of two crosses of v-c 5 strains by v-c 10 strains provides evidence that these two v-c groups are different at 5 or more v-c loci.     

Esterase Allozymes of Soybean Cyst Nematode,Heterodera glycines,from China,Japan, and the United States

Individual females from 19 populations of Heterodera glycines from China, Japan, and the United States were analyzed for esterase allozyme polymorphism. Eight esterase electrophoretic phenotypes were resolved. Four putative loci, est-1, est-2, est-3, and est-4, were identified, having one, one, two, and one allele, respectively. The four loci expressed six genotypes in the four Chinese populations. Loci est-2, est-3, and est-4 were identified in five Japanese populations and expressed five genotypes, whereas only loci est-2 and est-3 were identified in 10 populations from the United States and expressed four genotypes. Putative alleles at each locus were defined as characters for data analysis. Phylogenetic analysis using parsimony (PAUP) was utilized to determine relationships among the 19 populations. More loci and alleles in populations from China and phylogenetic similarities among populations from Japan and the United States are consistent with a founder effect resulting from dissemination of progenitor H. glycines from China to Japan and subsequent introductions of founder populations from Japan to the United States.     

Genetic diversification of the chestnut blight fungus Cryphonectria parasitica and its associated hypovirus in Germany

Chestnut blight in south-western Germany was first reported in 1992 and is since expanding in distribution. Here we investigated the invasion history of Cryphonectria parasitica and its associated hypovirus. For this, we characterized 284 isolates collected between 1992 and 2012 for hypovirulence, vegetative compatibility (vc), mating type, and microsatellite haplotype. A total of 27 haplotypes and 15 vc types were observed, although the C. parasitica population analyzed is currently dominated to 50 % by one haplotype and to 64 % by the vc type EU-2. Structure analysis indicated two divergent genetic pools. Over 66 % of the haplotypes belonged to a pool probably originating from northern Italy. Further diversification is expected due to ongoing sexual recombination, but also to new migration and additional introductions. Cryphonectria hypovirus 1 (CHV-1) was found in four of five C. parasitica populations from Baden-Württemberg. Genetic analysis of the 35 CHV-1 isolates obtained revealed that they all belong to the German subtype, although they have clearly diverged from the first German hypovirus isolated in 1992. Our study suggests that C. parasitica has been introduced into Germany several times from two different gene pools, whereas the hypovirus most probably has a single origin.     

Alleles at storage protein loci in Triticum spelta L. accessions and their occurrence in related wheats

The diversity at eight storage protein loci was analyzed in the collection of Triticum spelta accesssions from the National Center for Plant Genetic Resources of Ukraine (most of accessions were European spelts). Seven alleles at the Gli-B1 locus; five alleles at the Gli-A1 and Glu-B1 loci; three alleles at the Glu-B1 locus; and two alleles at the Gli-D1, Gli-B5, Glu-A1, and Glu-D1 loci were identified. Most alleles are found among common wheat cultivars; only five spelt-specific alleles were detected. The high frequency of the GliB1hs* and h alleles encoding the 45-type γ-gliadin among European spelt and durum wheat, as well as the occurrence of these alleles in T. dicoccum (particularly, in emmer accessions from Switzerland and Germany), are evidence in favor of von Büren’s hypothesis that the European spelt arose from the hybridization between tetraploid wheat with the 45-type γ-gliadin and hexaploid wheat. The analysis of genetic distances based on the genotypes at eight storage protein loci allowed differentiating the Asian spelt accession from European spelts.     

Genetic control of horizontal virus transmission in the chestnut blight fungus, Cryphonectria parasitica

Vegetative incompatibility in fungi has long been known to reduce the transmission of viruses between individuals, but the barrier to transmission is incomplete. In replicated laboratory assays, we showed conclusively that the transmission of viruses between individuals of the chestnut blight fungus Cryphonectria parasitica is controlled primarily by vegetative incompatibility (vic) genes. By replicating vic genotypes in independent fungal isolates, we quantified the effect of heteroallelism at each of six vic loci on virus transmission. Transmission occurs with 100% frequency when donor and recipient isolates have the same vic genotypes, but heteroallelism at one or more vic loci generally reduces virus transmission. Transmission was variable among single heteroallelic loci. At the extremes, heteroallelism at vic4 had no effect on virus transmission, but transmission occurred in only 21% of pairings that were heteroallelic at vic2. Intermediate frequencies of transmission were observed when vic3 and vic6 were heteroallelic (76 and 32%, respectively). When vic1, vic2, and vic7 were heteroallelic, the frequency of transmission depended on which alleles were present in the donor and the recipient. The effect of heteroallelism at two vic loci was mostly additive, although small but statistically significant interactions (epistasis) were observed in four pairs of vic loci. A logistic regression model was developed to predict the probability of virus transmission between vic genotypes. Heteroallelism at vic loci, asymmetry, and epistasis were the dominant factors controlling transmission, but host genetic background also was statistically significant, indicating that vic genes alone cannot explain all the variation in virus transmission. Predictions from the logistic regression model were highly correlated to independent transmission tests with field isolates. Our model can be used to estimate horizontal transmission rates as a function of host genetics in natural populations of C. parasitica.     

Potential diversity in vegetative compatibility groupings in the California population of Gibberella circinata

Pitch canker, caused by Gibberella circinata, is a disease affecting pines throughout the world. Although the pathogen is capable of sexual reproduction, natural populations are often comprised of very few vegetative compatibility groups (VCGs), which implies a predominance of asexual reproduction. However, even where outcrossing occurs, a population could have limited VCG diversity due to a low level of polymorphism at the loci governing vegetative compatibility (= vic loci). To determine whether this was the case for the California population of G. circinata, inter-fertile strains were crossed under laboratory conditions. Progeny of this cross included a minimum of 29 VCGs, which was consistent with segregation of vic alleles at five loci. Only two of these VCGs were known to occur naturally in California. Three VCGs accounted for 32.4 % of the progeny but 0 % of the California population. Overall, the results support the conclusion that outcrossing has been rare or absent in the California population of G. circinata.     

A minimum requirements method to isolate large quantities of highly purified DNA from one drop of poultry blood

Requirement of vernalization is an important factor which plays a crucial role in cereals to transit from vegetative to reproductive phase. There are three types of growth habit in barley: winter, spring and facultative types; in which spring type does not require vernalization but winter and facultative genotypes require full and partial vernalization, respectively. Combination of two loci, Vrn-h1 and Vrn-h2, regulates vernalization in barley genotypes. Specific DNA markers have been identified for growth habit regulator genes in barley. In this study, we examined 24 barley genotypes using specific primers for detecting Vrn-h1 and Vrn-h2 loci. Results showed that among all differently suggested primer combinations, a few markers were precisely correlated with seasonal growth habit in barley. The specific markers of 600, 600 and 200 bps were verified for ZCCT-Ha, ZCCT-Hb and ZCCT-Hc loci, respectively. Our field growth habit test showed that cultivar Bahman as a winter growth habit, where all the others genotypes exhibited spring growth habit. By using specific primers for Vrn-h1, only Bahman cultivar produced 616 bp and 830 bp fragments and spring genotypes showed 574 bp or 616 bp alleles without any amplification for 830 bp fragments. Therefore, presence of 616 bp and 830 bp alleles together in each genotype can be considered as an informative marker for winter growth habit in barley. These informative markers can be used easily in barley breeding programmes for detection of growth habit types in the seedling stage.     

Genetic Diversity of Some Pear Cultivars and Genotypes Using Simple Sequence Repeat (SSR) Markers

The genetic diversity and relationships among 47 pear cultivars and genotypes (Pyrus spp.), including 4 Japanese pears (Pyrus pyrifolia), 40 European pears (Pyrus communis), 1 Chinese pear (Pyrus bretschneideri) as well as 2 wild relatives (Pyrus salicifolia and Pyrus mazandaranica) were studied using 28 microsatellite primer pairs. A total of 174 alleles were produced at the 28 SSR loci with their sizes ranging from 81 to 290?bp. The number of observed alleles for each locus ranged from 3 (TsuENH014 and TsuENH046) to 12 (NB103a), with an average of 6.21 alleles per locus. In some SSR loci, more than two alleles were amplified in some cultivars and genotypes, suggesting that duplication has occurred in those accessions. This information suggests that at least two genomic regions exist for these loci in the pear genome. The observed heterozygosity (H _o) values of amplified loci ranged from 0.17 (TsuENH006) to 0.97 (NB103a). Shannon's information index (I) value was observed to be highest (2.14) in the NB103a locus, while the TsuENH006 locus had the lowest value with an average of 1.37 among SSR loci. The Dice genetic similarity coefficient ranged from 0.29 (??Nijisseiki?? and P. mazandaranica) to 0.91 (??Chojuro?? and ??Nijisseiki??) among samples. UPGMA cluster analysis showed two major groups corresponding to the Japanese and European pears.     

Heterokaryons and parasexual recombinants of Cryphonectria parasitica in two clonal populations in southeastern Europe

Evidence for parasexuality in natural populations of haploid fungi requires the demonstration of diploids or heterokaryons and recombinant genotypes in the absence of sex. We studied clonal populations of the chestnut blight fungus, Cryphonectria parasitica, in southeastern Europe and found evidence of parasexuality in two locations. In Osoj, Macedonia, we found one isolate (Os05-66) that had two alleles at six codominant loci, giving a haplotype that was a composite of two clones in this population. Six single-conidial isolates from Os05-66 had two alleles at some loci, suggesting partial diploidy or aneuploidy, and we found four recombinant haplotypes among single-conidial isolates from hyphal-tip isolates of the same isolate. In Teano, Italy, we found two heterokaryon isolates that were partial composites of two dominant clones. Single-conidial isolates from hyphal-tip isolates had recombinant haplotypes. These results provide evidence that is consistent with the hypothesis of parasexuality in C. parasitica in Europe, similar to an earlier report in a natural population in the USA.     

SSR polymorphism of modern cultivars and autochthonous forms of the pear tree from North Caucasus

Genetic similarity and relatedness within the set of pear genotypes including autochthonous Circassian cultivars from North Caucasus, European cultivars, accessions of Pyrus caucasica Fed., and modern Russian cultivars were estimated on the basis of analysis of SSR loci. The level of polymorphism for the studied loci varied from 11 to 15 alleles per locus in the set of 29 samples of pears. A higher level of allelic polymorphism of SSR loci was revealed for a set of P. caucasica samples in comparison with modern cultivated cultivars: from 9 to 12 alleles for P. caucasica and from 6 to 8 alleles for modern cultivars. Specific alleles for the mentioned groups of pears were identified. UPGMA clustering revealed two distinct groups: one includes P. caucasica accessions and autochthonous Caucasian cultivars and the other group includes all cultivated European and Russian pear cultivar. The results support the hypothesis of an isolated gene pool formation of autochthonous pear cultivars of the North Caucasus and their probable origin from the wild P. caucasica.     

Genealogical and statistical analyses of the inheritance of gliadin-coding alleles in a model set of common wheat <Emphasis Type="Italic">Triticum aestivum</Emphasis> L. cultivars

Allelic diversity of the gliadin-coding loci Gli-1 and Gli-2 was compared with the genealogical profiles of common wheat cultivars developed in Saratov. Allele tracking through their pedigrees and hierarchic cluster analysis associated 31 Gli alleles with groups of original ancestors. The cultivars Poltavka (12 alleles of six loci) and Selivanovskii Rusak (six alleles of six loci) were identified as sources of the majority of alleles. The results of the cluster analysis fully coincided with the results of allele tracking for alleles occurring at high frequencies. For rare alleles, the resolution of the cluster analysis was somewhat lower and depended on the similarity/distance measure. Thus, it proved possible to indirectly identify the donors of gene alleles by multidimensional statistics even when data on alleles identified in ancestors are unavailable. This approach to the analysis of inheritance has two limitations: detailed pedigree data should be known, and relatively high frequencies (no less than 15–20%) should be observed for the alleles in a sample under study. Cluster analysis was used to study the association of gliadin alleles with commercial quality classes. The most important gliadin-coding alleles, which mark strong cultivars, were identified. In the Saratov cultivars, such alleles include Gli-A1f, GliB1e, Gli-D1a, Gli-A2q, Gli-B2s, and Gli-D2e, which were inherited from the landrace Poltavka, and Gli-A1i, Gli-A2s, and Gli-B2q, which were inherited from the landrace Selivanovskii Rusak.     

Unraveling Multiple MHC Gene Associations with Systemic Lupus Erythematosus: Model Choice Indicates a Role for HLA Alleles and Non-HLA Genes in Europeans

We have performed a meta-analysis of the major-histocompatibility-complex (MHC) region in systemic lupus erythematosus (SLE) to determine the association with both SNPs and classical human-leukocyte-antigen (HLA) alleles. More specifically, we combined results from six studies and well-known out-of-study control data sets, providing us with 3,701 independent SLE cases and 12,110 independent controls of European ancestry. This study used genotypes for 7,199 SNPs within the MHC region and for classical HLA alleles (typed and imputed). Our results from conditional analysis and model choice with the use of the Bayesian information criterion show that the best model for SLE association includes both classical loci (HLA-DRB1^∗03:01, HLA-DRB1^∗08:01, and HLA-DQA1^∗01:02) and two SNPs, rs8192591 (in class III and upstream of NOTCH4) and rs2246618 (MICB in class I). Our approach was to perform a stepwise search from multiple baseline models deduced from a priori evidence on HLA-DRB1 lupus-associated alleles, a stepwise regression on SNPs alone, and a stepwise regression on HLA alleles. With this approach, we were able to identify a model that was an overwhelmingly better fit to the data than one identified by simple stepwise regression either on SNPs alone (Bayes factor [BF] > 50) or on classical HLA alleles alone (BF > 1,000).     

Application of gliadin polymorphism for pedigree analysis in common wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) from Northern Kazakhstan

Gliadins, seed storage proteins, are popular markers effectively employed for the analysis of common wheat. Gliadin electrophoretic patterns are genotype-specific, reproducible, not dependent on growing conditions and are suitable for germplasm identification complementary to molecular markers. Gliadins have been identified and used in wheat from various countries, but prior to this study little was known about gliadin polymorphism in wheat from Kazakhstan. In this study, 48 alleles of six gliadin-coding loci were identified in 43 cultivars of spring wheat from Northern Kazakhstan. The alleles Gli-A1 f , Gli-B1 e , Gli-D1 a , Gli-A2 p , Gli-B2 d and Gli-D2 e had maximal frequencies in each of the six loci. Identified Gli alleles in the loci formed ‘Gliadin Genetic Formula’ unique for each studied variety, and these were compared to the published data from previously analyzed wheat varieties. Pedigree analysis of 43 varieties studied for gliadin polymorphisms indicated that some Gli alleles were conserved and inherited from the progenitor cultivar Akmolinka 1. In contrast, other Gli alleles were replaced by those from modern germplasms. It is assumed that a higher frequency of gliadin alleles can be associated with the selection of genotypes with improved traits for yield and seed quality in the studied wheat cultivars from Northern Kazakhstan.     

High diversity of vegetative compatibility types in Cryphonectria parasitica in Japan and China

We found high diversity of vegetative compatibility (vc) types in native populations of the chestnut blight fungus, Cryphonectria parasitica, in Japan and China; almost every isolate was in a unique vc type. In Japan we found 71 vc types in a sample of 79 isolates pooled from six populations. Within two populations in China, all isolates (n=28 and 11) had unique vc types; we found 15 vc types among 25 isolates in a third Chinese population where multiple isolates were collected from some trees. None of the isolates from China and only three isolates in the 71 vc types from Japan were compatible with any of 64 vc type testers from Europe, which have known vegetative incompatibility genotypes. To our knowledge this is the first report of vc type diversity for C. parasitica in Japan or of any comparisons of vc types between Asia and Europe. The most significant result of this survey is the identification fungal isolates for expanding knowledge of the genetics of vegetative incompatibility.     

Identification of genomic regions involved in resistance against Sclerotinia sclerotiorum from wild Brassica oleracea

The lack of resistant source has greatly restrained resistance breeding of rapeseed (Brassica napus, AACC) against Sclerotinia sclerotiorum which causes severe yield losses in rapeseed production all over the world. Recently, several wild Brassica oleracea accessions (CC) with high level of resistance have been identified (Mei et al. in Euphytica 177:393–400, 2011), bringing a new hope to improve Sclerotinia resistance of rapeseed. To map quantitative trait loci (QTL) for Sclerotinia resistance from wild B. oleracea, an F2 population consisting of 149 genotypes, with several clones of each genotypes, was developed from one F1 individual derived from the cross between a resistant accession of wild B. oleracea (B. incana) and a susceptible accession of cultivated B. oleracea var. alboglabra. The F2 population was evaluated for Sclerotinia reaction in 2009 and 2010 under controlled condition. Significant differences among genotypes and high heritability for leaf and stem reaction indicated that genetic components accounted for a large portion of the phenotypic variance. A total of 12 QTL for leaf resistance and six QTL for stem resistance were identified in 2 years, each explaining 2.2–28.4 % of the phenotypic variation. The combined effect of alleles from wild B. oleracea reduced the relative susceptibility by 22.5 % in leaves and 15 % in stems on average over 2 years. A 12.8-cM genetic region on chromosome C09 of B. oleracea consisting of two major QTL intervals for both leaf and stem resistance was assigned into a 2.7-Mb genomic region on chromosome A09 of B. rapa, harboring about 30 putative resistance-related genes. Significant negative corrections were found between flowering time and relative susceptibility of leaf and stem. The association of flowering time with Sclerotinia resistance is discussed.     

Isolation and characterization of ten polymorphic microsatellite loci in Ixodes arboricola, and cross-amplification in three other Ixodes species

We characterized ten polymorphic microsatellite loci from the tree-hole tick, Ixodes arboricola. Loci were screened in 11–18 individuals from three Belgian populations and five to ten alleles were found at each locus. Seven loci did not show deviations from Hardy–Weinberg equilibrium conditions and there were no indications for null alleles at these loci. The three other loci showed significant heterozygote deficiencies in at least one population, and a high potential for the occurrence of null alleles. We observed no effect of potential host DNA on the scoring of the microsatellites. Cross-amplification of the microsatellites was tested in eight specimens of three congeneric species: I. ricinus, I. hexagonus and I. frontalis. Depending on the species, six or seven of the loci were amplified in ≥4 of the 8 specimens and were polymorphic in each of these species (except for Ixaf 11 in I. frontalis and I. ricinus). These loci thus provide a tool for population genetic analysis of I. arboricola. The suitability of these markers needs to be further investigated in its congeners.     

Biochemical Genetic Relationships Among Tunisian Hares (Lepus sp.), South African Cape Hares (L. capensis), and European Brown Hares (L. europaeus)

Tunisian hares (n = 45), currently assigned to Lepus capensis, were assayed for allelic variation at 40 allozyme loci, and allele frequencies at 32 loci were directly compared with earlier data of South African cape hares (L. capensis, n = 9) and European brown hares (L. europaeus, n = 244) to reveal genetic relationships among them. European mountain hares (L. timidus, n = 200) were used for outgroup comparison. In the Tunisian hares 27.5% of the loci were polymorphic with 2–4 alleles. Among all alleles at polymorphic loci, 15.1% occurred exclusively in Tunisian hares, 5.7% exclusively in cape hares, and 7.5% exclusively in brown hares at low frequencies. Not a single locus showed alternately fixed alleles between the samples of the L. capensis/L. europaeus complex. Levels of absolute and relative genetic differentiation among the samples of the L. capensis/ L. europaeus complex were low, relative to pairwise comparisons involving mountain hares. Diverse cluster analyses and multidimensional scaling of various pairwise genetic distance matrices concordantly grouped Tunisian hares with brown hares, and South African cape hares clustered only slightly farther apart, whereas mountain hares were distinctly separate. These results suggest regionally distinct phylogenetic units within an overall cohesive gene pool in the L. capensis/ L. europaeus complex, supporting Petter's view that all North African hares belong to L. capensis except for one local population of savanna hares, and that cape hares and brown hares are conspecific.     

Population genetic analysis of Helicobacter pylori by multilocus enzyme electrophoresis: extensive allelic diversity and recombinational population structure.

Genetic diversity and relationships in 74 Helicobacter pylori isolates recovered from patients assigned to distinct clinical categories were estimated by examination of allelic variation in six genes encoding metabolic housekeeping enzymes by multilocus enzyme electrophoresis. Seventy-three distinct allele profiles, representing multilocus chromosomal genotypes, were identified. All six loci were highly polymorphic, with an average of 11.2 alleles per locus. The mean genetic diversity in the sample was 0.735, a value that exceeds the level of diversity recorded in virtually all bacterial species studied by multilocus enzyme electrophoresis. A high frequency of occurrence of null alleles (lack of enzyme activity) was identified and warrants further investigation at the molecular level. Lack of linkage disequilibrium (nonrandom association (of alleles over loci) indicates that horizontal transfer and recombination of metabolic enzyme genes have contributed to the generation of chromosomal diversity in H. pylori. In this sample of isolates, there was no statistically significant association of multilocus enzyme electrophoretic types or cluster of related chromosomal types and disease category.