Brian J. Knaus

Genetic Variation within Clonal Lineages of Phytophthora infestans Revealed through Genotyping-By-Sequencing, and Implications for Late Blight Epidemiology

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.

Diversity of Foliar Phytophthora Species on Rhododendron in Oregon Nurseries

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.

Genomic Analyses of Dominant U.S. Clonal Lineages of Phytophthora infestans Reveals a Shared Common Ancestry for Clonal Lineages US11 and US18 and a Lack of Recently Shared Ancestry Among All Other U.S. Lineages

Populations of the potato and tomato late-blight pathogen Phytophthora infestans are well known for emerging as novel clonal lineages. These successions of dominant clones have historically been named US1 through US24, in order of appearance, since their first characterization using molecular markers. Hypothetically, these lineages can emerge through divergence from other U.S. lineages, recombination among lineages, or as novel, independent lineages originating outside the United States. We tested for the presence of phylogenetic relationships among U.S. lineages using a population of 31 whole-genome sequences, including dominant U.S. clonal lineages as well as available samples from global populations. We analyzed ancestry of the whole mitochondrial genome and samples of nuclear loci, including supercontigs 1.1 and 1.5 as well as several previously characterized coding regions. We found support for a shared ancestry among lineages US11 and US18 from the mitochondrial genome as well as from one nuclear haplotype on each supercontig analyzed. The other nuclear haplotype from each sample assorted independently, indicating an independent ancestry. We found no support for emergence of any other of the U.S. lineages from a common ancestor shared with the other U.S. lineages. Each of the U.S. clonal lineages fit a model where populations of new clonal lineages emerge via migration from a source population that is sexual in nature and potentially located in central Mexico or elsewhere. This work provides novel insights into patterns of emergence of clonal lineages in plant pathogen genomes.

Comparative transcriptomics among four white pine species

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.

Inferring Variation in Copy Number Using High Throughput Sequencing Data in R

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.

Grunwaldlab/Poppr: Release Candidate For Poppr Version 2.5.0

poppr 2.5.0 release candidatenThis is a pre-release of poppr version 2.5.0.nIt contains a major bug fix for those who deal with polyploid data and Bruvos distance with either genome addition or genome loss corrections (details below). We have ensured that all the new functionality is tested accurately, but nevertheless, if you run into any major bugs or otherwise unexpected results, please open an issue at https://github.com/grunwaldlab/poppr/issues nAttached are the windows (poppr_2.5.0.zip) and OSX (poppr_2.5.0.tgz) binaries. You can install them by downloading them and using:n install.packages(path/to/poppr_2.5.0.tgz, repos = NULL)n nor you can use devtools (Requires a C compiler):n if (!require(devtools) install.packages(devtools, repos = https://cran.rstudio.com)ndevtools::install_github(grunwaldlab/poppr@v.2.5.0rc)n nnALGORITHMIC CHANGEnnIdentified in https://github.com/grunwaldlab/poppr/issues/139, Bruvos ndistance will now consider all possible combinations of ordered alleles in thencalculation under the genome addition and loss models for missing data. Thisnwill affect those who have polyploid data that contain more than one missing nallele at any genotypenTo facilitate comparison, the global option old.bruvo.model, has been created.nBy default it is set to FALSE, indicating that poppr should use the ordered nallele combinations. If the user wants to use the method considering unorderd nallele combinations, they can set options(old.bruvo.model = TRUE)nIt must be repeated that this does not affect haploid or diploid comparisons, nthose that use the infinite alleles model, or those who do not have more than none missing allele at any genotype.nnnDEPRECATIONnnThe warning for a short repeat length vector for Bruvos distance is ndeprecated and will become an error in the futurennMISCnnThe internal plotting function for mlg.table now uses tidy evaluation forndplyr versions > 0.5.0nThe package reshape2 was removed from imports and replaced with base functionsn(see https://github.com/grunwaldlab/poppr/issues/144 for details)nnNEW IMPORTSnnDue to the migration to dplyr version 0.7.0, poppr now imports the !!noperator from the rlang package

Identification and Taxonomic Status of Cordylanthus tenuis SUBSP. Pallescens (Orobanchaceae)

Abstract Cordylanthus tenuis subsp. pallescens, Pallid Birds-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 its very limited range.