Downy mildew is a newly emergent disease of Impatiens walleriana Hook. f. (impatiens; Balsaminaceae), a high-value, flowering annual plant contributing $105 million annually to the horticulture industry in the United States alone. This destructive disease threatens the cultivation of impatiens worldwide (Brasier, 2008). In 2011, widespread outbreaks of impatiens downy mildew (IDM) disease were observed for the first time in the United States, affecting plants grown in greenhouses, nurseries, and landscapes (e.g., Wegulo et al., 2004; Baysal-Gurel et al., 2012; Palmateer et al., 2013; Crouch et al., 2014). Similar disease outbreaks have been reported through the United Kingdom, continental Europe, and Australia (e.g., Lane et al., 2005; Cunnington et al., 2008; Toppe et al., 2010). The causal agent of IDM, Plasmopara obducens (J. Schröt.) J. Schröt., is one of many obligate biotrophic plant pathogens in the Oomycota (Chromalveolata, Heterokontophyta) afflicting numerous economically important plants around the world (Kamoun et al., 2015). Impatiens infected with P. obducens are quickly defoliated, and death occurs within weeks of disease onset. Infected plants cannot be cured, and the pathogen might be capable of persisting in the soil from one season to the next.
Despite the global impact of IDM on cultivated impatiens, there is currently no information about pathogen population structure or the factors that led to the epidemics, delaying the development of effective mitigation strategies (Plantegenest et al., 2007). Downy mildew pathogens engage in classic gene-for-gene interactions with their hosts during the infection process, producing fast-evolving elicitor molecules that in turn give rise to diverse physiological races varying in their ability to infect a given plant (e.g., Lebeda and Cohen, 2011). As such, knowledge of pathogen variability provides key information required to develop durable host disease resistance. In this study, we developed 37 simple sequence repeat (SSR) markers from the genome of P. obducens to support investigations of population diversity, and demonstrate the utility of these markers in a sample of 96 P. obducens collected throughout the United States.
METHODS AND RESULTS
Genomic DNA from P. obducens sample H12.14-11 (Appendix 1) was extracted from a sporangial mass using the OmniPrep DNA Kit (G-Biosciences, St. Louis, Missouri, USA) following manufacturer's instructions, then purified using the Zymo DNA Clean and Concentrator kit (Zymo Research, Irvine, California, USA). DNA was sheared to 600 bp using the Covaris M220 ultrasonicator (Covaris, Woburn, Massachusetts, USA), and then used to construct a library with the Illumina TruSeq DNA LT Sample Prep kit (Illumina, San Diego, California, USA). Library sequencing was conducted on an Illumina MiSeq instrument (Illumina) using 600-cycle sequencing cartridges, run as 2 × 300-bp paired-end reads. Reads were processed using CLC Genomics Work-bench version 7.5.1 (CLC Bio, Boston, Massachusetts, USA), and a de novo assembly was performed after removal of adapters, indices, bases with low-quality scores (limit = 0.05), and runs of ambiguous bases >2 bp. The assembly measured 202 Mb, contained in 137,754 scaffolds (N50 = 1486), with an average depth of coverage of 120.76×.
Characteristics of the 37 novel genomic SSR loci developed for Plasmopara obducens.
Using PrimerPro version 1.0 ( http://webdocs.cs.ualberta.ca/~yifeng/primerpro/), the P. obducens H12.14-11 genome assembly was mined for SSR motifs, screened for optimal PCR primer pairs, and BLASTN searched to ensure unique priming sites. Motif size search ranged from mono- to tridecanucleotides, with minimum repeat units set as follows: mononucleotides ≥10; di-, tri-, tetra-, penta-, and hexanucleotides ≥5; the remaining repeat motifs ≥5. The genome assembly contained 13,483 SSR motifs. Dinucleotide repeats were the most abundant class, followed by mononucleotides and trinucleotides. SSRs averaged 17.8 bp in length, with 78% smaller than 21 bp. Repeats averaged 7.4 ± 4.15 units/SSR. SSR relative abundance (# SSRs/genome size [Mb]) was 66.9, and SSR density (combined length of SSRs [bp]/genome size [Mb]) was 1083.5.
From the set of candidate SSR loci suitable for marker development (e.g., those found as a single copy in the genome assembly, with repeat units of trinucleotide or greater, and unique priming sites), we identified loci that were heterozygous in the genome assembly of H12.14-11 by performing probabilistic variant detection using CLC Genomics, then visually inspecting candidate regions. Because P. obducens is an obligate biotroph and the H12.14-11 sporangial sample was collected directly from the surface of the host plant, candidate markers were further assessed by performing BLAST searches of the National Center for Biotechnology Information (NCBI) GenBank nonredundant (nr) database to ensure the sequences were not derived from the plant host or other environmental components. This filtering yielded 189 primer sets, from which 62 primer sets were tested for amplification using DNA extracted from P. obducens sporangial sample PA1-1 (Appendix 1). Twenty-five primer sets were discarded due to lack of amplification, or the production of stutter and/or multiple bands. The 37 remaining markers represented a wide variety of repeat motif and length (Table 1) and were located on 37 different contigs. All but three of the markers contained trinucleotide motifs. When tested on I. walleriana DNA, none of the markers produced an amplicon. The 37 microsatellite loci were used to perform BLAST searches against the NCBI GenBank database to determine putative functions, as summarized in Table 1. Sequence contigs containing microsatellite loci shared homology to predicted proteins of different oomycete plant pathogens (Table 1).
A total of 96 P. obducens samples collected between 2012 and 2014 from I. walleriana (n = 73) and from four additional Impatiens species (n = 23) at different localities in the United States were used for marker validation (Appendix 1). DNA was extracted from leaves visibly afflicted with downy mildew using the DNeasy Plant Kit (QIAGEN, Germantown, Maryland, USA). PCR amplifications were performed as described (Schuelke, 2000) in 10-µL volumes: 6.5 µL of 2× Mango Mix (Bioline Inc., Tauton, Massachusetts, USA), 1 µL of DNA (2–10 ng/µL), 7 µM of forward primer with 5′ M13 tail, 13 µM of reverse primer, 7 µM of dye-labeled M13 (FAM, PET, VIC, NED), and 25 mM of MgCl2. Fragment sizing was performed by adding 1 µL amplicon to 9 µL of Hi-Di Formamide (Applied Biosystems, Carlsbad, California, USA) containing GeneScan 500 LIZ Size Standard (Applied Biosystems), denaturing at 95°C for 2 min, then injecting onto an ABI 3730x1 DNA Analyzer (Applied Biosystems). Results were analyzed using GeneMarker version 2.6.3 (SoftGenetics, State College, Pennsylvania, USA); GenAlEx version 6.5 (Peakall and Smouse, 2012) was used to generate summary statistics. Allele frequencies were used to calculate polymorphism information content (PIC; Botstein et al., 1980).
Only three of the SSR markers (Pob3197, Pob7989, and Pob10169) were monomorphic across the 96 P. obducens samples. Marker Pob10169 could be amplified from just 8% of the P. obducens samples; therefore, the monomorphic data might be an artifact of the small sample size. The 34 polymorphic markers displayed 2–6 alleles, for a total of 104 alleles (Table 1). Observed heterozygosity ranged from 0.000–0.892 (mean = 0.355), while expected heterozygosity ranged from 0.023–0.746 (mean = 0.354) from polymorphic loci. The PIC ranged from 0.022–0.746 (mean = 0.354), with 18 of the markers moderately informative (PIC > 0.40) and one marker highly informative (PIC > 0.70; Pob11700). Analysis in GenClone version 2.0 ( http://www.ccmar.ualg.pt/maree/software.php?soft=genclon) showed that just 17 of the 37 SSR markers (45.9%) were sufficient to identify all multilocus genotypes.
The oomycete P. obducens is one of many obligate biotrophic plant pathogens currently impacting the health of economically important plants worldwide. The SSR markers developed here are the first molecular resource available for P. obducens. The high level of polymorphism present in these markers will enhance efforts to monitor pathogen population genetic structure and diversity over time, trace source populations, and understand the role of pathogen physiological races on host susceptibility.
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Plasmopara obducens samples collected from Impatiens and used to screen microsatellite markers developed in this study. Voucher specimens corresponding to the samples used in this study were deposited in the U.S. National Fungus Collections (Herbarium BPI), Beltsville, Maryland, USA.
 Funding was provided by the 2013–2015 U.S. Department of Agriculture–Animal and Plant Health Inspection Service (USDA-APHIS) Farm Bill 10201 and 10007 Programs and USDA–Agricultural Research Service (USDA-ARS); D.V. is supported through the USDA-ARS Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the USDA, managed under DOE contract number DE-AC05-06OR23100. We are grateful to Ed Ismaiel for technical assistance and Sonja Sheffer and Matt Lewis for the use of the ABI 3730xl instrument. All opinions expressed in this paper are the author's and do not necessarily reflect the policies and views of USDA, ARS, DOE, or Oak Ridge Associated Universities (ORAU)/ORISE. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. USDA is an equal opportunity provider and employer.