A protein interaction map of soybean mosaic virus strain G7H based on the yeast two-hybrid system

A protein interaction map of Soybean mosaic virus (SMV) strain G7H was generated by the yeast two-hybrid system. Clones encoding the genes P1, HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa, NIb, and CP were fused downstream of the GAL4 binding domain (GAL4-BD) and of the GAL4 activation domain (GAL4-AD). The GAL4-BD and GAL4-AD fusion derivatives of each gene were co-transformed into yeast and transformants in which interaction took place were identified on selective media. Interacting fusion proteins were extracted from the yeast cells, run on SDS-PAGE gels and finally checked by Western blotting with GAL4 polyclonal antibodies. Strong interactions were detected between the pairs CP/CP, HC-Pro/HC-Pro, NIa/NIa, and CP/HC-Pro. Relatively weak but significant interaction was detected between VPg and NIa. Although not all of the protein-protein interactions previously reported in other potyviruses were detected, the interactions revealed here were, in general, similar to those reported previously.     

Analysis of the autoproteolytic activity of the recombinant helper component proteinase from zucchini yellow mosaic virus

The multifunctional helper component proteinase (HC-Pro) of potyviruses contains an autoproteolytic function that, together with the protein 1 (P1) and NIa proteinase, processes the polyprotein into mature proteins. In this study, we analysed the autoproteolytic active domain of zucchini yellow mosaic virus (ZYMV) HC-Pro. Several Escherichia coli-expressed MBP:HC-Pro:GFP mutants containing deletions or point mutations at either the N- or C-terminus of the HC-Pro protein were examined. Our results showed that amino acids essential for the proteolytic activity of ZYMV HC-Pro are distinct from those of the tobacco etch virus HC-Pro, although the amino acid sequences in the proteolytic active domain are conserved among potyviruses.     

Internal cleavage and trans-proteolytic activities of the VPg-proteinase (NIa) of tobacco etch potyvirus in vivo.

The NIa protein of plant potyviruses is a bifunctional protein containing an N-terminal VPg domain and a C-terminal proteinase region. The majority of tobacco etch potyvirus (TEV) NIa molecules are localized to the nucleus of infected cells, although a proportion of NIa is attached covalently as VPg to viral RNA in the cytoplasm. A suboptimal cleavage site that is recognized by the NIa proteinase is located between the two domains. This site was found to be utilized in the VPg-associated, but not the nuclear, pool of NIa. A mutation converting Glu-189 to Leu at the P1 position of the processing site inhibited internal cleavage. Introduction of this mutation into TEV-GUS, an engineered variant of TEV that expresses a reporter protein (beta-glucuronidase [GUS]) fused to the N terminus of the helper component-proteinase (HC-Pro), rendered the virus replication defective in tobacco protoplasts. Site-specific reversion of the mutant internal processing site to the wild-type sequence restored virus viability. In addition, the trans-processing activity of NIa proteinase was tested in vivo after introduction of an artificial cleavage site between the GUS and HC-Pro sequences in the cytoplasmic GUS/HC-Pro polyprotein encoded by TEV-GUS. The novel site was recognized and processed in plants infected by the engineered virus, indicating the presence of excess NIa processing capacity in the cytoplasm. The potential roles of internal NIa processing in TEV-infected cells are discussed.     

Debilitation of plant potyvirus infectivity by P1 proteinase-inactivating mutations and restoration by second-site modifications.

Tobacco etch virus (TEV) encodes three proteinases that catalyze processing of the genome-encoded polyprotein. The P1 proteinase originates from the N terminus of the polyprotein and catalyzes proteolysis between itself and the helper component proteinase (HC-Pro). Mutations resulting in substitution of a single amino acid, small insertions, or deletions were introduced into the P1 coding sequence of the TEV genome. Deletion of the N-terminal, nonproteolytic domain of P1 had only minor effects on virus infection in protoplasts and whole plants. Insertion mutations that did not impair proteolytic activity had no measurable effects regardless of whether the modification affected the N-terminal nonproteolytic or C-terminal proteolytic domain. In contrast, three mutations (termed S256A, F, and delta 304) that debilitated P1 proteolytic activity rendered the virus nonviable, whereas a fourth proteinase-debilitating mutation (termed C) resulted in a slow-infection phenotype. A strategy was devised to determine whether the defect in the P1 mutants was due to an inactive proteinase domain or due simply to a lack of proteolytic maturation between P1 and HC-Pro. Sequences coding for a surrogate cleavage site recognized by the TEV NIa proteinase were inserted into the genome of each processing-debilitated mutant at positions that resulted in NIa-mediated proteolysis between P1 and HC-Pro. The infectivity of each mutant was restored by these second-site modifications. These data indicate that P1 proteinase activity is not essential for viral infectivity but that separation of P1 and HC-Pro is required. The data also provide evidence that the proteinase domain is involved in additional, nonproteolytic functions.     

Evidence that the potyvirus P1 proteinase functions in trans as an accessory factor for genome amplification.

The tobacco etch potyvirus (TEV) polyprotein is proteolytically processed by three viral proteinases (NIa, HC-Pro, and P1). While the NIa and HC-Pro proteinases each provide multiple functions essential for viral infectivity, the role of the P1 proteinase beyond its autoproteolytic activity is understood poorly. To determine if P1 is necessary for genome amplification and/or virus movement from cell to cell, a mutant lacking the entire P1 coding region (delta P1 mutant) was produced with a modified TEV strain (TEV-GUS) expressing beta-glucuronidase (GUS) as a reporter, and its replication and movement phenotypes were assayed in tobacco protoplasts and plants. The delta P1 mutant accumulated in protoplasts to approximately 2 to 3% the level of parental TEV-GUS, indicating that the P1 protein may contribute to but is not strictly required for viral RNA amplification. The delta P1 mutant was capable of cell-to-cell and systemic (leaf-to-leaf) movement in plants but at reduced rates compared with parental virus. This is in contrast to the S256A mutant, which encodes a processing-defective P1 proteinase and which was nonviable in plants. Both delta P1 and S256A mutants were complemented by P1 proteinase expressed in a transgenic host. In transgenic protoplasts, genome amplification of the delta P1 mutant relative to parental virus was stimulated five- to sixfold. In transgenic plants, the level of accumulation of the delta P1 mutant was stimulated, although the rate of cell-to-cell movement was the same as in nontransgenic plants. Also, the S256A mutant was capable of replication and systemic infection in P1-expressing transgenic plants. These data suggest that, in addition to providing essential processing activity, the P1 proteinase functions in trans to stimulate genome amplification.     

The helper component proteinase cistron of Potato virus Y induces hypersensitivity and resistance in Potato genotypes carrying dominant resistance genes on chromosome IV

The Nc(tbr) and Ny(tbr) genes in Solanum tuberosum determine hypersensitive reactions, characterized by necrotic reactions and restriction of the virus systemic movement, toward isolates belonging to clade C and clade O of Potato virus Y (PVY), respectively. We describe a new resistance from S. sparsipilum which possesses the same phenotype and specificity as Nc(tbr) and is controlled by a dominant gene designated Nc(spl). Nc(spl) maps on potato chromosome IV close or allelic to Ny(tbr). The helper component proteinase (HC-Pro) cistron of PVY was shown to control necrotic reactions and resistance elicitation in plants carrying Nc(spl), Nc(tbr), and Ny(tbr). However, inductions of necrosis and of resistance to the systemic virus movement in plants carrying Nc(spl) reside in different regions of the HC-Pro cistron. Also, genomic determinants outside the HC-Pro cistron are involved in the systemic movement of PVY after induction of necroses on inoculated leaves of plants carrying Ny(tbr). These results suggest that the Ny(tbr) resistance may have been involved in the recent emergence of PVY isolates with a recombination breakpoint near the junction of HC-Pro and P3 cistrons in potato crops. Therefore, this emergence could constitute one of the rare examples of resistance breakdown by a virus which was caused by recombination instead of by successive accumulation of nucleotide substitutions.     

Phylogeographic structure,demographic history and morph composition in a colour polymorphic lizard

In polymorphic species, population divergence in morph composition and frequency has the potential to promote speciation. We assessed the relationship between geographic variation in male throat colour polymorphism and phylogeographic structure in the tawny dragon lizard, Ctenophorus decresii. We identified four genetically distinct lineages, corresponding to two polymorphic lineages in the Northern Flinders Ranges and Southern Flinders Ranges/Olary Ranges regions respectively, and a monomorphic lineage in the Mt Lofty Ranges/Kangaroo Island region. The degree of divergence between these three lineages was consistent with isolation to multiple refugia during Pleistocene glacial cycles, whereas a fourth, deeply divergent (at the interspecific level) and monomorphic lineage was restricted to western New South Wales. The same four morphs occurred in both polymorphic lineages, although populations exhibited considerable variation in the frequency of morphs. By contrast, male throat coloration in the monomorphic lineages differed from each other and from the polymorphic lineages. Our results suggest that colour polymorphism has evolved once in the C. decresii species complex, with subsequent loss of polymorphism in the Mt Lofty Ranges/Kangaroo Island lineage. However, an equally parsimonious scenario, that polymorphism arose independently twice within C. decresii, could not be ruled out. We also detected evidence of a narrow contact zone with limited genotypic admixture between the polymorphic Olary Ranges and monomorphic Mt Lofty Ranges regions, yet no individuals of intermediate colour phenotype. Such genetic divergence and evidence for barriers to gene flow between lineages suggest incipient speciation between populations that differ in morph composition.     

版纳小耳猪近交系5家系35个微卫星座位的遗传分析

利用35个微卫星座位对版纳小耳猪近交系5个家系进行了遗传检测。统计各家系的等位基因组成,计算各家系的平均基因纯合率,利用基因频率计算出各家系的平均杂合度及品系间的遗传距离,并进行系统聚类。结果表明各家系的平均基因纯合度均较高,其151家系达到88.79％;PIC（多态信息含量）和平均杂合度均较普通商品猪低;各家系等位基因组成差别较大;各家系间亲缘关系与其近交过程一致,据此认为版纳小耳猪近交系5家系均具有较高的近交程度;其基因多态性和遗传多样性较普通商品猪低;各家系均已构成独立遗传群体。     

Functional Analysis of the Interaction between VPg-Proteinase (NIa) and RNA Polymerase (NIb) of Tobacco Etch Potyvirus, Using Conditional and Suppressor Mutants

The tobacco etch potyvirus (TEV) RNA-dependent RNA polymerase (NIb) has been shown to interact with the proteinase domain of the VPg-proteinase (NIa). To investigate the significance of this interaction, a Saccharomyces cerevisiae two-hybrid assay was used to isolate conditional NIa mutant proteins with temperature-sensitive (ts) defects in interacting with NIb. Thirty-six unique tsNIa mutants with substitutions affecting the proteinase domain were recovered. Most of the mutants coded for proteins with little or no proteolytic activity at permissive and nonpermissive temperatures. However, three mutant proteins retained proteolytic activity at both temperatures and, in two cases (tsNIa-Q384P and tsNIa-N393D), the mutations responsible for the ts interaction phenotype could be mapped to single positions. One of the mutations (N393D) conferred a ts-genome-amplification phenotype when it was placed in a recombinant TEV strain. Suppressor NIb mutants that restored interaction with the tsNIa-N393D protein at the restrictive temperature were recovered by a two-hybrid selection system. Although most of the suppressor mutants failed to stimulate amplification of genomes encoding the tsNIa-N393D protein, two suppressors (NIb-I94T and NIb-C380R) stimulated amplification of virus containing the N393D substitution by approximately sevenfold. These results support the hypothesis that interaction between NIa and NIb is important during TEV genome replication.     

Gain of virulence on Rsv1-genotype soybean by an avirulent Soybean mosaic virus requires concurrent mutations in both P3 and HC-Pro

In soybean, Rsv1, a single dominant resistance gene, invokes extreme resistance (ER) against most Soybean mosaic virus (SMV) strains, including SMV-N, but not SMV-G7, which provokes a virulent lethal systemic hypersensitive response (LSHR). The elicitor functions of the two viruses provoking Rsv1-mediated ER and LSHR have been mapped to the N-terminal 271 amino acids of P3 from SMV-N and SMV-G7, respectively, which differ by nine residues between the two strains. To identify amino acids of P3 from SMV-N provoking Rsv1-mediated ER, the unique residues of SMV-G7 were substituted with those of SMV-N. Of the mutants tested on Rsv1-genotype soybean, only SMV-G7(I788R) and SMV-G7(T948A) lost virulence. However, substitution of amino acids of SMV-N, individually or in combination, with the reciprocal residues from SMV-G7 at these two positions failed to confer virulence to SMV-N. In the search for additional virulence determinants, a series of SMV-N chimeras was generated in which fragments within a region from near the middle of the helper-component proteinase (HC-Pro) cistron to the 5' end of the cytoplasmic inclusion cistron, nucleotides 1,605 to 3,787, were replaced with those of SMV-G7. Only SMV-N-derived chimeras harboring the 3' region of HC-Pro, at least from nucleotide 2,013, and the entire 5' end of P3 (nucleotides 2,430 to 3,237) from SMV-G7 were virulent whereas reciprocal exchanges resulted in loss of SMV-G7 virulence. This region of HC-Pro differs by three amino acids between SMV-N and SMV-G7. Analyses of SMV-G7-derived HC-Pro site-directed mutants showed that only SMV-G7(M683R) lost virulence on Rsv1-genotype soybean; however, SMV-N(R682M) failed to gain virulence. Nevertheless, an SMV-N derived mutant with three concurrent substitutions, R682M+R787I+A947T, gained virulence. The data indicate that both P3 and HC-Pro are involved in virulence of SMV on Rsv1-genotype soybean.     

Degree of Selective Constraint as an Explanation of the Different Rates of Evolution of Gender-Specific Mitochondrial DNA Lineages in the Mussel Mytilus

Mussels of the genus Mytilus segregate for a maternally transmitted F lineage and a paternally transmitted M lineage of mitochondrial DNA. Previous studies demonstrated that these lineages are older than the species of the M. edulis complex and that the M lineage evolves faster than the F lineage. Here we show that the latter observation also applies to a region of the molecule with no assigned function. Sequence data for the mitochondrial COIII gene and the ``unassigned' region of the F and M lineages of M. edulis and M. trossulus are used to evaluate various hypotheses that may account for the faster rate of evolution of the M lineage. Tests based on the proportion of synonymous and nonsynonymous substitutions suggest that the M lineage experiences relatively relaxed selection. Further support for this hypothesis comes from an examination of COIII amino acid substitutions at sites defined as either conserved or variable based on the pattern of variation in other mollusks and Drosophila. Most substitutions in the M lineage occur in regions that are also variable among non-Mytilus taxa. We suggest that these differences in selection pressure are a consequence of doubly uniparental mitochondrial DNA transmission in Mytilus.     

Genetic relationships and species authentication of Boesenbergia (Zingiberaceae) in Thailand based on AFLP and SSCP analyses

Genetic relationships of 15 Boesenbergia species were examined using AFLP analysis. A total of 893 fragments were generated from six primer combinations; of these, 99.78% were polymorphic. The mean genetic distance between pairs of taxa ranged from 0.435 to 0.935. The neighbor-joining tree resolved investigated species into two separate lineages (corresponding to species possessing compact or elongated inflorescences) and suggests a rapid radiation in Boesenbergia. In addition, SSCP patterns of the partial psbA–trnH spacer were not overlapping between different species and therefore can be used for authentication of Boesenbergia species. Morphologically similar Boesenbergia longiflora and Boesenbergia sp., which showed three SNPs based on psbA–trnH polymorphism were successfully differentiated by SSCP. Moreover, Boesenbergia curtisii displayed intraspecific ecomorphological variation but exhibited the same SSCP pattern. Therefore, AFLP and SSCP are rapid and cost-effective methods for analysis of genetic similarity and species identification in Boesenbergia.     

Transposable elements reveal the impact of introgression,rather than transposition,in Pisum diversity,evolution, and domestication

The genetic structure and evolutionary history of the genus Pisum were studied exploiting our germplasm collection to compare the contribution of different mechanisms to the generation of diversity. We used sequence-specific amplification polymorphism (SSAP) markers to assess insertion site polymorphism generated by a representative of each of the two major groups of LTR-containing retrotransposons, PDR1 (Ty1/copia-like) and Cyclops (Ty3/gypsy-like), together with Pis1, a member of the En/Spm transposon superfamily. The analysis of extended sets of the four main Pisum species, P. fulvum, P. elatius, P. abyssinicum, and P. sativum, together with the reference set, revealed a distinct pattern of the NJ (Neighbor-Joining) tree for each basic lineage, which reflects the different evolutionary history of each species. The SSAP markers showed that Pisum is exceptionally polymorphic for an inbreeding species. The patterns of phylogenetic relationships deduced from different transposable elements were in general agreement. The retrotransposon-derived markers gave a clearer separation of the main lineages than the Pis1 markers and were able to distinguish the truly wild form of P. elatius from the antecedents of P. sativum. There were more species-specific and unique PDR1 markers than Pis1 markers in P. fulvum and P. elatius, pointing to PDR1 activity during speciation and diversification, but the proportion of these markers is low. The overall genetic diversity of Pisum and the extreme polymorphism in all species, except P. abyssinicum, indicate a high contribution of recombination between multiple ancestral lineages compared to transposition within lineages. The two independently domesticated pea species, P. abyssinicum and P. sativum, arose in contrasting ways from the common processes of hybridization, introgression, and selection without associated transpositional activity.     

Detection of point mutations in the chloroplast genome by single-stranded conformation polymorphism analysis

Single-stranded conformation polymorphism (SSCP) analysis was used to examine the mutations of the chloroplast 16S rRNA locus of streptomycin-resistant mutants in Nicotiana plumbaginifolia. DNA fragments of 121, 517, 968 and 1578 bp, each possessing a known point mutation, were generated by polymerase chain reaction (PCR). The resulting fragments were denatured and separated by nondenaturing polyacrylamide gel electrophoresis. Compared to the patterns of the wild-type DNA fragments, the bands of the single-stranded DNA fragments of 121 and 517 bp with base changes were shifted. However, no pattern variations were detected from the DNA fragments of 968 and 1578 bp generated from both wild-type and mutants.     

Polymorphism and evolution of vulval precursor cell lineages within two nematode genera, Caenorhabditis and Oscheius

BACKGROUND: The cell lineage of nematodes is mostly invariant for a given species, but varies between species. One can thus wonder how a cell lineage varies during evolution. We have started a microevolutionary approach within two genera by observing lineage variations of vulval precursor cells in different natural nematode populations of the same and closely related species. RESULTS: In Caenorhabditis elegans, the P3.p cell lineage is variable within a genetically homogeneous population and polymorphic between wild strains. Irrespective of its division pattern, P3.p is competent to form vulval tissue in different C. elegans strains, whereas it is not competent in C. briggsae. In Oscheius sp. 1, P4.p and P8.p lineages are strongly polymorphic. Within each genus, these intraspecies polymorphisms in cell lineages are amplified between closely related species. In Oscheius sp. 1, the large polymorphisms in P4.p and P8.p lineages allowed us to undertake a genetic analysis of the variation between two pairs of strains. Multiple loci are involved in cell lineage differences, and variation at one locus appears to have a relatively strong effect. In addition to these large lineage variations in cells that do not normally contribute to the vulva, we find minor variations (errors) in vulval lineages, which represent the precision level of the vulval-patterning process and point to a selection pressure for maintenance of a large vulval equivalence group. CONCLUSIONS: Polymorphisms in vulval cell lineage are found within a given nematode species, and could be instrumental in explaining evolutionary variations between closely related species.     

Amino acid changes in P3, and not the overlapping pipo-encoded protein, determine virulence of soybean mosaic virus on functionally immune Rsv1-genotype soybean

A small open reading frame, termed 'pipo', is embedded in the P3 cistron of potyviruses. Currently, knowledge on pipo and its role(s) in the life cycle of potyviruses is limited. The P3 and helper-component proteinase (HC-Pro) cistrons of Soybean mosaic virus (SMV) harbour determinants affecting virulence on functionally immune Rsv1-genotype soybeans. Interestingly, a key virulence determinant of SMV on Rsv1-genotype soybeans (i.e. soybeans containing the Rsv1 resistance gene) that resides at polyprotein codon 947 overlaps both P3 and a pipo-encoded codon. This raises the question of whether PIPO or P3 is the virulence factor. To answer this question, the corresponding pipo of an avirulent and two virulent strains of SMV were studied by comparative genomics, followed by syntheses and analyses of site-directed mutants. Our data demonstrate that the virulence of SMV on Rsv1-genotype soybeans is affected by P3 and not the overlapping pipo-encoded protein.     

Whole Genome Sequence and Characterization of a Novel Isolate of PVY Inducing Tuber Necrotic Ringspot in Potato and Leaf Mosaic in Tobacco

In a previous study on a Syrian isolate of Potato virus Y (PVY), namely PVY-12, a point mutation in the coat protein (CP) was detected. This mutation caused the double reactivity of this isolate to monoclonal antibodies specific to O and N serotypes. We report here the biological and molecular characteristics of PVY-12. In potato, PVY-12 behaved like a PVY^NTN isolate inducing potato tuber necrotic ringspot disease although it induced mosaic in tobacco like PVY^O. The genomic analysis grouped PVY-12 with the recombinant PVY^NTN isolates, which is consistent with the phenotype in potato. PVY-12 HC-Pro had the two amino acids K400 and E419 that were previously reported as determinant keys of the tobacco necrotic response. This indicates the involvement of other determinants in this phenotype yet to be determined. This is the first report on a PVY^NTN isolate that induces mosaic in tobacco, implying that the induction of potato tuber necrosis does not require the ability to induce the tobacco necrosis. PVY-12 genome had four recombinant points in the P1, HC-Pro/P3, 6K2/NIa and C terminal region of the CP gene identical to those of PVY^NTN isolates 12–94 and 34/01. The PVY-12 central genomic part flanked by nucleotide positions 2414 and 8604 had highest similarity with that of the Syrian isolate SYR-NB-16 suggesting a common origin of these isolates. This common origin was supported using the phylogenetic analysis of this region. In addition, the phylogenetic analysis of the whole genome of the reported North American PVY^N:O and the European PVY^NW along with other PVY isolates suggests that PVY^N:O might have descended from PVY^NW with the isolate SASA-207 as a nearest-known relative.     

Adaptation of Soybean mosaic virus avirulent chimeras containing P3 sequences from virulent strains to Rsv1-genotype soybeans is mediated by mutations in HC-Pro

In Rsv1-genotype soybean, Soybean mosaic virus (SMV)-N (an avirulent isolate of strain G2) elicits extreme resistance (ER) whereas strain SMV-G7 provokes a lethal systemic hypersensitive response (LSHR). SMV-G7d, an experimentally evolved variant of SMV-G7, induces systemic mosaic. Thus, for Rsv1-genotype soybean, SMV-N is avirulent whereas SMV-G7 and SMV-G7d are both virulent. Exploiting these differential interactions, we recently mapped the elicitor functions of SMV provoking Rsv1-mediated ER and LSHR to the N-terminal 271 amino acids of P3 from SMV-N and SMV-G7, respectively. The phenotype of both SMV-G7 and SMV-G7d were rendered avirulent on Rsv1-genotype soybean when the part of the genome encoding the N-terminus or the entire P3 cistron was replaced with that from SMV-N; however, reciprocal exchanges did not confer virulence to SMV-N-derived P3 chimeras. Here, we describe virulent SMV-N-derived P3 chimeras containing the full-length or the N-terminal P3 from SMV-G7 or SMV-G7d, with or without additional mutations in P3, that were selected on Rsv1-genotype soybean by sequential transfers on rsv1 and Rsv1-genotype soybean. Sequence analyses of the P3 and helper-component proteinase (HC-Pro) cistrons of progeny recovered from Rsv1-genotype soybean consistently revealed the presence of mutations in HC-Pro. Interestingly, the precise mutations in HC-Pro required for the adaptation varied among the chimeras. No mutation was detected in the HC-Pro of progeny passaged continuously in rsv1-genotype soybean, suggesting that selection is a consequence of pressure imposed by Rsv1. Mutations in HC-Pro alone failed to confer virulence to SMV-N; however, reconstruction of mutations in HC-Pro of the SMV-N-derived P3 chimeras resulted in virulence. Taken together, the data suggest that HC-Pro complementation of P3 is essential for SMV virulence on Rsv1-genotype soybean.     

Population structure suggests reproductively isolated lineages of Phaeocryptopus gaeumannii

A survey of the genetic diversity and population structure of the Douglas-fir Swiss needle cast pathogen Phaeocryptopus gaeumannii was conducted with single-strand conformational polymorphisms (SSCP) to screen for variability in mitochondrial and nuclear housekeeping genes. Thirty host populations representing the natural range of Douglas-fir as well as locations where the tree was planted as an exotic were sampled. Sequencing of SSCP variants revealed that the method accurately detected both single nucleotide and indel polymorphisms. Sequence information was used to construct multilocus gene genealogies and to test various hypotheses of recombination (outcrossing) and clonality (selfing). We found that P. gaeumannii in the region of Oregon's Swiss needle cast epidemic exhibits strong multilocus gametic phase disequilibrium and is subdivided into two reproductively isolated sympatric lineages. Low genotypic diversity together with the presence of overrepresented genotypes in both lineages suggests a predominantly selfing reproductive mode. Genotypes of one lineage were found in isolates from a widespread geographic distribution, occurring throughout much of the Pacific Northwest as well as nonindigenous populations abroad that have historical reports of disease. Genotypes of the second lineage were detected only in isolates from Oregon's coastal region. Within the main epidemic area, abundance of this second lineage in young plantations appeared to be correlated with disease severity.