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Hop powdery mildew (caused by Podosphaera macularis) was confirmed in the Pacific Northwest in 1996. Before 2012, the most common race of P. macularis was able to infect plants that possessed powdery mildew resistance based on the R-genes Rb, R3, and R5. After 2012, two additional races of P. macularis were discovered that can overcome the resistance gene R6 and the partial resistance found in the cultivar Cascade. These three races now occur throughout the region, which can complicate management and research efforts because of uncertainty on which race(s) may be present in the region and able to infect susceptible hop genotypes. Current methods for determining the races of P. macularis are labor intensive, costly, and typically require more than 14 days to obtain results. We sought to develop a molecular assay to differentiate races of the fungus possessing virulence on plants with R6, referred to as V6-virulent, from other races. The transcriptomes of 46 isolates of P. macularis were sequenced to identify loci and variants unique to V6 isolates. Fourteen primer pairs were designed for 10 candidate loci that contained single nucleotide polymorphisms (SNP) and short insertion-deletion polymorphisms. Two differentially labeled locked nucleic acid probes were designed for a contig that contained a conserved SNP associated with V6-virulence. The resulting duplexed real-time PCR assay was validated against 46 V6 and 54 non-V6 P. macularis isolates collected from the United States and Europe. The assay had perfect discrimination of V6-virulence among isolates of P. macularis originating from the western U.S. but failed to predict V6-virulence in three isolates collected from Europe. The specificity of the assay was tested with different species of powdery mildew fungi and other microorganisms associated with hop. Weak nonspecific amplification occurred with powdery mildew fungi collected from Vitis vinifera, Fragaria sp., and Zinnia sp.; however, nonspecification amplification is not a concern when differentiating pathogen race from colonies on hop. The assay has practical applications in hop breeding, epidemiological studies, and other settings where rapid confirmation of pathogen race is needed.

Obligately biotrophic plant pathogens pose challenges in population genetic studies due to their genomic complexities and elaborate culturing requirements with limited biomass. Hop powdery mildew (Podosphaera macularis) is an obligately biotrophic ascomycete that threatens sustainable hop production. P. macularis populations of the Pacific Northwest (PNW) United States differ from those of the Midwest and Northeastern United States, lacking one of two mating types needed for sexual recombination and harboring two strains that are differentially aggressive on the cultivar Cascade and able to overcome the Humulus lupulus R-gene R6 (V6), respectively. To develop a high-throughput marker platform for tracking the flow of genotypes across the United States and internationally, we used an existing transcriptome of diverse P. macularis isolates to design a multiplex of 54 amplicon sequencing markers, validated across a panel of 391 U.S. samples and 123 international samples. The results suggest that P. macularis from U.S. commercial hop yards form one population closely related to P. macularis of the United Kingdom, while P. macularis from U.S. feral hop locations grouped with P. macularis of Eastern Europe. Included in this multiplex was a marker that successfully tracked V6-virulence in 65 of 66 samples with a confirmed V6-phenotype. A new qPCR assay for high-throughput genotyping of P. macularis mating type generated the highest resolution distribution map of P. macularis mating type to date. Together, these genotyping strategies enable the high-throughput and inexpensive tracking of pathogen spread among geographical regions from single-colony samples and provide a roadmap to develop markers for other obligate biotrophs.

Powdery mildew, caused by Podosphaera macularis, is one of the most important diseases of hop. The disease was first reported in the Pacific Northwestern United States, the primary hop-growing region in this country, in the mid-1990s. More recently, the disease has reemerged in newly planted hopyards of the eastern United States, as hop production has expanded to meet demands of local craft brewers. The spread of strains virulent on previously resistant cultivars, the paucity of available fungicides, and the potential introduction of the MAT1-2 mating type to the western United States, all threaten sustainability of hop production. We sequenced the transcriptome of 104 isolates of P. macularis collected throughout the western United States, eastern United States, and Europe to quantify genetic diversity of pathogen populations and elucidate the possible origins of pathogen populations in the western United States. Discriminant analysis of principal components grouped isolates within three to five geographic populations, dependent on stringency of grouping criteria. Isolates from the western United States were phenotyped and categorized into one of three pathogenic races based on disease symptoms generated on differential cultivars. Western U.S. populations were clonal, irrespective of pathogenic race, and grouped with isolates originating from Europe. Isolates originating from wild hop plants in the eastern United States were genetically differentiated from all other populations, whereas isolates from cultivated hop plants in the eastern United States mostly grouped with isolates originating from the west, consistent with origins from nursery sources. Mating types of isolates originating from cultivated western and eastern U.S. hop plants were entirely MAT1-1. In contrast, a 1:1 ratio of MAT1-1 and MAT1-2 was observed with isolates sampled from wild plants or Europe. Within the western United States a set of highly differentiated loci were identified in P. macularis isolates associated with virulence to the powdery mildew R-gene R6. The weight of genetic and phenotypic evidence suggests a European origin of the P. macularis populations in the western United States, followed by spread of the pathogen from the western United States to re-emergent production regions in the eastern United States. Furthermore, R6 compatibility appears to have been selected from an extant isolate within the western United States. Greater emphasis on sanitation measures during propagation and quarantine policies should be considered to limit further spread of novel genotypes of the pathogen, both between and within production areas.

Botrytis cinerea is an important pathogen of vegetable and fruit crops but little is known about its population structure and genetics in China. We hypothesized that the geographic populations of B. cinerea in China would be genetically differentiated by host, geographic location, and/or year. In this study, we collected 393 B. cinerea isolates representing 28 populations from tomato, cherry, and nectarine from 2006 to 2014 in China. The isolates were analyzed using 14 microsatellite markers, including six new markers that provided more genotyping power than the eight previously published loci. We also investigated the B. cinerea population structure and inferred its mode of reproduction and dispersal based on genotype data. High genotypic diversity was detected in all populations, and clonal reproduction was dominant. Southern China populations harbored more genotypes than northern populations. Differentiation by host plant was evident. Between 2011 and 2012, genotypes changed only slightly among years for Liaoning populations, but they changed substantially among years for the Shanghai and Fujian populations. Clonal dispersal was detected and the farthest dispersal distance was estimated to be about 1,717 km. Two high-frequency genotypes were widely distributed in more than 10 populations and across several years. Our results provide useful, novel information for plant breeding programs and control of B. cinerea in China.

Verticillium wilt is the most important disease threatening the commercial production of mint grown for essential oil. An important long-term goal for mint breeders is the production of cultivars with resistance to Verticillium wilt. Before that can be accomplished, a better understanding of the genetic variation within and among populations of Verticillium dahliae is needed. We characterized the extent of phenotypic and genetic diversity present in contemporary and archival populations of V. dahliae from mint fields in Oregon and other production regions of the United States using genotyping by sequencing, PCR assays for mating type and pathogenic race, vegetative compatibility group (VCG) tests, and aggressiveness assays. We report that the population in the Pacific Northwest can be described as one common genetic group and four relatively rare genetic groups. Eighty-three percent of the isolates belonged to VCG2B, and all isolates possessed the MAT1-2 idiomorph and were characterized as pathogenic race 2. These results indicate low levels of genetic diversity and a negligible risk of sexual recombination in populations of this host-adapted pathogen population. Knowledge of the genetic structure of V. dahliae in the Pacific Northwest will inform breeders about the diversity of pathogenicity factors that may need to be considered in their breeding programs.

Downy mildew of grape caused by Plasmopara viticola is a global pathogen of economic importance to commercial viticulture. In contrast to populations in the northern hemisphere, few studies have investigated the population biology, genetic diversity, and origin of the pathogen in Australian production systems. DNA was extracted from 381 P. viticola samples from Vitis vinifera and alternate hosts collected via fresh and herbarium leaves from populations within Australia and Whatman FTA cards from North America, Brazil, and Uruguay. A total of 32 DNA samples were provided from a French population. The populations were genotyped using 16 polymorphic microsatellite markers. Representative samples from within Australia, Brazil, and Uruguay were also genotyped to determine which of the cryptic species (clades) within the P. viticola species complex were present. Our findings suggest the Australian and South American populations of P. viticola are more closely related to the European population than the North American population, the reported source of origin of the pathogen. The Western Australian population had similarities to the South Australian population, and the tight clustering of samples suggests a single introduction into Western Australia. P. viticola clade aestivalis was the only clade detected in Australian and South American populations. Analysis of the Western Australian population suggests that it is reproducing clonally, but additional research is required to determine the mechanism as to how this is occurring.

Pseudoperonospora humuli is the causal agent of downy mildew of hop, one of the most important diseases of this plant and a limiting factor for production of susceptible cultivars in certain environments. The degree of genetic diversity and population differentiation within and among P. humuli populations at multiple spatial scales was quantified using genotyping-by-sequencing to test the hypothesis that populations of P. humuli have limited genetic diversity but are differentiated at the scale of individual hop yards. Hierarchical sampling was conducted to collect isolates from three hop yards in Oregon, plants within these yards, and infected shoots within heavily diseased plants. Additional isolates also were collected broadly from other geographic regions and from the two previously described clades of the sister species, P. cubensis. Genotyping of these 240 isolates produced a final quality-filtered data set of 216 isolates possessing 25,227 variants. Plots of G’ST values indicated that the majority of variants had G’ST values near 0 and were scattered randomly across contig positions. However, there was a subset of variants that were highly differentiated (G’ST > 0.3) and reproducible when genotyped independently. Within P. humuli, there was evidence of genetic differentiation at the level of hop yards and plants within yards; 19.8% of the genetic variance was associated with differences among yards and 20.3% of the variance was associated with plants within the yard. Isolates of P. humuli were well differentiated from two isolates of P. cubensis representative of the two clades of this organism. There was strong evidence of linkage disequilibrium in variant loci, consistent with nonrandom assortment of alleles expected from inbreeding and/or asexual recombination. Mantel tests found evidence that the genetic distance between isolates collected from heavily diseased plants within a hop yard was associated with the physical distance of the plants from which the isolates were collected. The sum of the data presented here indicates that populations of P. humuli are consistent with a clonal or highly inbred genetic structure with a small, yet significant differentiation of populations among yards and plants within yards. Fine-scale genetic differentiation at the yard and plant scales may point to persistence of founder genotypes associated with planting material, and chronic, systemic infection of hop plants by P. humuli. More broadly, genotyping-by-sequencing appears to have sufficient resolution to identify rare variants that differentiate subpopulations within organisms with limited genetic variability.

Various hypotheses have been proposed regarding the origin of the plant pathogen Phytophthora cinnamomi. P. cinnamomi is a devastating, highly invasive soilborne pathogen associated with epidemics of agricultural, horticultural and forest plantations and native ecosystems worldwide. We conducted a phylogeographic analysis of populations of this pathogen sampled in Asia, Australia, Europe, southern and northern Africa, South America, and North America. Based on genotyping-by-sequencing, we observed the highest genotypic diversity in Taiwan and Vietnam, followed by Australia and South Africa. Mating type ratios were in equal proportions in Asia as expected for a sexual population. Simulations based on the index of association suggest a partially sexual, semi-clonal mode of reproduction for the Taiwanese and Vietnamese populations while populations outside of Asia are clonal. Ancestral area reconstruction provides new evidence supporting Taiwan as the ancestral area, given our sample, indicating that this region might be near or at the centre of origin for this pathogen as speculated previously. The Australian and South African populations appear to be a secondary centre of diversity following migration from Taiwan or Vietnam. Our work also identified two panglobal, clonal lineages PcG1-A2 and PcG2-A2 of A2 mating type found on all continents. Further surveys of natural forests across Southeast Asia are needed to definitively locate the actual centre of origin of this important plant pathogen. Agricultural Research Service, Grant/Award Number: 2072-22000-041-00-D; National Institute of Food and Agriculture, Grant/Award Number: 2011-68004-30154 and 2018-67013-27823 info:eu-repo/semantics/publishedVersion

Obligately biotrophic plant pathogens pose challenges in population genetic studies due to their genomic complexities and elaborate culturing requirements with limited biomass. Hop powdery mildew (

Hop powdery mildew (caused by

The plant pathogen implicated in the Irish potato famine, Phytophthora infestans, continues to reemerge globally. Understanding changes in the genome during emergence can provide insights useful for managing this pathogen. Previous work has relied on studying individuals from the United States, South America, Europe, and China reporting that these can occur as diploids, triploids, or tetraploids and are clonal. We studied variation in sexual populations at the pathogen’s center of origin, in Mexico, where it has been reported to reproduce sexually as well as within clonally reproducing, dominant clones from the United States and Europe. Our results newly show that sexual populations at the center of origin are diploid, whereas populations elsewhere are more variable and show genome-wide variation in gene copy number. We propose a model of evolution whereby new pathogen clones emerge predominantly by increasing the gene copy number genome-wide., The plant pathogen that caused the Irish potato famine, Phytophthora infestans, continues to reemerge globally. These modern epidemics are caused by clonally reproducing lineages. In contrast, a sexual mode of reproduction is observed at its center of origin in Mexico. We conducted a comparative genomic analysis of 47 high-coverage genomes to infer changes in genic copy number. We included samples from sexual populations at the center of origin as well as several dominant clonal lineages sampled worldwide. We conclude that sexual populations at the center of origin are diploid, as was the lineage that caused the famine, while modern clonal lineages showed increased copy number (3×). Copy number variation (CNV) was found genome-wide and did not to adhere to the two-speed genome hypothesis. Although previously reported, tetraploidy was not found in any of the genomes evaluated. We propose a model of dominant clone emergence supported by the epidemiological record (e.g., EU_13_A2, US-11, US-23) whereby a higher copy number provides fitness, leading to replacement of prior clonal lineages.

Powdery mildew, caused by

Conifers are the dominant plant species throughout the high latitude boreal forests as well as some lower latitude temperate forests of North America, Europe, and Asia. As such, they play an integral economic and ecological role across much of the world. This study focused on the characterization of needle transcriptomes from four ecologically important and understudied North American white pines within the Pinus subgenus Strobus. The populations of many Strobus species are challenged by native and introduced pathogens, native insects, and abiotic factors. RNA from the needles of western white pine (Pinus monticola), limber pine (Pinus flexilis), whitebark pine (Pinus albicaulis), and sugar pine (Pinus lambertiana) was sampled, Illumina short read sequenced, and de novo assembled. The assembled transcripts and their subsequent structural and functional annotations were processed through custom pipelines to contend with the challenges of non-model organism transcriptome validation. Orthologous gene family analysis of over 58,000 translated transcripts, implemented through Tribe-MCL, estimated the shared and unique gene space among the four species. This revealed 2025 conserved gene families, of which 408 were aligned to estimate levels of divergence and reveal patterns of selection. Specific candidate genes previously associated with drought tolerance and white pine blister rust resistance in conifers were investigated.

Genotyping by sequencing (GBS) is considered a powerful tool to discover single nucleotide polymorphisms (SNPs), which are useful to characterize closely related genomes of plant species and plant pathogens. We applied GBS to determine genome-wide variations in a panel of 187 isolates of three closely relatedAlternariaspp. that cause diseases on tomato and potato in North Carolina (NC) and Wisconsin (WI). To compare genetic variations, reads were mapped to bothA. alternataandA. solanidraft reference genomes and detected dramatic differences in SNPs among them. Comparison ofA. linariaeandA. solanipopulations by principal component analysis revealed the first (83.8% of variation) and second (8.0% of variation) components containedA. linariaefrom tomato in NC andA. solanifrom potato in WI, respectively, providing evidence of population structure. Genetic differentiation (Hedrick’s G’ST) inA. linariaepopulations from Haywood, Macon, and Madison counties in NC were little or no differentiated (G’ST0.0 - 0.2). However,A. linariaepopulation from Swain county appeared to be highly differentiated (G’ST> 0.8). To measure the strength of the linkage disequilibrium (LD), we also calculated the allelic association between pairs of loci. Lewontin’sD(measures the fraction of allelic variations) and physical distances provided evidence of linkage throughout the entire genome, consistent with the hypothesis of non-random association of alleles among loci. Our findings provide new insights into the understanding of clonal populations on a genome-wide scale and microevolutionary factors that might play an important role in population structure. Although we found limited genetic diversity, the threeAlternariaspp. studied here are genetically distinct and each species is preferentially associated with one host.

Verticillium wilt is the most important disease threatening the commercial production of mint grown for essential oil. An important long-term goal for mint breeders is the production of cultivars with resistance to Verticillium wilt. Before that can be accomplished, a better understanding of the genetic variation within and among populations of

The genus Phytophthora contains some of the most notorious plant pathogens affecting nursery crops. Given the recent emergence of the sudden oak death pathogen Phytophthora ramorum, particularly in association with Rhododendron spp., characterization of Phytophthora communities associated with this host in nursery environments is prudent. Many taxa may present symptoms similar to P. ramorum but we do not necessarily know their identity, frequency, and importance. Here, we present a survey of Phytophthora taxa observed from seven nurseries in the U.S. state of Oregon. Incidence and diversity of Phytophthora communities differed significantly among nurseries and among seasons within nursery. The taxa P. syringae and P. plurivora were widespread and detected at most of the nurseries sampled. Nine other taxa were also detected but were found either in a single nursery or were shared among only a few nurseries. Characterization of the Phytophthora communities present in nurseries is an important step toward understanding the ecology of these organisms as well as an aid to nursery managers in determining what risks may be present when symptomatic plants are observed. This study builds on an increasing literature, which characterizes Phytophthora community structure in nurseries.

Phytophthora infestans, the cause of the devastating late blight disease of potato and tomato, exhibits a clonal reproductive lifestyle in North America. Phenotypes such as fungicide sensitivity and host preference are conserved among individuals within clonal lineages, while substantial phenotypic differences can exist between lineages. Whole P. infestans genomes were aligned and single nucleotide polymorphisms (SNPs) identified as targets for the development of clonal-lineage-specific molecular diagnostic tools. Informative SNPs were used to develop high-resolution melt (HRM) assays and locked nucleic acid (LNA) probes to differentiate lineage US-23, the predominant lineage in the Eastern United States for the past several years, from three other U.S. lineages. Three different primer pairs targeting one to three SNPs were capable of separating lineage US-23 from lineages US-8, US-11, and US-24 using HRM analysis. A fourth HRM primer pair targeted a highly variable genomic region containing nine polymorphisms within 63 bp. These primers separated US-23, US-11, and US-8 plus US-24 into three separate groups following HRM analysis but did not separate US-8 from US-24. Additionally, two LNA probes were designed to target a portion of the P. infestans genome containing two SNPs diagnostic for US-23. A single multiplex quantitative polymerase chain reaction assay containing both differentially labeled LNA probes differentiated individuals belonging to lineage US-23 from those belonging to US-8, US-11, and US-24.

Background The interaction between oomycete plant pathogen Phytophthora sojae and soybean is characterized by the presence of avirulence (Avr) genes in P. sojae, which encode for effectors that trigger immune responses and resistance in soybean via corresponding resistance genes (Rps). A recent survey highlighted a rapid diversification of P. sojae Avr genes in soybean fields and the need to deploy new Rps genes. However, the full genetic diversity of P. sojae isolates remains complex and dynamic and is mostly characterized on the basis of phenotypic associations with differential soybean lines. Results We sequenced the genomes of 31 isolates of P. sojae, representing a large spectrum of the pathotypes found in soybean fields, and compared all the genetic variations associated with seven Avr genes (1a, 1b, 1c, 1d, 1k, 3a, 6) and how the derived haplotypes matched reported phenotypes in 217 interactions. We discovered new variants, copy number variations and some discrepancies with the virulence of previously described isolates with Avr genes, notably with Avr1b and Avr1c. In addition, genomic signatures revealed 11.5% potentially erroneous phenotypes. When these interactions were re-phenotyped, and the Avr genes re-sequenced over time and analyzed for expression, our results showed that genomic signatures alone accurately predicted 99.5% of the interactions. Conclusions This comprehensive genomic analysis of seven Avr genes of P. sojae in a population of 31 isolates highlights that genomic signatures can be used as accurate predictors of phenotypes for compatibility with Rps genes in soybean. Our findings also show that spontaneous mutations, often speculated as a source of aberrant phenotypes, did not occur within the confines of our experiments and further suggest that epigenesis or gene silencing do not account alone for previous discordance between genotypes and phenotypes. Furthermore, on the basis of newly identified virulence patterns within Avr1c, our results offer an explanation why Rps1c has failed more rapidly in the field than the reported information on virulence pathotypes. Electronic supplementary material The online version of this article (10.1186/s12915-018-0549-9) contains supplementary material, which is available to authorized users.

Inference of copy number variation presents a technical challenge because variant callers typically require the copy number of a genome or genomic region to be known a priori. Here we present a method to infer copy number that uses variant call format (VCF) data as input and is implemented in the R package vcfR. This method is based on the relative frequency of each allele (in both genic and non-genic regions) sequenced at heterozygous positions throughout a genome. These heterozygous positions are summarized by using arbitrarily sized windows of heterozygous positions, binning the allele frequencies, and selecting the bin with the greatest abundance of positions. This provides a non-parametric summary of the frequency that alleles were sequenced at. The method is applicable to organisms that have reference genomes that consist of full chromosomes or sub-chromosomal contigs. In contrast to other software designed to detect copy number variation, our method does not rely on an assumption of base ploidy, but instead infers it. We validated these approaches with the model system of Saccharomyces cerevisiae and applied it to the oomycete Phytophthora infestans, both known to vary in copy number. This functionality has been incorporated into the current release of the R package vcfR to provide modular and flexible methods to investigate copy number variation in genomic projects.

Podosphaera macularis, the causal agent of hop powdery mildew, is known to produce chasmothecia (formerly cleistothecia) in eastern North America and Europe. Ascocarps have not yet been reported from the Pacific Northwestern region of North America. Reasons for the apparent absence of chasmothecia in the Pacific Northwest were unknown. This study established that P. macularis is heterothallic and ascocarp ontogeny, maturation, dehiscence and ascospore infection proceed similarly to other powdery mildew fungi. Genome sequencing of a MAT1-1 isolate revealed the structure of the MAT1 locus and presence of MAT1-1-3, demonstrating further similarities to other powdery mildew fungi. PCR assays with primers designed from conserved domains of the MAT1 idiomorphs were developed to characterize the frequency of idiomorphs in populations of P. macularis. Amongst 317 samples of P. macularis collected during 2012 and 2013 from the Pacific Northwest only the MAT1-1 idiomorph was found. In contrast, among 56 samples from the eastern United States and Europe, MAT1-1 and MAT1-2 idiomorphs were detected at equivalent frequencies. At temperatures representative of late season conditions in the Pacific Northwest, chasmothecia formed readily when a Pacific Northwest MAT1-1 isolate was paired with a MAT1-2 isolate collected from outside the region. Although these findings do not encompass all climatic, geographic or temporal barriers that could inhibit the formation of chasmothecia, the current absence of the ascigerious stage of P. macularis in the Pacific Northwest could be explained by the absence of the MAT1-2 mating type idiomorph.

Population genetic analysis is a powerful tool to understand how pathogens emerge and adapt. However, determining the genetic structure of populations requires complex knowledge on a range of subtle skills that are often not explicitly stated in book chapters or review articles on population genetics. What is a good sampling strategy? How many isolates should I sample? How do I include positive and negative controls in my molecular assays? What marker system should I use? This review will attempt to address many of these practical questions that are often not readily answered from reading books or reviews on the topic, but emerge from discussions with colleagues and from practical experience. A further complication for microbial or pathogen populations is the frequent observation of clonality or partial clonality. Clonality invariably makes analyses of population data difficult because many assumptions underlying the theory from which analysis methods were derived are often violated. This review provides practical guidance on how to navigate through the complex web of data analyses of pathogens that may violate typical population genetics assumptions. We also provide resources and examples for analysis in the R programming environment.

Identifying and evaluating genetic diversity of culinary rhubarb (Rheum ×rhababarum) cultivars using morphological characteristics is challenging given the existence of synonyms and nomenclatural inconsistencies. Some cultivars with similar names are morphologically different, and seedlings may grow and become associated with the parental name. Morphological traits of one cultivar may vary when measured under different environmental conditions. Molecular markers are consistent for unique genotypes across environments and provide genetic fingerprints to assist in resolving identity issues. Microsatellite repeats, also called simple sequence repeats (SSRs), are commonly used for fingerprinting fruit and nut crops, but only 10 SSRs have previously been reported in rhubarb. The objectives of this study were to use short-read DNA sequences to develop new di-nucleotide-containing SSR markers for rhubarb and to determine if the markers were useful for cultivar identification. A total of 97 new SSR primer pairs were designed from the short-read DNA sequences. The amplification success rate of these SSRs was 77%, whereas polymorphism of those reached 76% in a test panel of four or eight rhubarb individuals. From the 57 potentially polymorphic primer pairs obtained, 25 SSRs were evaluated in 58 Rheum accessions preserved in the U.S. Department of Agriculture, National Plant Germplasm System. The primer pairs generated 314 fragments with an average of 12.6 fragments per pair. The clustering of many accessions in well-supported groups supported previous findings based on amplified fragment length polymorphisms (AFLPs). Cluster analysis, using the proportion of shared allele distance among the 25 SSRs, distinguished each of the 58 accessions including individuals that had similar names or the same name. Accessions that grouped in well-supported clusters previously belonged to similar clusters with high bootstrap support based on AFLP. In summary, our technique of mining short-read sequencing data was successful in identifying 97 di-nucleotide-containing SSR sequences. Of those tested, the 25 most polymorphic and easy-to-score primer pairs proved useful in fingerprinting rhubarb cultivars. We recommend the use of short-read sequencing for the development of SSR markers in the identification of horticultural crops.

Cordylanthus tenuis subsp. pallescens, Pallid Bird's-beak, is a rare plant of the Mount Shasta area of northern California. Recent reports of dozens of populations outside its limited expected range, observations of plants with morphology intermediate to other subspecies of C. tenuis, and populations that seemed to include individuals of more than one subspecies, raised questions about its rarity and taxonomic validity. Examining populations in the field suggested that many reported populations were misidentified because they were based on a single trait, often foliage color. The name C. t. subsp. pallescens should be restricted to populations in which all or most plants have the combination of traits expected of this taxon, including yellow-green foliage, four to eight flowers per cluster, and short, mostly non-glandular calyx hairs. In the context of other variation recognized at the subspecies level in C. tenuis, recognizing C. t. subsp. pallescens taxonomically is a reasonable choice, despite...

Summary Armillaria species display diverse ecological roles ranging from beneficial saprobe to virulent pathogen. Armillaria solidipes (formerly A. ostoyae), a causal agent of Armillaria root disease, is a virulent primary pathogen with a broad host range of woody plants across the Northern Hemisphere. This white-rot pathogen grows between trees as rhizomorphs and attacks sapwood as mycelial fans under the bark. Armillaria root disease is responsible for reduced forest productivity due to direct tree mortality and non-lethal infections that impact growth. Here, we characterize a transcriptome of a widespread, virulent genet (vegetative clone) of A. solidipes isolated from a mycelial fan on a natural grand fi r( Abies grandis) sapling in northern Idaho, USA. cDNA from polyA + -purified total RNA was sequenced using a singleend read approach on the Illumina GAIIx platform which generated 24 170 384 reads. A BLASTx search against the NCBI nr database using 39 943 de novo assembled contigs resulted in 24 442 sequences with significant hits (e-value < 1e 3 ), predominantly to fungi (85%). A filtered data set of 20 882 assembled transcripts that encoded putative homologous fungal proteins was created and used for all subsequent analyses. Signal P identified 10 668 putative signal peptides from these fungal transcripts, and 14 360 were annotated with gene ontology terms. Several sequences showed strong homology to annotated genes with functions in pathogenesis, specifically those involved in plant cell wall degradation and response to the post-infection host environment. This transcriptome contributes to the growing body of resources for studies on fungal pathogens of woody plants, and our results provide useful insights towards identifying specific genes with potential roles associated with pathogenesis and other metabolic functions.

Software to call single nucleotide polymorphisms or related genetic variants has converged on the variant call format (VCF) as the output format of choice. This has created a need for tools to work with VCF files. While an increasing number of software exists to read VCF data, many only extract the genotypes without including the data associated with each genotype that describes its quality. We created the R package vcfR to address this issue. We developed a VCF file exploration tool implemented in the R language because R provides an interactive experience and an environment that is commonly used for genetic data analysis. Functions to read and write VCF files into R as well as functions to extract portions of the data and to plot summary statistics of the data are implemented. VcfR further provides the ability to visualize how various parameterizations of the data affect the results. Additional tools are included to integrate sequence (FASTA) and annotation data (GFF) for visualization of genomic regions such as chromosomes. Conversion functions translate data from the vcfR data structure to formats used by other R genetics packages. Computationally intensive functions are implemented in C++ to improve performance. Use of these tools is intended to facilitate VCF data exploration, including intuitive methods for data quality control and easy export to other R packages for further analysis. VcfR thus provides essential, novel tools currently not available in R.

Software to call single-nucleotide polymorphisms or related genetic variants has converged on the variant call format (VCF) as the output format of choice. This has created a need for tools to work with VCF files. While an increasing number of software exists to read VCF data, many only extract the genotypes without including the data associated with each genotype that describes its quality. We created the r package vcfr to address this issue. We developed a VCF file exploration tool implemented in the r language because r provides an interactive experience and an environment that is commonly used for genetic data analysis. Functions to read and write VCF files into r as well as functions to extract portions of the data and to plot summary statistics of the data are implemented. vcfr further provides the ability to visualize how various parameterizations of the data affect the results. Additional tools are included to integrate sequence (fasta) and annotation data (GFF) for visualization of genomic regions such as chromosomes. Conversion functions translate data from the vcfr data structure to formats used by other r genetics packages. Computationally intensive functions are implemented in C++ to improve performance. Use of these tools is intended to facilitate VCF data exploration, including intuitive methods for data quality control and easy export to other r packages for further analysis. vcfr thus provides essential, novel tools currently not available in r.

Genotyping-by-sequencing (GBS) was performed on 257 Phytophthora infestans isolates belonging to four clonal lineages to study within-lineage diversity. The four lineages used in the study were US-8 (n = 28), US-11 (n = 27), US-23 (n = 166), and US-24 (n = 36), with isolates originating from 23 of the United States and Ontario, Canada. The majority of isolates were collected between 2010 and 2014 (94%), with the remaining isolates collected from 1994 to 2009, and 2015. Between 3,774 and 5,070 single-nucleotide polymorphisms (SNPs) were identified within each lineage and were used to investigate relationships among individuals. K-means hierarchical clustering revealed three clusters within lineage US-23, with US-23 isolates clustering more by collection year than by geographic origin. K-means hierarchical clustering did not reveal significant clustering within the smaller US-8, US-11, and US-24 data sets. Neighbor-joining (NJ) trees were also constructed for each lineage. All four NJ trees revealed evidence for pathogen dispersal and overwintering within regions, as well as long-distance pathogen transport across regions. In the US-23 NJ tree, grouping by year was more prominent than grouping by region, which indicates the importance of long-distance pathogen transport as a source of initial late blight inoculum. Our results support previous studies that found significant genetic diversity within clonal lineages of P. infestans and show that GBS offers sufficiently high resolution to detect sub-structuring within clonal populations.

Sedum section Gormania was restricted to Oregon, Nevada and California in the western United States. After extensive field work from 2011 to 2016, we revised 17 members of the group using floral and vegetative characters, resulting in the acceptance of four new taxa in California. A serpentine endemic from the mountains of western Tehama County was recognized as S. rubiginosum . It was separated from S. kiersteadiae by its dense rosettes, overlapping stem leaves and non-apiculate corolla. A serpentine endemic from low elevation canyons in Del Norte County was described as S. patens. It was distinguished from S. laxum by its white spreading petals and yellow anthers. A plant of high elevation, serpentine and non-serpentine sites in Siskiyou County was circumscribed as S. marmorense ; it differed from S. oregonense in its sepals and inflorescence with a thick granular waxy deposit, and leaves in dense rosettes . Sedum paradisum was segregated from S. obtusatum , raised to species level, and divided into two subspecies. Plants of the northern Sierra Nevada were newly defined as S. paradisum subsp. subroseum , separable with nodding young flowering shoots and a disjunct range in Butte, Plumas and Sierra counties. Sedum flavidum and Sedum eastwoodiae were removed from S. laxum sensu stricto , and raised to species rank, based on floral characters. We clarified the concept of S. obtusatum subsp. retusum , and restored it to the rank of species as S. sanhedrinum ; it was restricted to Glenn, Lake, Mendocino, and Tehama counties, California. Sedum flavidum and S. oregonense as defined here showed more morphological variation than previously understood. Finally, we remarked on hybridization and cleistogamy observed in the field.

Aims To design and validate a colorimetric loop-mediated isothermal amplification assay for rapid detection of Phytophthora infestans DNA. Methods and results Two sets of loop-mediated isothermal amplification (LAMP) primers were designed and evaluated for their sensitivity and specificity for P. infestans. ITSII primers targeted a portion of the internal transcribed spacer region of ribosomal DNA. These primers had a limit of detection of 2 pg P. infestans DNA and cross-reacted with the closely related species Phytophthora nicotianae. Rgn86_2 primers, designed to improve assay specificity, targeted a portion of a conserved hypothetical protein. These primers had a limit of detection of 200 pg P. infestans DNA and did not cross-react with P. nicotianae. The specificity of the Rgn86_2 assay was tested further using the closely related species P. andina, P. ipomoeae, P. mirabilis and P. phaseoli. Cross-reactions occurred with P. andina and P. mirabilis, but neither species occurs on tomato or potato. Both primer sets were able to detect P. infestans DNA extracted from tomato late blight leaf lesions. Conclusions Two colorimetric LAMP assays detected P. infestans DNA from pure cultures as well as infected leaf tissue. The ITSII primers had higher sensitivity, and the Rgn86_2 primers had higher specificity. Significance and impact of the study This is the first report of a LAMP assay for the detection of P. infestans, the causal organism of potato and tomato late blight. These assays have potential for immediate utility in plant disease research and diagnostic laboratories.

The U.S. Endangered Species Act (ESA) allows listing of subspecies and other groupings below the rank of species. This provides the U.S. Fish and Wildlife Service and the National Marine Fisheries Service with a means to target the most critical unit in need of conservation. Although roughly one-quarter of listed taxa are subspecies, these management agencies are hindered by uncertainties about taxonomic standards during listing or delisting activities. In a review of taxonomic publications and societies, we found few subspecies lists and none that stated standardized criteria for determining subspecific taxa. Lack of criteria is attributed to a centuries-old debate over species and subspecies concepts. Nevertheless, the critical need to resolve this debate for ESA listings led us to propose that minimal biological criteria to define disjunct subspecies (legally or taxonomically) should include the discreteness and significance criteria of distinct population segments (as defined under the ESA). Our subspecies criteria are in stark contrast to that proposed by supporters of the phylogenetic species concept and provide a clear distinction between species and subspecies. Efforts to eliminate or reduce ambiguity associated with subspecies-level classifications will assist with ESA listing decisions. Thus, we urge professional taxonomic societies to publish and periodically update peer-reviewed species and subspecies lists. This effort must be paralleled throughout the world for efficient taxonomic conservation to take place.

Knaus, B. J. (Oregon State University, Department of Botany and Plant Pathology, 2082 Cordley Hall, Corvallis, OR, 97331-2902, U.S.A.; knausb@science.oregonstate.edu), R. C. Cronn (USDA Forest Service Pacific Northwest Research Station, 3200 SW Jefferson Way, Corvallis, OR, 97331, U.S.A.; rcronn@fs.fed.us) & A. Liston (Oregon State University, Department of Botany and Plant Pathology, 2082 Cordley Hall, Corvallis, OR, 97331-2902, U.S.A.; listona@science.oregonstate.edu). Genetic characterization of three varieties of Astragalus lentiginosus (Fabaceae). Brittonia 57: 334–344. 2005.—Astragalus lentiginosus is a polymorphic species that occurs in geologically young habitats and whose varietal circumscription implies active morphological and genetic differentiation. In this preliminary study, we evaluate the potential of amplified fragment length polymorphism (AFLP) markers to resolve infraspecific taxa in three varieties of Astragalus lentiginosus. Distance-based principle coordinate and neighbor-jo...

Nursery plants are important vectors for plant pathogens. Understanding what pathogens occur in nurseries in different production stages can be useful to the development of integrated systems approaches. Four horticultural nurseries in Oregon were sampled every 2 months for 4 years to determine the identity and community structure of Phytophthora spp. associated with different sources and stages in the nursery production cycle. Plants, potting media, used containers, water, greenhouse soil, and container yard substrates were systematically sampled from propagation to the field. From 674 Phytophthora isolates recovered, 28 different species or taxa were identified. The most commonly isolated species from plants were Phytophthora plurivora (33%), P. cinnamomi (26%), P. syringae (19%), and P. citrophthora (11%). From soil and gravel substrates, P. plurivora accounted for 25% of the isolates, with P. taxon Pgchlamydo, P. cryptogea, and P. cinnamomi accounting for 18, 17, and 15%, respectively. Five species (P. plurivora, P. syringae, P. taxon Pgchlamydo, P. gonapodyides, and P. cryptogea) were found in all nurseries. The greatest diversity of taxa occurred in irrigation water reservoirs (20 taxa), with the majority of isolates belonging to internal transcribed spacer clade 6, typically including aquatic opportunists. Nurseries differed in composition of Phytophthora communities across years, seasons, and source within the nursery. These findings suggest likely contamination hazards and target critical control points for management of Phytophthora disease using a systems approach.

Emergent diseases are an ever-increasing threat to forests and forest ecosystems and necessitate the development of research tools for species that often may have few pre-existing resources. We sequenced the mRNA expressed by the sudden oak death pathogen Phytophthora ramorum and its most susceptible forest host, tanoak, within the same tissue at two time points after inoculation, and in uninfected tanoak controls. Using the P. ramorum genome to differentiate host and pathogen transcripts, we detected more than 850 P. ramorum transcripts at 5 days post-inoculation and a concurrent upregulation of host genes usually associated with pathogenicity. At 1 day, in contrast, we did not detect pathogen expression or significant enrichment of functional categories of host transcripts relative to controls, highlighting the importance of sequencing depth for in planta studies of host-pathogen interactions. This study highlights processes in molecular host-pathogen interactions in forest trees and provides a first reference transcriptome for tanoak, allowing the preliminary identification of disease-related genes in this study and facilitating future work for this and other members of the family Fagaceae.

SSR_pipeline is a flexible set of programs designed to efficiently identify simple sequence repeats (e.g., microsatellites) from paired-end high-throughput Illumina DNA sequencing data. The program suite contains 3 analysis modules along with a fourth control module that can automate analyses of large volumes of data. The modules are used to 1) identify the subset of paired-end sequences that pass Illumina quality standards, 2) align paired-end reads into a single composite DNA sequence, and 3) identify sequences that possess microsatellites (both simple and compound) conforming to user-specified parameters. The microsatellite search algorithm is extremely efficient, and we have used it to identify repeats with motifs from 2 to 25 bp in length. Each of the 3 analysis modules can also be used independently to provide greater flexibility or to work with FASTQ or FASTA files generated from other sequencing platforms (Roche 454, Ion Torrent, etc.). We demonstrate use of the program with data from the brine fly Ephydra packardi (Diptera: Ephydridae) and provide empirical timing benchmarks to illustrate program performance on a common desktop computer environment. We further show that the Illumina platform is capable of identifying large numbers of microsatellites, even when using unenriched sample libraries and a very small percentage of the sequencing capacity from a single DNA sequencing run. All modules from SSR_pipeline are implemented in the Python programming language and can therefore be used from nearly any computer operating system (Linux, Macintosh, and Windows).

Aspen (Populus tremuloides) is a temperate North American tree species with a geographical distribution more extensive than any other tree species on the continent. Because it is economically important for pulp and paper industries and ecologically important for its role as a foundation species in forest ecosystems, the decline of aspen in large portions of its range is of serious concern. The availability and annotation of the black cottonwood (Populus trichocarpa) genome enables a range of high throughput sequencing approaches that can be used to understand rangewide patterns of genetic variation, adaptation, and responses to environmental challenges in other Populus species, including aspen. Gene expression studies are particularly useful for understanding the molecular basis of ecological responses, but are limited by the availability of transcriptome data. We explored the aspen transcriptome through the use of high-throughput sequencing with two main goals: (1) characterization of the expressed portion of the P. tremuloides genome in leaves and (2) assessment of variation in gene expression among genets collected from distinct latitudes but reared in a common garden. We also report a large single nucleotide polymorphism dataset that provides the groundwork for future studies of aspen evolution and ecology, and we identify a set of differentially expressed genes across individuals and population boundaries for the leaf transcriptome of P. tremuloides.

 Premise of the study: Microsatellite primers were developed for the Pacifi c Northwest conifer, Chamaecyparis lawsoniana (Cupressaceae), to enhance effi ciencies in disease-resistance breeding and germplasm screening for gene conservation of this rare species.  Methods and Results: Using multiplexed massively parallel Illumina sequencing, we identifi ed over 300 000 microsatellitecontaining sequences from 2 million paired-end microreads. After stringent fi ltering and primer evaluation, we selected 11 primer pairs and used these to screen variation in four populations of C. lawsoniana. Loci show between three and 10 repeats per locus, with an average of eight. Screening of these markers in the North American relative Callitropsis nootkatensis demonstrated limited marker transferability, but these markers could have utility in Asian species of Chamaecyparis .  Conclusions: These microsatellite primers show high polymorphism and should provide a high level of individual discrimination for paternity analysis in defi ned pedigrees, and routine screening of wild variation in Chamaecyparis lawsoniana .

Conservation and management of natural populations requires accurate and inexpensive genotyping methods. Traditional microsatellite, or simple sequence repeat (SSR), marker analysis remains a popular genotyping method because of the comparatively low cost of marker development, ease of analysis and high power of genotype discrimination. With the availability of massively parallel sequencing (MPS), it is now possible to sequence microsatellite-enriched genomic libraries in multiplex pools. To test this approach, we prepared seven microsatellite-enriched, barcoded genomic libraries from diverse taxa (two conifer trees, five birds) and sequenced these on one lane of the Illumina Genome Analyzer using paired-end 80-bp reads. In this experiment, we screened 6.1 million sequences and identified 356 958 unique microreads that contained di- or trinucleotide microsatellites. Examination of four species shows that our conversion rate from raw sequences to polymorphic markers compares favourably to Sanger- and 454-based methods. The advantage of multiplexed MPS is that the staggering capacity of modern microread sequencing is spread across many libraries; this reduces sample preparation and sequencing costs to less than $400 (USD) per species. This price is sufficiently low that microsatellite libraries could be prepared and sequenced for all 1373 organisms listed as ‘threatened’ and ‘endangered’ in the United States for under $0.5 M (USD).

 Premise of the study : The study of infra-taxa has historically been considered the study of incipient species. Astragalus lentiginosus Douglas ex Hooker (Fabaceae) is the most taxonomically diverse species in the U. S. fl ora. To gain insight into the nature of infra-taxa, I ask whether an optimal number of groups exists within A. lentiginosus , whether the varieties represent discrete entities, and whether morphological diversity within A. lentiginosus can be explained by climate.  Methods : Morphometric measurement was made on herbarium specimens. Seasonality was inferred by fi sine waves to climatic data. Statistical clustering, ordination, and multiple regression analyses were employed to test hypotheses.  Key results : K -means clustering resulted in no clear optimal number of groups within the species. Principal components analysis demonstrated that taxa occupy contiguous regions of morphospace but do not form distinct clusters. Multiple regression demonstrated signifi cant relations among morphology and climatic parameters, which are generally stronger than geographic relations.  Conclusions : The bewildering amount of diversity contained within A. lentiginosus begs for decomposition, yet its clinal nature precludes it from division into discrete groups. As a species, A. lentiginosus exists as an array of populations in a delicate balance between homogenizing forces, such as gene fl ow and retention of ancestral characters, and diversifying forces, such as local adaptation and drift. The effect of these forces has become evident in the phenotype of A. lentiginosus , resulting in more infra-taxa than any other species in the U. S. fl ora, but has failed to fracture this group into distinct entities.

Background Science-based wildlife management relies on genetic information to infer population connectivity and identify conservation units. The most commonly used genetic marker for characterizing animal biodiversity and identifying maternal lineages is the mitochondrial genome. Mitochondrial genotyping figures prominently in conservation and management plans, with much of the attention focused on the non-coding displacement ("D") loop. We used massively parallel multiplexed sequencing to sequence complete mitochondrial genomes from 40 fishers, a threatened carnivore that possesses low mitogenomic diversity. This allowed us to test a key assumption of conservation genetics, specifically, that the D-loop accurately reflects genealogical relationships and variation of the larger mitochondrial genome. Results Overall mitogenomic divergence in fishers is exceedingly low, with 66 segregating sites and an average pairwise distance between genomes of 0.00088 across their aligned length (16,290 bp). Estimates of variation and genealogical relationships from the displacement (D) loop region (299 bp) are contradicted by the complete mitochondrial genome, as well as the protein coding fraction of the mitochondrial genome. The sources of this contradiction trace primarily to the near-absence of mutations marking the D-loop region of one of the most divergent lineages, and secondarily to independent (recurrent) mutations at two nucleotide position in the D-loop amplicon. Conclusions Our study has two important implications. First, inferred genealogical reconstructions based on the fisher D-loop region contradict inferences based on the entire mitogenome to the point that the populations of greatest conservation concern cannot be accurately resolved. Whole-genome analysis identifies Californian haplotypes from the northern-most populations as highly distinctive, with a significant excess of amino acid changes that may be indicative of molecular adaptation; D-loop sequences fail to identify this unique mitochondrial lineage. Second, the impact of recurrent mutation appears most acute in closely related haplotypes, due to the low level of evolutionary signal (unique mutations that mark lineages) relative to evolutionary noise (recurrent, shared mutation in unrelated haplotypes). For wildlife managers, this means that the populations of greatest conservation concern may be at the highest risk of being misidentified by D-loop haplotyping. This message is timely because it highlights the new opportunities for basing conservation decisions on more accurate genetic information.

Premise of the study: Microsatellite primers were developed for Nootka cypress (Callitropsis nootkatensis) to provide quantitative measures for gene conservation that can assist in guiding management decisions for a species experiencing climate-induced decline. Methods and Results: Using multiplexed massively parallel sequencing, we identified 136,785 microsatellite-containing sequences from 489,625 Illumina paired-end 80-bp reads. After stringent filtering, we selected 144 primer pairs and screened variation at these loci in five populations of C. nootkatensis. Loci show between three and 36 dinucleotide repeats per locus, with an average of 13. Screening of these markers in the Pacific Northwest relative Chamaecyparis lawsoniana demonstrated no marker transferability. This finding highlights the narrow taxonomic utility of microsatellite markers in Callitropsis. Conclusions: These microsatellites show high polymorphism and can be used for routine screening of natural variation in Callitropsis nootkatensis, and will be particularly helpful in identifying clones and inbred relatives at the stand-level.