INTRODUCTION

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Sphaeropsis sapinea is a fungal endophyte of Pinus spp. that is associated with symptomless infections and that was introduced into the Southern Hemisphere along with its hosts. Predisposition due to a variety of biotic and abiotic stress factors can result in this normally benign fungus causing substantial deaths in exotic pine plantations (12, 29). In South Africa, for example, significant economic losses in pine plantations occur due to shoot and crown dieback after hail (29, 35). S. sapinea is thought to reproduce solely by asexual mitospores, as no known sexual stage has ever been found in this well-studied fungus. Phylogenetic studies based on internal transcribed spacer sequence data group this fungus with species of Botryosphaeria that have Sphaeropsis anamorphs, most closely related to Botryosphaeria obtusa (11).

In the mid 1980s, two morphotypes (A and B) of S. sapinea from the United States were described, based on spore morphology and culture characteristics (19). This division was confirmed using randomly amplified polymorphic DNA (RAPD) markers (27). Two recent studies have provided evidence for a third and possibly a fourth morphotype. De Wet et al. (5) used the RAPD markers and morphological characters and showed the existence of a C morphotype of S. sapinea among isolates from Indonesia that had spores larger than those of the A morphotype. Likewise, restriction fragment length polymorphism (RFLP) fingerprinting of ribosomal DNA (DNA) coupled with morphological characters led Hausner et al. (9) to propose an I morphotype among Canadian isolates that had spores intermediate in size between those of the A and B morphotypes. RAPD analysis of New Zealand isolates of S. sapinea has also indicated high genomic variability and possibly the existence of more than just the A and B morphotypes (S. J. Kay, R. L. Farrell, D. Hofstra, T. Harrington, S. Duncan, A. Ah Chee, E. Hadar, Y. Hadav, and R. Blanchette, APPS, 12th Biennial Conf. Asia-Pacific Plant Pathol. New Millennium, p. 348).

RAPD-PCR uses short primers (10 bp) and low annealing temperatures to produce differential banding patterns between individuals, usually due to mutations at the primer binding sites. This results in dominant markers (presence or absence of a band) and occasionally codominant length polymorphism markers (23). With diploid organisms, dominant markers can be a problem because homozygous and heterozygous alleles cannot be distinguished, which results in biased gene diversity estimates (10). Many fungi are haploid, and distinguishing homozygous and heterozygous alleles is not necessary; however, some isolates will have null alleles, and this makes analysis difficult. In addition, the low annealing temperatures used for RAPD-PCR often cause problems with repeatability and reproducibility in other laboratories (2).

Codominant markers are powerful tools for genetic analysis of populations. RFLP analysis, which involves cutting genomic DNA with restriction enzymes to produce a complex DNA profile, does produce codominant markers, but it requires large quantities of DNA (23). RFLP analysis following the amplification of a known region of genomic DNA, such as the IGS region of stet rDNA (9), requires less DNA than RFLP analysis of complete genomic DNA (15). However, as only a few bands are produced, this technique can be used to identify isolates but is not suitable for population studies.

Techniques such as sequence characterized amplified regions PCR and simple sequence repeat (SSR), or microsatellite, PCR, where known DNA sequences are amplified, provide codominant Mendelian markers. These are much more powerful than dominant markers and can be used to determine population genetic structure, kinship, reproductive mode, and genetic isolation (21, 33). SSR markers are usually found by probing partial or enriched genomic libraries with di- or trinucleotide repeats (18). However, polymorphic markers, often rich in microsatellite repeats, have been developed by sequencing fragments amplified by RAPD-PCR (3, 4, 6) and inter-SSR (ISSR) PCR (2, 6, 34). During the last decade, SSR markers have been extensively used in population studies of many plants and animals (31), although to date there have been few studies of fungi (16). Fungal studies have generally used only a few markers, predominantly for genotyping (7, 8, 14, 17). Recently, however, Epichloë endophytes have been identified in planta using 11 polymorphic microsatellite markers (16).

The aim of this study was to develop polymorphic SSR markers for the identification of different S. sapinea morphotypes which could also ultimately be used in population studies. The robustness of the markers was tested on the previously described morphotypes. Polymorphic alleles at each locus were sequenced to establish the specific base changes causing the length polymorphisms and also to determine whether alleles were identical in length through mutation or by descent.

	MATERIALS AND METHODS

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Fungal cultures. Forty single conidial isolates of S. sapinea were used in this study: 13 of the A morphotype, 14 of the B morphotype, 11 of the C morphotype and 2 of the I morphotype (4, 9, 19, 27) (Table 1). A morphotype isolates were from the United States, Australia, New Zealand, and South Africa. B morphotype isolates were from the United States and Mexico. Isolates from the United States included CMW 190 and 189, first described as representative of the A and B morphotypes, respectively, by Palmer et al. (19). Many other isolates had been characterized in previous studies (Table 1) (4, 9, 19, 27). C morphotype isolates were collected from four plantations within 20 km of each other in Indonesia (4). I morphotype isolates were from Canada (9). Two isolates of a closely related species, B. obtusa, were included for comparison. The isolates were maintained on malt extract agar and are all stored in the culture collection at the Forestry and Agriculture Biotechnology Institute, University of Pretoria.

DNA extraction. A small plug (4 mm²) of actively growing mycelium from the edge of 7-day-old cultures was transferred to Eppendorf tubes containing 0.5 ml of malt extract broth. The tubes were inverted and incubated at 25°C for 3 days. The tubes were then centrifuged, the broth was removed, and the mycelium was freeze-dried and stored at 20°C until it was required. The mycelium (approximately 10 mg) was ground to a fine powder with a pestle in the same Eppendorf tube, using liquid nitrogen to keep the mycelium frozen, and DNA was extracted as previously described (22). The DNA concentration was estimated by comparing the intensity of ethidium bromide fluorescence of the DNA sample to a known concentration of lambda DNA marker on agarose gels using a UV transilluminator imaging system (UVP, Cambridge, United Kingdom).

Development of SSR markers. ISSR-PCR of S. sapinea isolate CMW 5977 (belonging to the A morphotype) was conducted with seven primers, 5'DDB(CCA)₅, 5'DHB(CGA)₅, 5'YHY(GT)₅G, 5'HVH(GTG)₅, 5'NDB(CA)₇C, 5'NDV(CT)₈, and 5'HBDB(GACA)₄, as previously described (34) except that an annealing temperature of 49°C was used for all of the primers. The amplification products from each ISSR primer were purified with the High Pure PCR product purification kit (Roche Diagnostics, Mannheim, Germany). The purified products were ligated overnight at 10°C into the pGEM-T vector using the pGEM-T Easy Vector System (Promega, Madison, Wis.). The ligation products were transformed into competent Escherichia coli JM109 (Promega) and screened on Luria-Bertani medium containing 80 µg of X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside) ml¹, 0.5 mM IPTG (isopropyl--D-thiogalactopyranoside), and 100 µg of ampicillin ml¹. Positive clones were grown overnight in Luria-Bertani medium containing 100 µg of ampicillin ml¹, and the plasmids were recovered by alkaline lysis (25). Plasmid DNA was digested with EcoRI to release the insert and determine its size. Inserts were sequenced with the BigDye terminator cycle sequencing kit (Perkin-Elmer Applied Biosystems) using the T7 and Sp6 universal primers. The products were separated by polyacrylamide gel electrophoresis (PAGE) on an ABI Prism 377 DNA sequencer (Perkin-Elmer Corp.). Electropherograms were analyzed using Sequence Navigator software (Perkin-Elmer Corp.).

Genome walking. For some sequences, the microsatellite region of interest was at the beginning or end of the insert in the region recognized by the ISSR primer. In order to obtain the full repeat sequence, genome walking was performed using a modification of the method previously described (26). Genomic DNA (1.2 µg) of isolate CMW 5977 was digested in separate tubes with 10 U of one of three blunt-ended restriction enzymes (HaeIII, EcoRV, and ScaI). The cut DNA was extracted as described above, and the adapter DNA (Fig. 1) was then ligated to each restriction digest, creating three libraries (26). Each library was used as a template in primary PCRs, using a gene-specific primer from the sequence of interest and the first adapter-specific primer, AP1 (Fig. 1). The PCR mixture contained 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂, 50 mM KCl, 50 µM (each) deoxynucleoside triphosphate, 300 nM (each) primer, 2 ng of DNA, 5 U of Taq DNA polymerase, and water to a final volume of 50 µl. The reactions were carried out in an Eppendorf (Hamburg, Germany) thermocycler programmed for an initial denaturization of 1 min at 95°C, followed by 35 cycles of 30 s at 95°C and 6 min at 68°C, and a final extension of 15 min at 68°C. The lower strand of the adapter has an amine group that blocks polymerase extension, preventing the generation of the primer binding site, unless a defined distal gene-specific primer extends a DNA strand opposite the upper strand of the adapter.

View larger version (6K): [in this window] [in a new window]   FIG. 1.   Alignment of adapter and primer sequences used in genome walking as given by Siebert et al. (26).

PCR amplification of SSR loci. Specific primers were designed to flank microsatellite-rich regions. Particular care was taken to identify primer pairs that would amplify different-size fragments. This allows multiplexing of more than one reaction per lane during analysis of the fragments. Twenty-two primer pairs were constructed to amplify microsatellite-rich regions or SSRs in S. sapinea. Primer pairs were designed with a melting temperature between 58 and 66°C (Table 2). SSR-PCR was conducted with a PCR mixture containing 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂, 50 mM KCl, 100 µM (each) deoxynucleoside triphosphate, 300 nM (each) primer, 2 ng of DNA template, 0.5 U of Taq DNA polymerase, and water to a final volume of 25 or 50 µl. The reactions were carried out in either a HYBAID (Teddington, United Kingdom) or an Eppendorf thermocycler programmed for an initial denaturization of 2 min at 95°C, followed by 40 cycles of 30 s at 95°C, 40 s at the annealing temperature (Table 3), and 1 min at 72°C, and a final extension of 7 min at 72°C.

Separation of SSR-PCR products. Fluorescence-labeled SSR-PCR products (0.5 µl containing approximately 1.5 ng of DNA from each amplification product) and 0.5 µl of the internal standard GS-500 TAMRA (Perkin-Elmer Corp.) were added to 1.5 µl of loading buffer. The mixture was heated to 95°C for 3 min. One microliter of this mixture was separated by PAGE (4.25% acrylamide) on an ABI Prism 377 DNA sequencer. The allele size was estimated by comparing the mobility of the SSR products to that of the internal size standard as determined by GeneScan 2.1 analysis software (Perkin-Elmer Corp.) in conjunction with Genotyper 2 (Perkin-Elmer Corp.). Repeatibility was confirmed by running the same reference samples (from S. sapinea isolate CMW 5977) on every gel.

Data analysis. Data from Genotyper were compiled in Excel files (Microsoft). For each isolate, a data matrix of characters was compiled by scoring the presence or absence of each allele at each locus. For simplicity, these data are presented as multistate characters (Table 1). Parsimony analysis was performed on the data set using PAUP* (30). The most parsimonious trees were obtained by using heuristic searches with random addition in 1,000 replicates, with the tree bisection-reconnection branch-swapping option on and the steepest-descent option off. Bootstrap consensus trees were constructed using the same conditions.

Nucleotide sequence accession numbers. The sequences for each of the SSR loci have been deposited in the GenBank database with the accession numbers AF263294 to 304 for SS1 to SS11, respectively.

	RESULTS

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Evaluation of SSR markers. The ISSR primers each amplified 5 to 10 bands that ranged in size between 300 and 2,500 bp. A total of 42 cloned inserts were sequenced, 6 from each of the primers. Nineteen SSR primer pairs were designed to flank microsatellite-rich regions found within the sequence.

Identification of the putative I morphotype. Two isolates of the I morphotype were examined. Phylogenetic analysis grouped these isolates with B. obtusa, and their separation from the other S. sapinea morphotypes had strong bootstrap support (Fig. 2). At SS2, SS5, SS8, and SS10, alleles were shared by B. obtusa and the I isolates which were not present in the other morphotypes (Table 3). Both B. obtusa and the putative I isolates shared alleles with the B isolates at SS1 and with the A isolates at SS9 and SS11. I isolates also shared alleles with the B isolates at SS3 and SS6 and with the A isolates at SS4. Interestingly, sequences of SS5, SS8, SS10, and SS11 were identical for the putative I isolates and B. obtusa (Fig. 3). Allele size differed between the putative I isolates and B. obtusa at SS7 and SS9. However, in both cases this was due to a mutation in a microsatellite region, and all other substitutions and small indels were identical (Fig. 3).

View larger version (30K): [in this window] [in a new window]   FIG. 2.   Unrooted most parsimonious tree generated from SSR polymorphic data showing bootstrap values for the major branches. The solid arrows indicate the strongly supported branches separating the A, B, and C morphotypes of S. sapinea from the putative I morphotype and the closely related B. obtusa. The open arrows indicate the moderately supported branches separating geographically isolated populations of the B morphotype.

View larger version (80K): [in this window] [in a new window]   FIG. 3.   Aligned nucleotide sequences indicating polymorphism among different isolates of S. sapinea at loci SS5 (a), SS7 (b), SS8 (c), SS9 (d), SS10 (e), and SS11 (f). *, unspecified length of homologous sequence; , gaps that indicate variation between aligned sequences. The isolate code is given at the beginning, and the length of the fragment is shown at the end of each sequence. The morphotype of the isolate is given in parentheses after the code. The isolate without a morphotype, CMW5991, is B. obtusa.

Segregation of SSR alleles. C isolates were monomorphic at 8 of the 11 loci, producing a total of only 15 alleles across all loci (Tables 1 and 3). A isolates were monomorphic at 5 of the 11 loci but produced 23 alleles across all loci. B isolates were only monomorphic at three loci (SS2, SS6, and SS10) and were the most diverse, with 38 alleles across all loci (Table 3).

Parsimony analysis of SSR markers. The data matrix comprised 65 characters, each character representing an individual allele at one of the 11 polymorphic SSR loci. Of the 65 characters, 55 were parsimony informative. Heuristic searches using parsimony resulted in 362 trees of 114 steps, one of which is shown in Fig. 2. Bootstrap analysis supported strong branches separating each of the morphotypes.

Source of polymorphisms. For each locus, two or more alleles were sequenced. There were many transitions and transversions that altered the sequence but not the size of the fragment (Fig. 2). Polymorphisms at loci SS5 (Fig. 3a), SS7 (Fig. 3b), SS8 (Fig. 3c), SS9 (Fig. 3d), and SS10 (Fig. 3e) were due to a mutation within a repeat motif. Polymorphism in locus SS1 resulted from a 36-bp indel that had been inserted between one and five times (Table 3). Polymorphisms at the remaining loci were due to indels of various sizes (Table 3 and Fig. 3).

	DISCUSSION

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In this study, ISSR-PCR was effectively used to produce SSR markers for S. sapinea. From 42 sequences amplified by ISSR, 22 primer pairs were designed, and of these, 15 produced polymorphic amplification products. This is a success rate of 35% compared with less than 10% for more traditional methods of obtaining polymorphic markers, such as screening genomic libraries with a microsatellite probe (2).

The SSR markers developed in this study clearly distinguished among the morphotypes of S. sapinea. Isolates previously designated as representing the A and B morphotypes using RAPD and morphological data were once again found within these groups using SSR-PCR (19, 27, 28). There were 16 common isolates for the current study and that of de Wet et al. (5), with the resultant classification of the morphotypes being the same for all but 2 isolates. These isolates, CMW 4886 and 4899, had been difficult to classify using RAPD (J. de Wet, personal communication). Isolate CMW 4899 was designated as belonging to the C morphotype by de Wet et al. (5), while SSR-PCR placed it firmly in the A morphotype group. CMW 4886 was designated as an A morphotype isolate by de Wet et al. (5), but using SSR-PCR it was shown to represent the C morphotype, along with all the other Indonesian isolates. The five isolates designated as belonging to the A and B morphotypes using RFLP (9) also fell into those groups using SSR-PCR. Overall, characterization of isolates by SSR-PCR gave the same results as those previously derived using the RAPD and RFLP techniques (4, 9, 27, 28).

The use of higher annealing temperatures and automated analysis resulted in highly reproducible SSR markers that are much more robust than the RAPD markers used previously (4, 27, 28). The SSR markers are also more useful than the RFLP profiles generated from rDNA (9). These RFLP profiles can be used to distinguish between morphotypes, but compared with polymorphic markers, they are of little use in population studies. Three of the SSR markers (SS7, SS9, and SS10) produced different-size alleles for each of the morphotypes and could be used for a simple diagnostic test to distinguish morphotypes of S. sapinea.

An interesting result of this study was that the two isolates described by Hausner et al. (9) as belonging to a putative I morphotype were found to be identical to B. obtusa. B. obtusa is closely related to S. sapinea and has a Sphaeropsis anamorph (11). B. obtusa is a cosmopolitan fungus that is commonly found in temperate areas on numerous woody hosts, including Pinus spp. (20). The two isolates used in this study were collected in Ontario, Canada, from Picea glauca and Pinus banksiana (9). Examination of SSR marker sequences for representative isolates of all morphotypes of S. sapinea and B. obtusa revealed that while some substitutions and small indels are unique to B. obtusa, many are identical to those found in either the A or B morphotype.

Parsimony analysis of SSR markers generated in this study strongly supported the divisions among the three S. sapinea morphotypes. The C morphotype, however, clustered closely with the A morphotype. This confirmed previous observations based on culture characteristics, spore morphology, and internal transcribed spacer sequence data (5). The isolates of the B morphotype were much more polymorphic than those of the A or C morphotype. This is particularly interesting, as the markers were developed using isolate CMW 5977, which is a representative of the A morphotype. As a general principle, markers are usually more diverse in the population for which they are designed.

The 14 isolates of the B morphotype separated into three groups based on geographic location. At five loci, different alleles were fixed in these geographically isolated populations. Genetic isolation can lead to the loss of shared polymorphisms (32, 33). Thus, when all isolates are examined together, it would appear that the population consisted of a number of clones when in fact each genetically isolated population could be undergoing recombination. In order to ascertain the mode of reproduction, geographically isolated populations will need to be examined separately (32, 33).

Genomic regions containing microsatellites are evolving and mutating more rapidly than other areas due to slipped-strand mispairing during replication, with the slippage rate dependent upon the length of the repeat (13). Thus, the longer the repeat the more likely there is to be slippage. This is supported by our sequence data. We observed that the most polymorphic of the 11 loci examined were those where mutations occurred in a repeat motif (SS7, SS8, SS9, and SS10).

In this study, we have clearly demonstrated sequence differences between alleles of the same size. At loci SS5 and SS11, isolates of the A and B morphotypes of S. sapinea all shared an allele. Sequencing of these fragments showed that they are very different. Although microsatellite alleles are considered to be codominant markers, differences in alleles are measured based solely on size. There is the possibility of single point mutations within the flanking sequence that do not result in a change in the fragment length. It is also possible that different indels could result in fragments of the same size that have different sequences. Thus, the genotypic diversity in a population will always be underestimated using such markers. Additional information could be obtained from loci such as SS11, using single-strand conformation polymorphisms of same-size alleles to confirm their similarity in sequence as well as size (6, 24).

The SSR markers developed in this study can be used to distinguish morphotypes of S. sapinea. However, the markers are more powerful than a simple diagnostic tool. Interesting results will emerge from comparing populations of this asexual fungus from distinct geographic locations. Thus, future studies will focus on the population diversity and recombination within and between native and introduced populations of S. sapinea.