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Department of Plant Pathology, Washington State. University, Pullman, Washington 99164-6430. Abstract: .... level fungal systematics (Pryor and Gilbertson 2000;. Kang et al. ..... while two others (A16.2 and P18.1) were less variable than the ...
Mycologia, 105(4), 2013, pp. 1077–1086. DOI: 10.3852/12-287 # 2013 by The Mycological Society of America, Lawrence, KS 66044-8897

Development of sequence characterized amplified genomic regions (SCAR) for fungal systematics: proof of principle using Alternaria, Ascochyta and Tilletia Jane E. Stewart1 Marion Andrew2 Xiaodong Bao3 Martin I. Chilvers4 Lori M. Carris Tobin L. Peever5

species (Burnett 2003, Freeman and Herron 2004). DNA sequence data have provided large numbers of variable, highly robust and reproducible characters that can be used for phylogenetic analyses and may serve to discriminate among rapidly evolving organisms where phenotypic traits are insufficiently variable or environmentally plastic (Taylor et al. 2000). In many instances, phylogenetic lineages are correlated to previously unknown or undetected morphological or physiological traits allowing the detection of cryptic species (Kaiser et al. 1997, Geiser et al. 1998). The protein-coding genes typically used for phylogenetic analyses of fungi are not sufficiently informative to address some systematic questions (Paavanen-Huhtala et al. 2000, Randig et al. 2002, Bailey et al. 2004, Lardner et al. 2005) and this is especially true for closely related taxa at or near the population level (Andrew et al. 2009; Pimentel et al. 2000a, b; Peever et al. 2004, 2007). In many fungal systematics studies and DNA barcoding efforts, the nuclear ribosomal internal transcribed spacer (ITS) region has been considered the de facto diagnostic genomic region for specieslevel differentiation (Bruns et al. 1991, Gardes and Bruns 1993, Hopple and Vilgalys 1994, Bruns et al. 1998, Skouboe et al. 2000, Martin and Rygiewicz 2005, O’Brien et al. 2005, Nilsson et al. 2009, Seifert 2009, Bellemain et al. 2010, Schoch et al. 2012). However, some fungal taxa with invariant ITS sequences can be considered distinct species based on other criteria, such as host specificity, lack of gene flow, deficits in post zygotic fitness etc. (Levy et al. 2001, Peever et al. 2007). Summerbell et al. (1999) showed that the ITS was not variable among species of Trichophyton, although these taxa could be distinguished easily based on morphological, physiological and epidemiological differences. Similarly ITS lacked variation among closely related species of Tilletia (Levy et al. 2001), even though some of these taxa generally can be distinguished on the basis of host specificity and disease phenotype (Pimentel et al. 2000a, b). Phylogenetic analyses of Tilletia spp. based on the second largest subunit of RNA polymerase II (RPB2), the nuclear large rDNA subunit and translation elongation factor 1-a (EF) sequence data produced more resolved tree topologies that generally correlated with host specificity (Castlebury et al. 2005; Carris et al. 2006, 2007; Bao et al. 2010). These studies suggest that choice of locus (or loci) is important in resolving

Department of Plant Pathology, Washington State University, Pullman, Washington 99164-6430

Abstract: SCARs were developed by cloning RAPDPCR amplicons into commercially available vectors, sequencing them and designing specific primers for PCR, direct sequencing and phylogenetic analysis. Eighteen to seventy percent of cloned RAPD-PCR amplicons were phylogenetically informative among closely related small-spored Alternaria spp., Ascochyta spp. and Tilletia spp., taxa that have been resistant to phylogenetic analysis with universally primed, protein-coding sequence data. Selected SCARs were sequenced for larger, population-scale samples of each taxon and demonstrated to be useful for phylogenetic inference. Variation observed in the cloned SCARs generally was higher than variation in nuclear ribosomal internal transcribed spacer (ITS) and several protein-coding sequences commonly used in lower level fungal systematics. Sequence data derived from SCARs will provide sufficient resolution to address lower level phylogenetic hypotheses in Alternaria, Ascochyta, Tilletia and possibly many other fungal groups and organisms. Key words: anonymous loci, Ascochyta blight, bunt fungi, Didymella, Phoma, phylogeny, population-level systematics, speciation INTRODUCTION Molecular data, primarily DNA sequence data, has become the gold standard for resolving evolutionary relationships among organisms and for delimiting Submitted 14 Aug 2012; accepted for publication 17 Dec 2012. 1 Present address: Horticultural Crops Research Laboratory, USDAARS, Corvallis, OR 97330. 2 Present address: Department of Ecology & Evolutionary Biology, University of Toronto, Mississauga, ON, L5L 1C6, Canada. 3 Present address: Center for Infectious Disease Dynamics, Pennsylvania State University, University Park, PA 16802. 4 Present address: Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, MI 48824. 5 Corresponding author. E-mail: [email protected]

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species trees but also demonstrate the need for additional polymorphic loci to estimate phylogenetic relationships at or near the population level. Molecular systematic studies of small-spored Alternaria spp. revealed little or no variation for the protein-coding genes b-tubulin, actin, calmodulin, 1,3,8-trihydroxynaphthalene reductase, EF, chitin synthase (CHS) and ribosomal and mitochondrial DNA sequence data commonly employed in lowerlevel fungal systematics (Pryor and Gilbertson 2000; Kang et al. 2002; Pryor and Bigelow 2003; Chou and Wu 2002; Peever et al. 2004, 2007). To date, the only protein-coding gene with sufficient variation to be useful in phylogenetic studies of small-spored Alternaria spp. has been an endopolygalacturonase gene (endoPG) that originally was cloned to study the function of this enzyme in fruit rot (Isshiki et al. 2001, Peever et al. 2004). Simmons and Freudenstein (2002) suggest that biased genome sampling may be an important issue when employing protein-coding loci for phylogenetics, given the limited number of loci for which universal primers are available. There is interest in the use of noncoding genomic regions for molecular phylogenetic analyses because they are potentially more informative, given a reduced degree of functional constraint and higher rate of neutral evolution (Small et al. 1998). For example, Cronn et al. (2002) used six anonymous nuclear loci to resolve the branching order among taxa in the plant genus Gossypium and to improve the relatively weak phylogenetic signal in trees inferred from ndhf (chloroplast NAD[P]H dehydrogenase) and ITS. Bailey et al. (2004) specifically argued for the use of SCARs to resolve phylogenetic uncertainties in the plant genus Leucaena. This approach has been demonstrated to be particularly useful for phylogenetic studies of small-spored Alternaria spp. where universally primed protein coding gene data are uniformly invariant (Peever et al. 2004, Andrew et al. 2009) and may be useful for other organisms such as nematodes (Zijlstra et al. 2000). Phylogenetic studies of Ascochyta spp. infecting cool-season food legumes found little to no ITS sequence variation among host-specific isolates infecting different host legumes (Hernandez-Bello et al. 2006, Peever et al. 2007). These taxa are morphologically indistinguishable but have distinct RAPD-PCR fingerprints and each formed a well supported monophyletic group in a phylogeny estimated from combined G3PD, EF and CHS data (Kaiser et al. 1997, Peever et al. 2007). Crosses among these closely related but host-specific taxa have revealed no evidence for intrinsic pre- or post-zygotic mating defects (Hernandez-Bello et al. 2006), arguing for

conspecificity as biological species. However, the progeny of these crosses were unable to infect either host infected by the parents, indicating extrinsic or ecological post-zygotic fitness defects. The availability of additional fast-evolving loci would greatly facilitate evolutionary studies of these and other closely related taxa infecting legumes. In an effort to identify loci with sufficient resolution to infer phylogenetic relationships among closely related small-spored Alternaria taxa, Peever et al. (2004) cloned and sequenced a large number of putatively noncoding genomic regions amplified by RAPD-PCR (Williams et al. 1990). Peever et al. (2004) identified several regions that had higher sequence variation compared to endoPG that were used to estimate phylogenies among small-spored Alternaria spp. This suggests that the use of SCARs may prove a highly efficient and inexpensive method for generating genomic sequences for phylogenetic and phylogeographic studies of other closely related fungi. The objective of this research was to test the hypothesis that SCARs could be effective in generating DNA sequence characters appropriate for phylogenetic analyses of a wider range of fungal taxa including both ascomycetes and basidiomycetes. We chose to compare such regions developed for two ascomycetes and a basidiomycete using Alternaria, Ascochyta and Tilletia as our model genera. Our results suggest that putatively noncoding genomic regions identified via RAPD-PCR (Williams et al. 1990), and possibly other anonymous marker techniques, are a quick and effective method of generating genome sequences that can be used to increase the resolution of phylogenies where whole genome sequences for multiple, closely related sets of taxa are not currently available. MATERIALS AND METHODS Fungal isolates and DNA extraction.— Representative isolates of each genus used for SCAR development and screening for variation are provided (TABLE I). For Ascochyta and Alternaria, single-conidial isolates were grown in 2-YEG medium (10 g dextrose and 2 g yeast extract per liter) on an orbital shaker at 100 rpm 4–6 d. DNA was extracted with a phenol/chloroform method (Peever et al. 1999). For Tilletia, cultures were derived from single, germinating teliospores on 1.5% water agar. Tilletia DNA was extracted from approximately 5 mg lyophilized mycelia scraped from M-19 agar plates (Trione 1964) with either the DNeasy Plant Mini Kit (QIAGEN, Valencia, California) or directly from 1–5 mg teliospores as described by Shi et al. (1996), modified to replace CTAB with poly-vinyl pyrrolidone (PVP40). DNA concentrations were estimated with a NanoDrop ND1000 spectrophotometer (Thermo Scientific, Wilmington, Delaware) and adjusted to 10–30 ng/mL in 10 mM Tris-HCl (pH 8.0) or sterile water.

STEWART ET AL.: DEVELOPMENT OF ANONYMOUS REGIONS TABLE I.

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Isolates of Alternaria, Ascochyta and Tilletia used for SCAR locus development and screening

Genus

Species

Isolate code

Collector

Reference

Alternaria

Alternaria citrimacularis Alternaria gaisen Alternaria limoniasperae Alternaria sp. Ascochyta fabae Ascochyta fabae Ascochyta lentis Ascochyta lentis Tilletia caries Tilletia contraversa Tilletia laevis

BC2-RLR-32s EGS 90-0512 BC2-RLR-1s EGS 39-192 AF1 AF4 AL1 AL2 WSP 72049 WSP 72108 WSP 72068

T.L. Peever E.G. Simmons T.L. Peever L.W. Timmer B. Vandenberg C. Bernier W.J. Kaiser W.J. Kaiser B. Goates L.M. Carris G. Murray

Peever et al. 2004 Peever et al. 2004 Peever et al. 2004 Peever et al. 2004 Peever et al. 2007 Peever et al. 2007 Peever et al. 2007 Peever et al. 2007 Bao et al. 2010 Bao et al. 2010 Bao et al. 2010

Ascochyta

Tilletia

RAPD-PCR, cloning, screening and primer design.—RAPDPCR (Williams et al. 1990) for Alternaria and Ascochyta was performed with 20 primers from each of Operon 10-mer kits OPA and OPB (Eurofins MWG Operon, Huntsville, Alabama). PCR conditions were as described by Peever et al. (2004) using a single primer per reaction. Four isolates of small-spored Alternaria spp., Al. gaisen (EGS 90-0512), Al. sp. (EGS 39-192), Al. limoniasperae (BC2-RLR-1s) and Al. citrimacularis (BC2-RLR-32s) (TABLE I), were used as screening isolates to detect RAPD-PCR polymorphisms and quantify sequence divergence. These isolates were selected based on Andrew et al. (2009) and Peever et al. (2004) to maximize genetic diversity among the described small-spored Alternaria taxa. RAPD-PCR (Williams et al. 1990) for Ascochyta similarly employed 20 primers from each of Operon kits OPA and OPB and PCR conditions as described by Peever et al. (2004) using a single primer per reaction. Two isolates each of As. fabae (isolates AF1 and AF4) and As. lentis (AL1 and AL2) (TABLE I) were selected as the screening taxa. These taxa are morphologically identical (Gossen et al. 1986) and closely related (Peever et al. 2007) but host specific (Kaiser et al. 1997, HernandezBello et al. 2006). RAPD-PCR for Tilletia used one isolate of T. caries (WSP 72049), one isolate of T. contraversa (WSP 72108) and one isolate of T. laevis (WSP 72068) (TABLE I), which were selected based on differences in morphology and geographic location (Bao 2010). Thirty primers were screened from Operon kits OPA, OPB, OPE and OPP using PCR conditions described by Peever et al. (2004) with one primer per reaction. For all genera, amplified products were visualized in 1.2% ethidium bromide-stained agarose gels. All visible and unambiguously scoreable fragments amplified were scored as present or absent among the screening isolates and approximate size recorded. Strategies for isolation and cloning of polymorphic RAPD-PCR amplicons varied slightly for each genus. For Alternaria, reactions that yielded polymorphic amplicons among the four screening isolates were cut from the gel, purified with QIAquick purification columns (QIAGEN), cloned into pGEM Easy Vector (Promega, Madison, Wisconsin) and transformed into One Shot TOP10 Competent Cells (Invitrogen, Carlsbad, California) following the manufacturer’s instructions. For Ascochyta and Tilletia, RAPD-PCR amplicons that

yielded polymorphic banding patterns among the screening isolates were cloned directly from the PCR, bypassing the gel purification step described above. Following cloning of amplicons from Ascochyta and Tilletia, clones with inserts were prescreened with restriction enzymes MspI, HaeIII and TaqI (New England BioLabs [NEB], Ipswich, Massachusetts) to identify polymorphic inserts before sequencing. Plasmids with different restriction digestion patterns were selected for sequencing. Plasmids were sequenced on one strand as described by Peever et al. (2004) using the universal primer M13F. Sequence reads were performed by the Laboratory for Biotechnology and Bioanalysis, School of Molecular Biosciences, Washington State University, or by the USDA-ARS Systematic Mycology and Microbiology Laboratory in Beltsville, Maryland, using Applied Biosystems model 373A Automated DNA Sequencing Systems (Life Technologies, Carlsbad, California). Specific SCAR primers were designed to each cloned insert using Primer 3 software (Rozen and Skaletsky 2000). Screening of SCAR loci.—Primers designed to the cloned inserts were used in PCR, the amplicons were directsequenced on both strands and consensus sequences obtained. Variation at each locus and percent divergence was assessed initially with the same set of screening isolates used to develop the SCAR loci (TABLE I). SCAR primers were designed with higher annealing temperatures compared to RAPD-PCR primers, and subsequent PCR protocols were altered to accommodate this change. For Alternaria, the standard PCR mix included 13 PCR buffer with 0.6 mM MgCl2 (with Ficoll and tartrazine; Idaho Technologies, Salt Lake City, Utah); 10 mM deoxyribonucleotide triphosphate (NEB); 0.2 m M each primer (TABLE II); 1 U Taq polymerase (NEB); 10–20 ng DNA template and water to a total volume of 25 mL per tube. Ascochyta and Tilletia PCR mixes contained 13 PCR buffer (NEB); 10 mM deoxyribonucleotide triphosphate (NEB); 0.2 mM each primer (TABLES III, IV); and 1 U Taq polymerase (NEB). All PCR was performed in a BioRad MyCycler thermal cycler (BioRad, Hercules, California) with cycling conditions of 95 C for 1 min, followed by 35 cycles of 95 C for 30s, 55 to 65 C (specific for each primer pair, TABLES II–IV) for 30 s and 72 C for 30 s. Cycling was

1080 TABLE II.

MYCOLOGIA Summary of variation in sequence-characterized amplified regions (SCARs) for Alternaria

SCAR locus

Primer sequencea

AA-SCAR-5

TGACCGTGACTCAGGTGAAC GGTGACCGTATCGGTACTAGTGATT CAAACGTCGGTGTCATAAACA CAAACGTCGGCAACTGTAGTG CAAACGTCGGGCACACAA CAAACGTCGGCTGGACAGT GATTCGCAGCAGGGAAACTA TCGCAGTAAGACACATTCTACG CAAACGTCGGCTGGACAGTTGGAC CAAACGTCGGGCACACAAGTGTG GGAGGGTGTTGAGGCAGAGT GGAGGGTGTTCAACAGCAAG

AA-SCAR-9 AA-SCAR-11 OPA10-2d OPA19-650 OPB15-2

Annealing temp (C) 59 59 59 59 59 59

Variation among screening isolatesb 281/712 38/712 15/717 3/717 21/653 1/653 32/691 10/691 22/609 1/609 20/596 10/596

(39.0%)T (5.3%)P (2.1%)T (0.4%)P (3.2%)T (0.2%)P (4.6%)T (1.4%)P (3.6%)T (0.2%)P (3.4%)T (1.6%)P

Variation among samplesc N/A N/A N/A 94/662 (14.2%)d N/A N/A

a

Forward and reverse primers in 59–39 orientation. Number of polymorphic sites/total sites (T) and number of parsimonious sites/total sites (P) and percent variation among screening isolates EGS 90-0512, EGS 39-192, BC2-RLR-1s and BC2-RLR-32s (Peever et al. 2004). c Number of polymorphic sites/total sites and percent variation among population sample of 141 isolates from diverse geographic locations and host associations (Andrew et al. 2009). d Andrew et al. (2009). b

followed by a final 5 min elongation cycle at 72 C. A temperature gradient was employed to identify the optimal annealing temperature for each locus. PCR amplicons were purified through QIAQuick Columns (QIAGEN) or with ExoSAP-IT (Affymetrix, Cleveland, Ohio), following the manufacturer’s directions. DNA concentrations were estimated visually in 1.5% ethidium-bromide-stained agarose gels by comparing band intensity to a quantitative 100 bp DNA ladder (NEB). Sequencing reactions were carried out with a standard mixture that contained 10 ng of DNA template, 480 nM primer, 4 mL of Big Dye Terminator Cycle Sequencing Ready Reaction Mix (Applied Biosystems, Foster City, California) and sterile distilled water to a total TABLE III.

Summary of variation in sequence characterized amplified regions (SCARs) for Ascochyta

SCAR locus OPA1.J3 OPA4.1J4 OPA1.E1 OPA1.R1 OPA1.T1 a

volume of 15 mL per tube. All sequencing reactions were performed in a BioRad MyCycler thermal cycler with 35 cycles of 96 C for 15 s, 50 C for 15 s and 60 C for 4 min. The sequencing products were purified using Performa gel filtration cartridges (EdgeBio, Gaithersburg, Maryland), following manufacturer’s directions, and dried at 60 C in a vacufuge before sequencing. All sequence reads were performed on an Applied Biosystems Model 373A automated DNA Sequencing System (Life Technologies, Grand Island, New York) in the Laboratory for Biotechnology and Bioanalysis, School of Molecular Biosciences, Washington State University. GenBank accession numbers for each cloned sequence are provided (SUPPLEMENTARY TABLE I).

Primer sequencea GCTCGCTCAAGTATTCCTAG TGTACTAGCGTATCTACTGC CTGGCATGCTCTTGATTTGAAC CAAGACGTTGATCAGTGCG TAAGGTGCCGAGCGAGTCAG CACTGGGTGTGTTGGGGAAC GGTGTTGTACAGCTCCAGTG CGACTGACGTGTAGATCTAG TCTCACTGTGTGTGGGTATG ACCTAACGCAGTATACCCAG

Annealing temp (C) 57 57 59 59 59

Variation among screening isolatesb 18/332 8/332 7/342 7/342 1/331 0/331 8/240 8/240 14/467 12/467

(5.4%)T (2.4%)P (2.0%)T (2.0%)P (0.3%)T (0.0%)P (3.3%)T (3.3%)P (3.0%)T (2.6%)P

Variation among species samplesc 19/328 (5.8%) 25/342 (7.3%) 6/336 (1.8%) 19/245 (7.8%) 38/471 (8.1%)

Forward and reverse primers in 59–39 orientation. Number of polymorphic sites/total sites (T) and number of parsimonious sites/total sites (P) and percent variation among two isolates of As. lentis (AL1 and AL2) and 2 isolates of As. fabae (AF1 and AF4). c Number of polymorphic sites/total sites and percent variation among one isolate each of As. fabae, As. lentis, As. pisi, As. viciae-villosae and Ascochyta sp. b

STEWART ET AL.: DEVELOPMENT OF ANONYMOUS REGIONS

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TABLE IV. Summary of variation in sequence characterized amplified regions (SCARs) for Tilletia SCAR locus A13.1 A16.2 P18.1

Primer sequencea GCAAGCGTGGGCCTCARTA TCAAGTAAGTACGGTAGAAGGGTGC AGGAGGGTTGTAGCAGCGAGCGA CCCCATTATTATTGCTGCTGACTG CGCCCTTTTCCTGGTAGTTCA GCGGAGCATCCCACCAACT

Annealing temp (C) 64 64 64

Variation among screening isolatesb 9/705 0/705 1/535 0/535 2/400 1/400

(1.3%)T (0.0%)P (0.2%)T (0.0%)P (0.5%)T (0.03%)P

Variation among species samplec 20/705 (2.8%) 6/414 (1.5%) 15/403 (3.7%)

a

Forward and reverse primers in 59–39 orientation. Number of polymorphic sites/total sites (T) and number of parsimonious sites/total sites (P) and percent variation estimated among one representative isolate each of Tilletia contraversa (WSP 72108), T. caries (WSP 72049), and T. laevis (WSP 72068). c Number of polymorphic sites/total sites and percent variation estimated among a sample of 75 isolates of T. contraversa (n 5 41), T. caries (n 5 20) and T. laevis (n 5 13) from diverse geographic locations. b

Sequence fragments for each locus were aligned in Clustal X 1.81 (Thompson et al. 1997) and further edited by eye. Sequence divergence, measured as the number of polymorphic sites including indels among the screening isolates, was compared to divergence in ITS and several protein-coding loci among the same screening isolates for each genus. This facilitated all pairwise comparisons between the most closely and most distantly related isolates in the screening set. Sequences for each SCAR locus was assessed for similarity to known protein coding genes with BLASTx query tool against the non-redundant protein database of the National Center for Biotechnology Information (Benson et al. 2007). BLAST expected (E) values less than e250 were considered significant.

screened against nine Ascochyta isolates comprising seven species, including As. fabae (isolates AF1 and AF4), As. lentis (isolates AL1 and AL2), As. pisi (isolate AP1), As. viciae-villosae (isolate AV11), As. pinodella (isolate PMP3), As. pinodes (isolate MP1) and an undescribed Ascochyta sp. from big flower vetch (Vicia grandiflora) (isolate Georgia3) (Peever et al. 2007). All three SCAR loci developed for Tilletia were screened against 75 isolates of T. caries (n 5 20) and T. laevis (n 5 13) from wheat (Triticum aestivum) and T. contraversa (n 5 41) from wheat and other grass hosts (Castlebury et al. 2005, Carris et al. 2007).

Sequencing of ITS and protein coding loci.—For all taxa three or more nuclear loci were sequenced for the same set of screening isolates used in SCAR marker development and polymorphism screening (TABLE I) to provide a direct comparison with the SCAR markers. A portion of the nuclear ribosomal internal transcribed spacer 1 (ITS) was sequenced on both strands for all three genera with universal primers (White et al. 1990). Alternaria screening isolates also were sequenced at the EF, endoPG, and G3PD loci with primers and methods described in Peever et al. (1999, 2007). For Ascochyta, EF, G3PD and CHS sequences were retrieved from GenBank for screening isolates AF1, AF4, AL1 and AL2 (TABLE I). Tilletia screening isolates T. contraversa (WSP 72108), T. caries (WSP 72049) and T. laevis (WSP 72068) were sequenced at the EF locus and a portion of the second largest subunit of RNA polymerase II (RPB2) (Carris et al. 2007). GenBank accession numbers are provided (SUPPLEMENTARY TABLE I).

Six of 19 SCARs highly polymorphic in Alternaria.— Nineteen SCARs were developed and screened for variation among the small-spored Alternaria tester isolates EGS 90-0512, EGS 39-192, BC2-RLR-1s and BC2-RLR-32s. Six SCARs (32%) had significant hits to demonstrated or putative proteins in the NCBI database. Thirteen of nineteen loci (68%) showed little or no variation among taxa and were deemed not useful for phylogenetic analyses of these taxa. The remaining six loci revealed 2–39.5% variation among the tester isolates (TABLE II, FIG. 1). AA-SCAR-5 was significantly more variable (39.5%) than the others, and as such its sequence was difficult to align and it proved difficult to successfully amplify this locus from all tester isolates. The remaining five SCAR loci (OPA 10-2, OPA 19-650, AA-SCAR-11, AA-SCAR-9, OPB 152) were easily aligned. All Alternaria SCARs were more variable than ITS (0%) and most were equally or more variable than the protein coding loci G3PD (1.1%), EF (2.6%) and endoPG (3.4%) (FIG. 1A). Four of the five SCAR loci displayed equivalent or slightly elevated polymorphism compared to the protein-coding endoPG locus previously used for phylogenetic study of these taxa (FIG. 1A; Peever et al. 2004). OPA10-2 and OPA15-2 were significantly

Suitability of SCARs for phylogenetic studies of closely related Alternaria, Ascochyta and Tilletia spp.—The phylogenetic utility of selected SCAR loci was determined by screening the SCAR primers against a wider range of taxa for all three genera. For Alternaria, SCAR locus OPA10-2 was screened against a population sample of 141 small-spored Alternaria isolates from diverse locations and host associations as described by Andrew et al. (2009) (TABLE II). All seven SCAR loci developed for Ascochyta (TABLE III) were

RESULTS

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MYCOLOGIA isolates, whereas three protein-coding loci demonstrated similar variation to the SCARs with 1.5, 4.3 and 2.1% for G3PD, EF and CHS respectively (FIG. 1B). Three of the SCARs, OPA4.1J4, OPA1.R1 and OPA.T1, revealed similar variation as the protein coding loci with approximately 2–5% polymorphism, while SCAR OPA1.J3 was more variable than the protein coding loci (FIG. 1B). BLASTx searches revealed that OPA1.E1 was 90% similar to the 18S small subunit ribosomal RNA from Cochliobolus kusanoi strain NBRC 100198 (E-value 5 1 e2163). The other four SCAR loci had no significant similarity to any entry in the NCBI protein database. The Ascochyta SCAR markers were phylogenetically informative among a wider collection of Ascochyta taxa comprising nine isolates and seven Ascochyta spp. Percent divergence was 1.8–8.1% (TABLE III).

FIG. 1. Comparison of sequence divergence (percent nucleotide difference) of nuclear ribosomal internal transcribed spacer (ITS), selected protein-coding loci and sequence characterized amplified region (SCAR) among three fungal taxa. Divergence at each locus was estimated using four screening isolates each of Alternaria (A), Ascochyta (B) and three screening isolates for Tilletia (C) (TABLE I). Percent divergence in ITS is in black, selected protein-coding loci in gray, and SCARs in white.

similar to a FAD binding-domain protein from Pyrenophora tritici-repentis (E-value 5 2 e272) and to P. tritici-repentis Pt-1C-BFP tubulin alpha chain mRNA (E-value 5 7 e259) respectively (TABLE II). Four of seven SCARs highly polymorphic in Ascochyta.—Seven SCARs were developed and screened for variation among Ascochyta tester isolates AF1, AF4, AL1 and AL2 representing two Ascochyta spp., As. fabae and As. lentis (TABLE III). Two of seven loci (29%) were invariant among taxa and were deemed not useful for differentiating the screening taxa. The remaining five loci (71%) were polymorphic, 0.3– 5.4% among the screening isolates (TABLE III). The ITS region was invariant among the screening

Three of fourteen SCARs highly polymorphic in Tilletia.— Seventeen SCARs were developed and screened for variation among the Tilletia tester isolates T. contraversa (WSP 72108), T. caries (WSP 72049) and T. laevis (WSP 72068) (TABLE I). Fourteen SCAR loci (82%) were invariant among taxa and three loci (18%) were variable among the three tester isolates (TABLE IV). Divergence among the three polymorphic loci was 0.2–1.3% (TABLE IV). One SCAR (A13.1) was slightly more variable than the ITS region while two others (A16.2 and P18.1) were less variable than the ITS (FIG. 1C). All three SCARs were less variable than the protein coding loci RPB2 and EF, loci which varied by 2.2 and 2.9% respectively (FIG. 1C). BLASTx queries of the Tilletia SCAR revealed no similarities to known proteins in the NCBI database. SCAR loci for Tilletia were phylogenetically informative when amplified and sequenced among a population sample of 75 total isolates from T. caries, T. contraversa and T. laevis with the most polymorphic SCAR marker displaying 3.7% variation (TABLE IV). DISCUSSION In the absence of sequenced genomes, SCAR markers offer an efficient, inexpensive and rapid method to interrogate a genome and identify rapidly evolving loci useful for phylogenetic inferences among closely related fungal taxa. The limited availability of universal primers for phylogenetically informative nuclear loci has made it difficult to resolve phylogenetic relationships among closely related organisms such as the taxa studied here (Summerbell et al. 1999; Levy et al. 2001; Peever et al. 2004, 2007). Our results highlight the utility of this method for the development of polymorphic loci for three genera of

STEWART ET AL.: DEVELOPMENT OF ANONYMOUS REGIONS important plant pathogens, Alternaria, Ascochyta and Tilletia. Although direct comparisons of SCAR variation among these genera are difficult due to differences in locus selection and relatedness among screening isolates, 18–70% of randomly selected clones revealed SCARs that were useful for phylogenetic analyses of these genera. Loci with higher levels of polymorphism were obtained for Alternaria compared to Ascochyta and Tilletia, which may have been a result of the initial gel purification step of polymorphic amplicons prior to cloning. However, direct cloning of RAPD-PCR amplicons either with or without gel visualization or prescreening with restriction enzymes resulted in a significant number of phylogenetically useful SCARs from Ascochyta and Tilletia spp. from a very minimal cloning and sequencing effort. All polymorphic SCARs developed for the three genera were as variable or more variable than the nuclear ribosomal ITS region and several proteincoding genes. The SCAR loci developed for Ascochyta and Alternaria had greater sequence variation than the generally invariant ITS locus and had similar or greater variation than several protein coding loci. Although the three SCAR loci developed for Tilletia showed levels of divergence comparable to the ITS among the three test isolates, these loci will be useful for analyses in conjunction with ITS data to produce a more reliable estimate of species relationships in Tilletia. The Tilletia isolates used in this study represented three morphospecies, but a growing body of data support the conspecificity of these morphospecies (Russell and Mills 1993, 1994; Carris and Castlebury 2008; Bao et al. 2010). Therefore the reduced level of variation seen in the Tilletia SCARs relative to the Alternaria and Ascochyta SCARs was likely due to the biased selection of a more closely related set of screening isolates compared to the ascomycete taxa and the observed divergence likely represents intraspecific rather than interspecific variability. An additional challenge for lower-level systematic research concerns differences in evolutionary histories among individual gene trees caused by lineage sorting and recombination, which complicates phylogenetic inference (Redecker and Raab 2006, Andrew et al. 2009, Bischoff et al. 2009). These differences result in incongruent phylogenies estimated from each locus (Felsenstein 2004, Degnan and Rosenberg 2009) and can be due to several processes (Maddison and Knowles 2006, Kubatko et al. 2009). For closely related species, the discovery of sufficient phylogenetic signal in a set of loci is inherently difficult because of the lack of variation associated with most protein-coding regions. Townsend (2007) suggests

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that when low data informativeness is paired with poor phylogenetic resolution, as is commonly observed in closely related taxa, more data is needed to resolve relationships. We have demonstrated that SCAR loci offer a straightforward method to address this issue by increasing the availability of variable loci that can be used to address resolution problems for closely related organisms. Nucleotide sequence data provided by SCARs provides a means of rapidly identifying genomic regions suitable for phylogenetic study or the design of taxon-specific markers (Nielsen et al. 2002). As whole genome sequences become increasingly available for fungi, the design of primers for multiple regions of the genome including non-coding loci will become routine. However, this will require comparative genomics analysis of multiple, closely related species, which are still unavailable for most taxa. For well studied taxa where such sequences are available, comparative genomic analysis can be used to target regions with the appropriate phylogenetic signal for the study at hand (Townsend 2007). Until such extensive genomic data is available, SCARs developed from RAPD-PCR or AFLP are a rapid and easy method for the development of sequence loci for fungi that lack extensive genomic resources. In summary, randomly amplified and cloned RAPD-PCR amplicons were used to develop sequence-characterized amplified regions (SCAR) for lower-level systematic study of three fungal taxa including two ascomycetes and one basidiomycete. Eighteen to seventy percent of cloned loci were sufficiently variable for phylogenetic analyses and most displayed no significant similarity to any known sequences in the databases. SCARs developed for Alternaria and Ascochyta were more variable than nuclear ribosomal internal transcribed spacer (ITS) sequences and as variable or more variable than several fast-evolving protein coding genes typically used in lower-level fungal systematics. SCARs developed for Tilletia displayed similar variation to ITS and were slightly less variable than two protein-coding loci. These loci were demonstrated to be useful for lower-level phylogenetic analyses of these taxa, and it is expected that similar loci can be easily discovered and developed for any fungal taxon using this approach. These loci should prove particularly useful for phylogenetic and phylogeographic analyses of closely related fungal taxa for which genome sequences are not yet available. ACKNOWLEDGMENTS The authors thank Drs E. Njambere, R. Attanayake, T. Drader and Ms. K.A. Thomas for excellent laboratory

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assistance. We acknowledge the Derek Pouchnik Laboratory for Biotechnology and Bioanalysis, Washington State University and Lisa Castlebury, USDA-ARS Systematic Mycology and Microbiology Lab in Beltsville, Maryland, for sequencing support. This work was partially supported by National Science Foundation grant DEB-0416314.

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