Phenotypic and genotypic analysis of variability in Aspergillus fumigatus

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Phenotypic and Genotypic Analysis of Variability

in Aspergillus fumigatus

EDIT RINYU, JA´ NOS VARGA,*ANDLAJOS FERENCZY

Department of Microbiology, Attila Jo´zsef University, H-6701 Szeged, Hungary

Received 9 March 1995/Returned for modification 8 April 1995/Accepted 1 July 1995

Sixty-one isolates and collection strains ofAspergillus fumigatuswere compared for their phenotypic

(mor-phological features and isoenzyme profiles) and genotypic (restriction enzyme-generated mitochondrial DNA and ribosomal DNA profiles and random amplified polymorphic DNA patterns) features. The examined strains exhibited highly variable colony morphologies and growth rates at different temperatures, but their micro-morphologies and conidial diameters were characteristic of the species. Of the isoenzymes studied, the

b-arylesterase and phosphatase patterns were the most divergent, and the 61 strains could be classified into

seven groups. The glucose 6-phosphate dehydrogenase and catalase isoenzyme patterns displayed only a limited variability, while the profiles of superoxide dismutase, lactate dehydrogenase, and glutamate

dehydro-genase were highly conserved. TheHaeIII-generated mitochondrial DNA patterns andSmaI-digested repetitive

DNA and ribosomal DNA hybridization patterns of almost all strains were also invariable. The level of variation was much higher when random amplified polymorphic DNA analysis was applied. Although the patterns of the strains were very similar with most of the primers, the application of some primers made it

possible to cluster theA. fumigatusisolates into several groups. The results indicate that the random amplified

polymorphic DNA technique could be used more efficiently than isoenzyme analysis for typingA. fumigatus

isolates. A good correlation was found between the dendrograms obtained from the isoenzyme and random amplified polymorphic DNA data, but the isoenzyme and amplified DNA patterns did not correlate with the

pathogenicity, pigment production, or geographical origin of the strains. One ‘‘A. fumigatus’’ strain (strain FRR

1266) exhibited unique isoenzyme, mitochondrial DNA, ribosomal DNA, and random amplified polymorphic

DNA patterns; it is proposed that this strain represents a new species of the sectionFumigati.

Aspergillus fumigatus Fresenius is a ubiquitous filamentous fungus which plays an important role under natural conditions in the aerobic decomposition of organic materials. At the same time, this species is an important human pathogen which may cause several diseases like allergic bronchopulmonary aspergil-losis, aspergilloma, and invasive aspergilaspergil-losis, a usually fatal disease, particularly in immunocompromised patients. A. fu-migatus is generally regarded as a single homogeneous species, but strains may vary in their cultural characteristics and, to a lesser degree, in their micromorphologies. Several studies have been performed by examining phenotypic and genotypic char-acters to demonstrate strain variability in A. fumigatus. Strains were compared with regard to their cultural and serological features (28), and immunochemical studies were carried out by using various immunoelectrophoretic techniques to examine the antigenic variability of the species (17, 23, 55). Isoenzyme analysis has also been applied (13, 32). The genotypic charac-ters examined include restriction fragment length polymor-phism (RFLP) (7, 11, 12, 15, 45) and amplified fragment length polymorphism (to be described in detail in the Discussion section) (2, 29, 44, 46, 47).

In our study, A. fumigatus isolates and collection strains of different origins were compared. Morphological characteristics were examined, and isoenzyme and DNA polymorphisms were analyzed. Our goal was to examine the applicability of pheno-typic or genopheno-typic analysis for distinguishing and typing A. fumigatus strains.

(Preliminary data from this project were presented at the

11th Congress of the Hungarian Society for Microbiology [52] and at the Aspergillus Symposium in Canterbury, United King-dom (69th FEMS Symposium) [9].)

MATERIALS AND METHODS

Strains.The A. fumigatus strains examined in the study are listed in Table 1. The strains were maintained on malt extract agar slants.

Morphological studies.The A. fumigatus strains were grown for 4 days on Czapek-Dox minimal medium and malt extract medium at 30, 37, and 458C. The macro- and micromorphologies of the strains were recorded, and conidium diameters were measured by using an eyepiece micrometer.

Isoenzyme analysis.The strains were grown in the minimal medium of Pon-tecorvo et al. (39) at 308C for 2 days. Mycelia were harvested and lyophilized overnight. Crude protein extracts were prepared basically as described previously (19). Briefly, 200 mg of lyophilized mycelia ground in a mortar was suspended in 2 ml of protein extraction buffer (0.37 M Tris-HCl [pH 7.5], 1 mM disodium EDTA, 5 mMb-mercaptoethanol, 1% Triton X-100). After extraction for 45 min on ice, the cell debris was removed by centrifugation at 15,0003g for 30 min at 48C. The supernatant was mixed with one-third volume of 0.05% bromophenol blue in 70% glycerol, and the mixture was applied to the polyacrylamide gels or stored at2208C. Polyacrylamide slab gel electrophoresis was carried out as described previously (10), except that 3% (wt/vol) stacking gels and 5.6, 7.5, or 10% separation gels were used. After a 10-min prerun, the samples were loaded and run at a constant current of 40 mA in the stacking gel and 60 mA during the separation at 58C for 4 to 5 h. A Pharmacia GE-4 vertical gel slab electrophoretic apparatus was used with four 3-mm slabs. Isoenzymes were detected by the procedures described in Table 2.

Isolation and characterization of nucleic acids.Nucleic acids were isolated as described in the literature (25). Mitochondrial DNA (mtDNA) patterns were analyzed by digesting the total DNA samples with HaeIII or double digesting them with HaeIII-EcoRI, HaeIII-HindIII, or HaeIII-BglII restriction enzymes as described earlier (51). SmaI-generated repetitive DNA patterns were examined by digesting the total DNA samples with SmaI and separating the fragments by electrophoresis. The rRNA gene cluster (ribosomal DNA [rDNA]) of the strains was analyzed by hybridizing the ribosomal repeat unit of Aspergillus nidu-lans(pMN1) (5) to the filters obtained after Southern blotting of the SmaI-digested DNA smears. Hybridization experiments were performed as described previously (41).

Fungal DNA sequences were amplified by using the primers of the Operon

* Corresponding author. Mailing address: Department of Microbi-ology, Attila Jo´zsef University, P.O. Box 533, H-6701 Szeged, Hungary. Phone: (36)62-310-011/3964. Fax: (36)62-432-488. JVARGA@bio.u-szeged.hu.

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TABLE 1. A. fumigatus strains examined in our study

Strain No. Origin

A. fumigatus 1 3085, 1012; D. Kerridge, Cambridge, United Kingdom

A. fumigatus 2 31, 1058; L. Manczinger, from strain 1 (ade mutant)

A. fumigatus 3 12, 1059; L. Manczinger, from strain 1 (lys mutant)

A. fumigatus 4 FK2; F. Kevei, Szeged, Hungary

A. fumigatus 5 FK3; F. Kevei, Szeged, Hungary

A. fumigatus 6 NCAIM F 0673; Horticultural University, Budapest, Hungary

A. fumigatus 7 NCAIM F 056; Horticultural University, Budapest, Hungary

A. fumigatus mut. helvola 8 NCAIM F 00733; Horticultural University, Budapest, Hungary

A. fumigatus 9 FM-2; F. Kevei, UV (lys mutant)

A. fumigatus 10 FM-5; F. Kevei, UV (bio mutant)

A. fumigatus 11 16914; Plant Pathology Culture Collection, Brisbane, Australia

A. fumigatus 12 0886; Agricult. Service Be´ke´s-Csongra´d, Hungary

A. fumigatus 13 FRR 581; Food Research Laboratory, CSIRO, North Ryde, Australia

A. fumigatus 14 FRR 623; Food Research Laboratory, CSIRO, North Ryde, Australia

A. fumigatus 15 FRR 582; Food Research Laboratory, CSIRO, North Ryde, Australia

A. fumigatus 16 FRR 1266; Food Research Laboratory, CSIRO, North Ryde, Australia

A. fumigatus 17 FRR 3031; Food Research Laboratory, CSIRO, North Ryde, Australia

A. fumigatus 18 ATCC 42824, G. Szaka´cs, Technical University, Budapest, Hungary

A. fumigatus 19 ATCC 42826, G. Szaka´cs, Technical University, Budapest, Hungary

A. fumigatus 20 ATCC 58128, G. Szaka´cs, Technical University, Budapest, Hungary

A. fumigatus 21 ATCC 58129, G. Szaka´cs, Technical University, Budapest, Hungary

A. fumigatus 22 2708E; Glaxo Group Research Ltd., Greenford, United Kingdom

A. fumigatus 23 D142; strain with septate phialides; F. Staib, Berlin, Germany

A. fumigatus 24 3512E; Glaxo Group Research Ltd., Greenford, United Kingdom

A. fumigatus 25 NZFS 88; Forest Research Institute, Rotorna, New Zealand

A. fumigatus 26 UC-2431; Universidad Catholica, Santiago, Chile

A. fumigatus 27 FSC 522; Forestry Canada, Fredericton, Canada

A. fumigatus 28 CN(M)10; Wellcome Biotech, London, United Kingdom

A. fumigatus 29 CN(M)154; Wellcome Biotech, London, United Kingdom

A. fumigatus 30 CN(M)156; Wellcome Biotech, London, United Kingdom

A. fumigatus 31 CN(M)311; Wellcome Biotech, London, United Kingdom

A. fumigatus 34 UAMH 2978; University of Alberta, Edmonton, Alberta, Canada

A. fumigatus 35 UAMH 5878; University of Alberta, Edmonton, Alberta, Canada

A. fumigatus 36 BIK; Res. Institute of Leather Industry, Budapest, Hungary

A. fumigatus 37 FK1; F. Kevei, Szeged, Hungary

A. fumigatus 38 VJ1; J. Varga, Szeged, Hungary

A. fumigatus 39 CCF 547; Charles University, Prague, Czech Republic

A. fumigatus 40 CCF 1292; Charles University, Prague, Czech Republic

A. fumigatus 41 CCF 1294; Charles University, Prague, Czech Republic

A. fumigatus 42 231/56; NCTC, Prague, Czech Republic

A. fumigatus 43 237/90; NCTC, Prague, Czech Republic

A. fumigatus 44a ATCC 32722; M115, University of Guelph, Guelph, Ontario, Canada

A. fumigatus 44b Isolated as a contaminant from a culture of 44a

A. fumigatus 45 Adelaide Children’s Hospital, Adelaide, Australia

A. fumigatus 46 Adelaide Children’s Hospital, Adelaide, Australia

A. fumigatus 47 Adelaide Children’s Hospital, Adelaide, Australia

A. fumigatus 48 ICMP 434; International Collection of Microorganisms from Plants, Auckland, New Zealand

A. fumigatus 49 ICMP 1046; International Collection of Microorganisms from Plants, Auckland, New Zealand

A. fumigatus 50 ICMP 1061; International Collection of Microorganisms from Plants, Auckland, New Zealand

A. fumigatus 51 NRRL 163; ATCC 1022

A. fumigatus var. acolumnaris

52 NRRL 5587; CMI 174456

A. fumigatus var. ellipticus

53 NRRL 5109

A. fumigatus mut. helvola 54 NRRL 174; ATCC 16907

A. fumigatus 55 1180; Chinoin Pharmaceutical Company, Budapest, Hungary

A. fumigatus 56 CCF 1293; Charles University, Prague, Czech Republic

A. fumigatus 57 CCF 817; Charles University, Prague, Czech Republic

A. fumigatus 58 CCF 1059; Charles University, Prague, Czech Republic

A. fumigatus 59 CCF 1187; Charles University, Prague, Czech Republic

A. fumigatus 60 IAM 2004; IAM Culture Collection, Tokyo, Japan

A. fumigatus 61 IAM 2046; IAM Culture Collection, Tokyo, Japan

A. fumigatus 62 IAM 3006; IAM Culture Collection, Tokyo, Japan

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random primer kit C (Operon Technologies, Inc., Alameda, Calif.) according to the literature (18, 56). The primers used were OPC-01 (59-TTCGAGCCAG-39), OPC-04 (59-CCGCATCTAC-39), OPC-05 (59-GATGACCGCC-39), OPC-06 (59-GAACGGACTC-39), OPC-07 (59-GTCCCGACGA-39), OPC-08 (59-ACCT

GGCCAC-39), OPC-10 (59-TGTCTGGGTG-39), OPC-11 (59-AAAGCTGC

GG-39), OPC-12 (59-TGTCATCCCC-39), OPC-13 (59-AAGCCTCGTC-39), OPC-14 (59-TGCGTGCTTG-39), and OPC-18 (59-TGAGTGGGTG-39). The Taq polymerase used in these experiments was supplied by Amersham Interna-tional (Taq DNA polymerase cloned for PCR; catalog no. T0303Z). The MJ Research programmable thermal controller (model PTC-100-60; MJ Research, Inc., Watertown, Mass.) was programmed for 45 cycles (1 min at 928C, 1 min at 358C, and 2 min at 728C). The amplification products were separated by elec-trophoresis in 1.2% agarose gels, stained with ethidium bromide, and visualized under UV light. All amplifications were repeated three times. The faint bands which did not appear in all repeated experiments were not counted during cluster analysis.

Statistical analysis.Statistical analysis of the isoenzyme, RFLP, and random amplified polymorphic DNA (RAPD) data was carried out by using the SYN-TAX-pc version 5.0 software package (37). The binomial matrix obtained from the data was used to calculate the simple matching coefficients (SSM) (43). The

coefficient matrices were analyzed by an unweighted pair-group method by using arithmetic averages (UPGMA) (43). Cophenetic correlation coefficients were

determined as described previously (42). All calculations were performed on an International Business Machines-compatible AT computer.

RESULTS

Morphological studies. The macromorphologies of the

strains were examined at different temperatures and on differ-ent media. The strains exhibited highly variable colony mor-phologies, growth rates, and levels of pigment production (data not shown). Concerning the micromorphologies of the A. fu-migatus isolates, one strain (strain 23) had septate phialides, while all the other strains displayed the characteristics of A. fumigatus conidial heads. The conidia of three strains (strains 34, 45, and 53) were found to be elliptical in shape. All strains had conidia with diameters in the range of 2.5 to 3.5mm (data not shown).

Isoenzyme analysis.The isoenzyme patterns of 11 enzyme

systems were studied (Table 2). Staining for alcohol

dehydro-Enzyme EC no. Abbreviation Reference Pattern Strains showing these patterns

Acid and alkaline phosphatases

3.1.3.1, 3.1.3.2 PHO 16 A 4–6, 11–13, 15, 18, 19, 21, 22, 24, 25, 28–36, 38–40, 42–44b, 46–50, 53, 54, 56, 62

B 1–3, 7–10, 14, 17, 20, 23, 26, 27, 37, 41, 45, 51, 52, 55, 57–61

C 16, N.ps. D N.f.A, N.f.a

Catalase 1.11.1.6 CAT 57 A Most A. fumigatus strains

B 16

C 23, 53

D 57, 58

E N.f.A, N.f.a

F N.ps.

b-Arylesterase 3.1.1.2 EST 16 A 5, 11, 12, 18, 19, 22–24, 28–30, 34–38, 45, 48–51, 56, 57, 62

B 1–3, 6–10, 13, 15, 17, 26, 27, 31, 39, 40, 44b, 46, 47, 52, 55, 58–61

C 4, 14, 20, 21, 25, 41–44a, 53, 54

D 16

E N.f.a

F N.f.A

G N.ps.

Glucose 6-phosphate dehydrogenase

1.1.1.49 G6P 35 A Most A. fumigatus strains

B 16

C 22, 24–26 D N.f.A, N.f.a

E N.ps.

Glutamate dehydrogenase

1.4.1.4 GDH

(NADP)

1 A Most A. fumigatus strains, N.f.A, N.f.a., N.ps.

B 15, 44b

C 16

Lactate dehydrogenase 1.1.1.27 LDH 6 A Most A. fumigatus strains

B 16

C 20

D N.f.A, N.f.a

E N.ps.

Superoxide dismutase 1.15.1.1 SOD 4 A Most A. fumigatus strains, N.ps.

B 11

C N.f.A, N.f.a

aThe isoenzyme patterns observed are shown in Fig. 1. N.ps., N. pseudofischeri NCAIM F 00757; N.f.A and N.f.a, N. fennelliae NRRL 5534 and NRRL 5535, respectively.

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genase (20) and NAD-dependent glutamate dehydrogenase (GDP) (1) activities was unsuccessful. The NAD- and NADP-dependent malate dehydrogenase activity stains (6) resulted in diffuse bands which were difficult to interpret. Only a limited variation was observed for superoxide dismutase (SOD), lac-tate dehydrogenase (LDH), GDH (NADP), glucose 6-phos-phate dehydrogenase (G6P), and catalase (CAT) isoenzymes (Fig. 1 and Table 2; the isoenzyme patterns of the two mating type strains of Neosartorya fennelliae NRRL 5534 and NRRL 5535 and Neosartorya pseudofischeri NCAIM F 00757 are also given for comparison). The acid and alkaline phosphatase (PHO) andb-arylesterase (EST) patterns were more variable (Fig. 1 and 2). Two main groups were distinguished on the basis of the PHO patterns (A and B electromorphs in Fig. 1 and Table 2). Although only a small difference was observed between PHO patterns A and B, the strains consistently exhib-ited patterns A or B in the repeated experiments. Strains belonging in these groups could not be differentiated morpho-logically. Concerning the EST patterns, three groups were rec-ognized (group A, B, and C electromorphs in Fig. 1 and Table 2). The upper bands observed in the EST patterns of the strains were too faint and were difficult to reproduce in the repeated experiments, and accordingly, they were not taken into account. The two mating-type strains of N. fennelliae gave the same isoenzyme patterns in all cases with the exception of EST (patterns E and F in Fig. 1 and 2). The EST patterns of

the two strains differed even after repeated protein extraction and electrophoresis.

The dendrogram constructed from the UPGMA clustering of the similarity matrix of the simple matching coefficients of 44 characters originating from the isoenzyme data is shown in Fig. 3. Twenty-nine of these characters were available for A. fumigatus strains. The cophenetic correlation coefficient of this dendrogram was 0.9787.

RFLP analysis. The EcoRI-digested repetitive DNA

pat-terns of all of the examined strains except strain 16 were the same (data not shown). The patterns of the mtDNAs of the A. fumigatus strains generated by HaeIII digestion were also in-variable except for that for strain 16; all strains gave three bands at 25, 6.2, and 3.9 kb, while strain 16 gave two bands at 27 and 3.7 kb. DNA samples of some strains which differed in their micromorphologies and isoenzyme patterns and which represented the type strains of the A. fumigatus species and its ‘‘subspecies’’ (strains 51, 52, 53, and 54) were double digested with various combinations of restriction enzymes. These strains revealed identical mtDNA patterns with all enzyme combina-tions (HaeIII-BglII, HaeIII-EcoRI, and HaeIII-HindIII).

The repetitive DNA banding patterns of the A. fumigatus strains obtained by digesting their total DNA samples with SmaI were the same (four bands with sizes of 2.1, 1.7, 1.4, and 0.9 kb) except for those of strain 44b, which displayed a 1.1-kb band instead of the 0.9-kb fragment present in all the other FIG. 1. Isoenzyme patterns of the A. fumigatus strains. For the abbreviations used and for the strains showing these patterns, see Table 2. Patterns belonging to N. pseudofischeri or N. fennelliae strains are labelled (■) for clarity.

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strains, and strain 16, which showed four repetitive DNA bands at 2.4, 1.77, 1.7, and 0.8 kb. Hybridization of the A. nidulans ribosomal repeat unit to the filters obtained after blotting resulted in the same pattern for all strains except strain 16. Hybridization to the smallest bands was not observed (1.1 kb in strain 44b, 0.75 kb in strain 16, and 0.9 kb in all the other strains [data not shown]).

RAPD analysis.Twelve primers were tested for their ability

to detect variability among the A. fumigatus strains. OPC-01 did not amplify A. fumigatus DNA properly (only faint bands were observed). Primers OPC-05, OPC-11, OPC-12, OPC-14, and OPC-18 revealed only a limited amount of polymorphisms. Primers OPC-04 and OPC-13 gave different amplified DNA patterns only for some strains. When OPC-08 was used as the primer, one strong band at 1.23 kb was amplified in all A. fumigatus strains except strain 16 and many other Aspergillus species examined (data not shown). Faint bands also appeared consistently in the gels after repeated amplifications. The ap-plication of some primers (OPC-06, OPC-07, and OPC-10; Fig. 4) resulted in RAPD patterns sufficiently polymorphic for the A. fumigatus strains to be classified into different groups. The dendrogram based on the UPGMA clustering of the similarity matrix of 89 characters obtained from analysis of the RAPD patterns produced by using 05, 06, 07, OPC-08, OPC-10, and OPC-13 as primers is shown in Fig. 5. The cophenetic correlation coefficient of this dendrogram was 0.9230. The numbers of different RAPD patterns obtained by using each of the primers OPC-05, OPC-06, OPC-07, OPC-08, OPC-10, and OPC-13 were 6, 13, 11, 10, 21, and 6, respectively.

DISCUSSION

Several studies have been carried out recently on A. fumiga-tus mainly for the identification of this opportunistic pathogen mold in diseased tissues and the typing of clinical isolates (7, 8, 11, 12, 15, 17, 38). Our study has focused on a comparison of A. fumigatus strains of different origins at the protein and DNA levels to ascertain whether the widespread morphological, physiological, and immunochemical variabilities observed ear-lier are accompanied by differences that are detectable at a molecular level. We also wished to evaluate the applicability of

phenotypic and genotypic approaches for typing A. fumigatus strains.

The macromorphologies of the A. fumigatus strains were highly variable; extreme differences were observed in their conidium production, pigmentation, and growth rates at dif-ferent temperatures. In spite of the high degree of macromor-phological variability, the A. fumigatus strains revealed a quite uniform picture concerning their micromorphologies. All strains had conidial diameters of 2.5 to 3.5mm; this range is in agreement with data published earlier for A. fumigatus (2.1 to 3.5mm) (40). Strains with elliptical conidia (strains 34, 45 and 53), septate phialides (strain 23), and acolumnar heads (strain 52) have also been observed.

Concerning the isoenzyme profiles, polymorphisms were ob-served for all isoenzymes studied. The LDH, SOD, and GDH (NADP) isoenzyme patterns were very consistent; only one, one, and two strains, respectively, exhibited different patterns (Fig. 1). Similar results were achieved earlier by other investi-gators (32), who proposed the application of GDH and G6P isoenzyme patterns for the distinction of A. fumigatus strains from other aspergilli. Further experiments are in progress to evaluate the applicability of these isoenzyme patterns for the identification of A. fumigatus isolates. The CAT and G6P isoenzyme patterns were more variable, but isoenzyme poly-morphisms were mainly observed in the cases of PHOs and esterases. The minor differences observed between PHO pat-terns A and B were reproducible and could be used to cluster FIG. 2. EST patterns of some A. fumigatus strains. For descriptions of the

strain numbers above the lanes, see Table 1. Lane A, N. fennelliae mating type A; lane a, N. fennelliae mating type a. The upper faint bands of these patterns were difficult to reproduce and are not depicted in Fig. 1.

FIG. 3. UPGMA dendrogram of the A. fumigatus strains examined. The dendrogram was based on their isoenzyme patterns. The scale represents dis-similarity (squared distance). The strain numbers on the left are those listed in Table 1. N. pseudofischeri NCAIM F 00757 (N.ps.) and two strains of N. fennel-liae, NRRL 5535 and NRRL 5534, representing the two mating types (N.f.a and N.f.A, respectively) are also included for comparison.

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the A. fumigatus strains. However, these patterns are not dis-tinctive enough to be used as a routine typing technique. The two electromorphic groups observed in the case of PHO did not correspond strictly to any of the three groups observed for

EST isoenzyme patterns, although most strains with type A EST patterns also displayed type A PHO patterns (19 strains), and those with type B EST patterns displayed type B PHO patterns (16 strains). Their EST and PHO patterns indicated that the 61 A. fumigatus strains examined belonged in seven groups. No correlation was observed between the electromor-phic group to which the given strain belongs and the source of the strain (saprophytic or pathogenic) or between growth rates, macromorphologies, and isoenzyme patterns (data not shown). However, these enzyme polymorphisms could be useful for clustering A. fumigatus strains, even if the degree of discrimi-nation is low.

Other systems which detect phenotypic variability were also examined earlier in the case of A. fumigatus (e.g. morphology [28], susceptibility to yeast killer toxins [38], immunoblot fin-gerprinting [8]). As a result of their phenotypic characters, all of these methods have some disadvantages, e.g., they are de-pendent on gene expression, and it is difficult to standardize the procedures, because different culture conditions might re-sult in different isoenzyme patterns. The application of geno-type (DNA)-based systems could overcome these limitations. Cellular DNA can be characterized in numerous ways, includ-ing physical methods, restriction enzyme analysis, PCR-based techniques, and sequencing. We carried out the restriction enzyme analysis of DNA of nuclear and mitochondrial origin to distinguish A. fumigatus strains, since this method was suc-cessfully applied earlier to characterize black Aspergillus iso-lates (49–51). The EcoRI-generated repetitive DNA patterns of all A. fumigatus strains with the exception of strain 16 were the same. This observation is in agreement with earlier findings (7, 15). The SmaI-generated repetitive DNA patterns of all except two strains (strains 16 and 44b) were the same, while the rDNA patterns obtained by hybridizing the A. nidulans ribo-somal repeat unit to the SmaI-digested DNA did not display variability (except for strain 16). Hybridization with non-rDNA probes was found to be a more promising approach for typing A. fumigatus isolates (15).

The mtDNAs of the strains were examined by using HaeIII which has a 4-bp GC-rich recognition site. The application of such enzymes made it possible to examine mtDNA polymor-FIG. 4. RAPD patterns of some of the A. fumigatus strains with OPC-10 used as the primer. For descriptions of the strain numbers above the lanes, see Table 1. Lane M, EcoT14I-digested bacteriophage lambda DNA (Amersham, International). During cluster analysis, strains 40, 41, and 45; strains 43, 48, 49, and 50; and strains 46 and 47, respectively, were considered to have the same pattern. Strain 51 differs from strain 53 in the presence of a faint, but reproducible band.

FIG. 5. UPGMA dendrogram of the A. fumigatus strains examined. The dendrogram was based on their RAPD patterns. The scale represents dissimi-larity (squared distance). The strain numbers on the left are those listed in Table 1.

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Aspergillus genus. The repetitive DNA fragments observed in the gels were shown to be of mitochondrial origin by compar-ison with HaeIII-digested purified mtDNA patterns (51) and by hybridization with whole purified mtDNA (50). The HaeIII-generated mtDNA patterns of the A. fumigatus strains were invariable except for that of strain 16. mtDNA variability was not observed, even when HaeIII was used in combination with other restriction enzymes (EcoRI, HindIII, and BglII). Using any of these enzyme combinations, we could detect high levels of variability in the mtDNAs of Aspergillus niger and Aspergillus tubingensis strains (50, 51). More enzymes should be tested to seek intraspecific mtDNA polymorphisms in A. fumigatus. Re-striction enzymes with AT-rich recognition sequences were successfully applied in order to detect intraspecific variability in species of the section Flavi of the Aspergillus genus (34). These enzymes could also be useful for studying the mtDNAs of the A. fumigatus species. In conclusion, by using restriction enzymes such as EcoRI, SmaI, or HaeIII, rDNA and mtDNA RFLPs were not appropriate for distinguishing A. fumigatus strains. This observation is not in contrast to the earlier finding that restriction enzymes could successfully be used for typing (11, 12). In those experiments, very large DNA molecules (.50 kb) obtained by a protoplast lysis method were digested, and differences in the mobilities of 10- to 50-kb bands distinguished the isolates. The restriction enzymes used (SalI and XhoI) were also different from those used in the present study (HaeIII, SmaI, EcoRI, and HindIII).

Other genotypic methods based on the detection of ampli-fied fragment length polymorphisms have successfully been used to identify A. fumigatus in clinical samples (44, 46) and to type A. fumigatus isolates by using an interrepeat PCR (47) and the RAPD technique (2, 29). Those investigators (2, 29) found that only a small proportion of the examined primers could detect variability among the A. fumigatus strains. The same phenomenon has been observed in our laboratory. On use of most of the primers, only a limited number of strains gave different RAPD patterns. Although all primers detected some polymorphism among the A. fumigatus strains, only 3 of the 12 primers examined (OPC-06, OPC-07, and OPC-10) revealed high variability. With OPC-10 and OPC-07 as primers, the 61 A. fumigatus strains could be grouped into 37 slots. This ob-servation indicates that the application of only a limited num-ber of primers (two to three) might be sufficient for typing A. fumigatus isolates. The use of OPC-08, which amplified one strong band in all A. fumigatus strains but in none of the other aspergilli tested, might be useful for distinguishing A. fumigatus isolates from other potentially pathogenic Aspergillus species, such as A. flavus, A. niger, or A. terreus (data not shown).

The UPGMA dendrograms obtained from the statistical analysis of the isoenzyme and RAPD data of the A. fumigatus strains are depicted in Fig. 3 and 5, respectively. The cophen-etic analysis revealed a good correlation between the two den-drograms on the basis of isoenzyme and RAPD data (the cophenetic correlation coefficient was 0.9086 between the two dendrograms on the basis of different character sets, although we should mention that this high value is mainly due to the high number of negative matches, since on use of the Jaccard coefficient, which does not take into account the negative matches, the cophenetic correlation coefficient of the two den-drograms decreased to 0.75). The A. fumigatus strains are well separated from the N. pseudofischeri and N. fennelliae strains, which were used as outgroups in our experiments. In both dendrograms, the A. fumigatus strains showed homogeneity (at least 80% similarity). This result is in agreement with earlier

(32). The positions of A. fumigatus var. ellipticus (strain 53) and A. fumigatus var. acolumnaris (strain 52) in the dendrograms suggest that these ‘‘subspecies’’ of A. fumigatus do not deserve the subspecies status. The isoenzyme, mtDNA, and rDNA patterns of these strains were the same, and their RAPD pat-terns were also similar to those of the other A. fumigatus strains. This observation supports the earlier proposal, based on an examination of the antigens (21), micromorphologies and secondary metabolite profiles (14), and DNA reassocia-tion kinetics (36) of strains of the A. fumigatus species and its ‘‘subspecies’’ (A. fumigatus var. ellipticus and A. fumigatus var. acolumnaris), that these strains are morphological variants of the A. fumigatus species. These results lead us to disagree with earlier attempts to classify A. fumigatus var. ellipticus and A. fumigatus var. acolumnaris as new species (A. neoellipticus and A. acolumnaris, respectively) on the basis of morphological and physiological criteria (22, 54).

There was no strict correlation between the two dendro-grams, i.e., many strains which were found to be closely related by isoenzyme analysis gave different RAPD patterns. Such a lack of a strict correlation between isoenzyme and RAPD data has also been observed among Phaffia rhodozyma strains (53), which revealed similar isoenzyme profiles and highly variable RAPD patterns. In Kluyveromyces marxianus var. marxianus strains (27) and in Candida albicans and Candida stellatoidea type I strains (26), isoenzyme analysis indicated the close re-lationship of the strains, while DNA-based techniques (elec-trophoretic karyotyping, rDNA hybridization analysis and ri-boprinting) detected high levels of polymorphisms (27, 30, 31, 33). Likewise, a strict correlation was not observed between the types on the basis of phenotypic (immunoblotting and sodium dodecyl sulfate-polyacrylamide gel electrophoresis) approaches and the groupings based on the XbaI-generated DNA patterns of the A. fumigatus isolates (7). A similar situation has been found in our case, although most strains with type A and type B EST patterns also belonged in the same cluster in the den-drogram on the basis of the RAPD data (compare the data in Table 2 and those in Fig. 5). It would be interesting to examine whether these subgroups correspond to the vegetative incom-patibility groups of A. fumigatus. The groups determined by analysis of the molecular data were consistent with genetically determined incompatibility groups in A. flavus (3) and, at least partly, in A. nidulans (9). Determination of the genetic relat-edness of A. fumigatus strains by vegetative compatibility is in progress in our laboratory.

Both dendrograms (Fig. 3 and 5) reveal the very distant relationship of strain 16 (FRR 1266) to other A. fumigatus strains. Both the mtDNA and rDNA patterns of this strain also differed from those of all other species belonging in the section Fumigati (48). This Aspergillus strain was received from J. I. Pitt (Food Research Laboratories, Commonwealth Scientific and Industrial Research Organisation (CSIRO), North Ryde, Australia). This isolate has also been morphologically reexam-ined by other Aspergillus taxonomists and found to be a slightly unusual A. fumigatus isolate showing a floccose colony mor-phology and producing an orange mycelium on Czapek-Dox medium. Such a phenomenon, i.e., the fact that the molecular data are inconsistent with morphologically based taxonomic criteria, has been observed earlier in the section Nigri of the Aspergillus genus. The A. niger aggregate was divided into two species, A. niger and A. tubingensis, on the basis of nuclear DNA and mtDNA RFLP data (24, 49–51), while these species are morphologically indistinguishable. Later, a third group which differs significantly from these two species in its rDNA

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and mtDNA patterns (50) and secondary metabolite profiles (48) was also found. Our results prompt us to propose that strain 16 (FRR 1266) represents a new species in the section Fumigati; this strain could be asexual or a mating-type strain of a heterothallic Neosartorya species, since this strain was found to be reminiscent of the anamorph of some Neosartorya species in its morphology, although fruiting body or ascospore produc-tion has never been observed. More ‘‘A. fumigatus’’ isolates originating from the same area (Warrumbungle Mountains, New South Wales, Australia) should be examined to clarify the situation.

In conclusion, among the techniques tested, RAPD analysis was found to be the most useful method for typing A. fumigatus isolates; the 61 strains could be classified into 54 groups. The efficiency of RAPD analysis was much higher than that of isoenzyme analysis. In the latter case, the 61 A. fumigatus strains could be grouped into 19 slots on the basis of the analysis of seven enzyme systems (Fig. 3). RFLP analysis of the mtDNA and the rRNA gene cluster revealed no variability among the A. fumigatus isolates.

ACKNOWLEDGMENTS

We are indebted to James H. Croft (University of Birmingham, Birmingham, United Kingdom) for valuable help and discussions. We thank Z. Kozakiewicz (IMI, Egham, United Kingdom), R. A. Samson (CBS, Baarn, The Netherlands), and S. W. Peterson (ARS, Peoria, Ill.) for reexamining strain 16 (strain FRR 1266). We also thank S. Uda-gawa, M. Dick, L. Singler, L. P. Magasi, K. Yasamoto, J. I. Pitt, J. L. Alcorn, E. Ward, D. Flett, D. H. Ellis, F. Kevei, E. Nova´k, J. Sourek, F. Staib, G. Pe´ter, L. Manczinger, and G. Szaka´cs for providing us with

Aspergillus and Neosartorya strains.

This study was supported financially by Hungarian Scientific Re-search Fund (OTKA) grants F014641 and T013044.

REFERENCES

1. Anne´, J., and J. F. Peberdy. 1981. Characterisation of interspecific hybrids between Penicillium chrysogenum and P. roqueforti by iso-enzyme analysis. Trans. Br. Mycol. Soc. 77:401–408.

2. Aufauvre-Brown, A., J. Cohen, and D. W. Holden. 1992. Use of randomly amplified polymorphic DNA markers to distinguish isolates of Aspergillus fumigatus. J. Clin. Microbiol. 30:2991–2993.

3. Bayman, P., and P. J. Cotty. 1993. Genetic diversity in Aspergillus flavus: association with aflatoxin production and morphology. Can. J. Bot. 71:23–31. 4. Beauchamp, C., and J. Fridovitch. 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44:276– 287.

5. Borsuk, P. A., M. M. Nagiec, P. P. Stepien, and E. Bartnik. 1982. Organi-zation of the ribosomal RNA gene cluster of Aspergillus nidulans. Gene

17:147–152.

6. Brewer, G. J. 1970. An introduction to isoenzyme techniques. Academic Press, Inc., New York.

7. Burnie, J. P., A. Coke, and R. C. Matthews. 1992. Restriction endonuclease analysis of Aspergillus fumigatus DNA. J. Clin. Pathol. 45:324–327. 8. Burnie, J. P., R. C. Matthews, I. Clark, and L. J. R. Milne. 1989. Immunoblot

fingerprinting Aspergillus fumigatus. J. Immunol. Methods 118:179–186. 9. Croft, J. H., and J. Varga. 1994. Application of RFLPs in systematics and

population genetics of Aspergilli, p. 277–289. In K. A. Powell, A. Renwick, and J. F. Peberdy (ed.), The genus Aspergillus: from taxonomy and genetics to industrial applications (FEMS Symposium No. 69). Plenum Press, New York.

10. Davis, B. J. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121:404–423.

11. Denning, D. W., K. V. Clemmons, L. H. Hanson, and D. A. Stevens. 1990. Restriction endonuclease analysis of total cellular DNA of Aspergillus fu-migatus isolates of geographically and epidemiologically diverse origin. J. Infect. Dis. 162:1151–1158.

12. Denning, D. W., G. S. Shankland, and D. A. Stevens. 1991. DNA finger-printing of Aspergillus fumigatus isolates from patients with aspergilloma. J. Med. Vet. Mycol. 29:339–342.

13. Duriez, T., S. Walbaum, R. Tailliez, and J. Biguet. 1976. Etude enzy-mologique comparee de souches de Aspergillus fumigatus et de A. fischeri d’origine saprophytique ou isolees de lesions humaines ou animales. Reper-cussions practiques d’ordre diagnostique. Mycopathologia 59:81–90. 14. Frisvad, J. C., and R. A. Samson. 1990. Chemotaxonomy and morphology of

Aspergillus fumigatus and related taxa, p. 201–208. In R. A. Samson and J. I. Pitt (ed.), Modern concepts in Penicillium and Aspergillus classification. Ple-num Press, New York.

15. Girardin, H., J.-P. Latge´, T. Srikantha, B. Morrow, and D. R. Soll. 1993. Development of DNA probes for fingerprinting Aspergillus fumigatus. J. Clin. Microbiol. 31:1547–1554.

16. Harris, H., and D. A. Hopkinson. 1976. Handbook of enzyme electrophoresis in human genetics. Elsevier North-Holland Biomedical Press, Amsterdam. 17. Hearn, V. M., M. Moutaouakil, and J.-P. Latge´. 1990. Analysis of compo-nents of Aspergillus and Neosartorya mycelial preparations by gel electro-phoresis and Western blotting procedures, p. 235–247. In R. A. Samson and J. I. Pitt (ed.), Modern concepts in Penicillium and Aspergillus classification. Plenum Press, New York.

18. Hu, J., and C. F. Quiros. 1991. Identification of broccoli and cauliflower cultivars with RAPD markers. Plant Cell Rep. 10:505–511.

19. Ka´lma´n, E. T., J. Varga, and F. Kevei.1991. Characterization of interspecific hybrids within the Aspergillus nidulans species group by isoenzyme analysis. Can. J. Microbiol. 37:391–396.

20. Kelly, J. M., M. R. Drysdale, H. M. Sealy-Lewis, I. G. Jones, and R. A.

Lockington.1990. Alcohol dehydrogenase III in Aspergillus nidulans is anaer-obically induced and post-transcriptionally regulated. Mol. Gen. Genet. 222: 323–328.

21. Kim, S. J., and S. D. Chaparas. 1979. Characterization of antigens from Aspergillus fumigatus. III. Comparison of antigenic relationships of clinically important aspergilli. Am. Rev. Respir. Dis. 120:1297–1303.

22. Kozakiewicz, Z. 1989. Aspergillus species on stored products. Mycol. Papers

161:1–188.

23. Kurup, V. P., J. N. Fink, G. H. Scribner, and M. J. Falk. 1977. Antigenic variability of Aspergillus fumigatus strains. Microbios 19:191–204. 24. Kusters-van Someren, M. A., R. A. Samson, and J. Visser. 1991. The use of

RFLP analysis in classification of the black Aspergilli: reinterpretation of Aspergillus niger aggregate. Curr. Genet. 19:21–26.

25. Leach, J., D. B. Finkelstein, and J. A. Rambosek. 1986. Rapid miniprep of DNA from filamentous fungi. Fungal Genet. Newsl. 33:32–33.

26. Lehmann, P. F., B. J. Kemker, C.-B. Hsiao, and S. Dev. 1989. Isoenzyme biotypes of Candida species. J. Clin. Microbiol. 27:2514–2521.

27. Lehmann, P. F., U. Khazan, L.-C. Wu, B. I. Wickes, and K. J. Kwon-Chung. 1992. Karyotype and isozyme profiles do not correlate in Kluyveromyces marxianus var. marxianus. Mycol. Res. 96:637–642.

28. Leslie, C. E., B. Flannigan, and L. J. R. Milne. 1988. Morphological studies on clinical isolates of Aspergillus fumigatus. J. Med. Vet. Mycol. 26:335–341. 29. Loudon, K. W., J. P. Burnie, A. P. Coke, and R. C. Matthews. 1993. Appli-cation of polymerase chain reaction to fingerprinting Aspergillus fumigatus by random amplification of polymorphic DNA. J. Clin. Microbiol. 31:1117– 1121.

30. Magee, B. B., T. M. D’Souza, and P. T. Magee. 1987. Strain and species identification by restriction fragment length polymorphisms in the ribosomal DNA repeat of Candida species. J. Bacteriol. 169:1639–1643.

31. Magee, B. B., and P. T. Magee. 1987. Electrophoretic karyotypes and chro-mosome numbers in Candida species. J. Gen. Microbiol. 133:425–430. 32. Matsuda, H., S. Kohno, S. Maesaki, H. Yamada, H. Koga, M. Tamura, H.

Kuraishi, and J. Sugiyama.1992. Application of ubiquinone systems and electrophoretic comparison of enzymes to identification of clinical isolates of Aspergillus fumigatus and several other species of Aspergillus. J. Clin. Micro-biol. 30:1999–2005.

33. Molina, F. I., T. Inoue, and S.-C. Jong. 1992. Determination of infraspecific relationships in Kluyveromyces marxianus by riboprinting. Mycotaxon 43:49–60. 34. Moody, S. F., and B. M. Tyler. 1990. Restriction enzyme analysis of mito-chondrial DNA of the Aspergillus flavus group: A. flavus, A. parasiticus, and A. nomius. Appl. Environ. Microbiol. 56:2441–2452.

35. Mulvey, M., and R. C. Vrijenhoek. 1981. Genetic variation among laboratory strains of the planorbid snail Biomphalaria glabrata. Biochem. Genet. 19: 1169–1182.

36. Peterson, S. W. 1992. Neosartorya pseudofischeri sp. nov. and its relationship to other species in Aspergillus section Fumigati. Mycol. Res. 96:547–554. 37. Podani, J. 1993. SYN-TAX-pc. Computer programs for multivariate data

analysis in ecology and systematics. Version 5.0 user’s guide. Scientia Pub-lishing, Budapest.

38. Polonelli, L., S. Conti, L. Campani, and F. Fanti. 1990. Biotyping of As-pergillus fumigatus and related taxa by the yeast killer system, p. 225–233. In R. A. Samson and J. I. Pitt (ed.), Modern concepts in Penicillium and Aspergillus classification. Plenum Press, New York.

39. Pontecorvo, G., J. A. Roper, L. M. Hemmons, K. D. MacDonald, and A. W. J.

Bufton.1953. The genetics of Aspergillus nidulans. Adv. Genet. 5:141–238. 40. Raper, K. B., and D. I. Fennell. 1965. The genus Aspergillus. The Williams &

Wilkins Co., Baltimore.

41. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

42. Sneath, P. H. A. 1972. Computer taxonomy. Methods Microbiol. 7A:29–98. 43. Sneath, P. H. A., and R. R. Sokal. 1973. Numerical taxonomy. W. H.

Free-man & Co., San Francisco.

on May 15, 2020 by guest

http://jcm.asm.org/

(9)

J. Clin. Microbiol. 31:615–621.

45. Spreadbury, C. L., B. W. Bainbridge, and J. Cohen. 1990. Restriction frag-ment length polymorphisms in isolates of Aspergillus fumigatus probed with part of the intergenic spacer region from the ribosomal RNA gene complex of Aspergillus nidulans. J. Gen. Microbiol. 136:1991–1994.

46. Tang, C. M., D. W. Holden, A. Aufauvre-Brown, and J. Cohen. 1993. The detection of Aspergillus spp. by the polymerase chain reaction and its eval-uation in bronchoalveolar lavage fluid. Am. Rev. Respir. Dis. 148:1313–1317. 47. van Belkum, A., W. G. W. Quint, B. E. de Pauw, W. J. G. Melchers, and J. F.

Meis.1993. Typing of Aspergillus species and Aspergillus fumigatus by inter-repeat polymerase chain reaction. J. Clin. Microbiol. 31:2502–2505. 48. Varga, J. Unpublished data.

49. Varga, J., C. Fekete, F. Kevei, and J. H. Croft. 1989. Studies on mitochon-drial DNA polymorphism and protoplast fusion in black Aspergilli, abstr. MO1. In Abstracts of the 19th FEBS Meeting. Studio EGA, Rome. 50. Varga, J., F. Kevei, F. Debets, Z. Kozakiewicz, and J. H. Croft. 1994.

Mito-chondrial DNA restriction fragment length polymorphisms in field isolates of the Aspergillus niger aggregate. Can. J. Microbiol. 40:612–621. 51. Varga, J., F. Kevei, C. Fekete, A. Coenen, Z. Kozakiewicz, and J. H. Croft.

52. Varga, J., and E. Rinyu. 1991. Characterization of Aspergillus fumigatus strains by isoenzyme analysis. Acta Microbiol. Hung. 38:225.

53. Varga, J., C. Va´gvo¨lgyi, A´. Nagy, I. Pfeiffer, and L. Ferenczy. 1995.

Isoen-zyme, restriction fragment length polymorphism, and random amplified polymorphic DNA characterization of Phaffia rhodozyma Miller et al. Int. J. Syst. Bacteriol. 45:173–177.

54. Varshney, J. L., and A. K. Sarbhoy. 1981. A new species of Aspergillus fumigatus group and comments upon its taxonomy. Mycopathologia 73:89– 92.

55. Wallenbeck, I., L. Aukrust, and R. Einarsson. 1984. Antigenic variability of different strains of Aspergillus fumigatus. Int. Arch. Allergy Appl. Immunol.

73:166–172.

56. Williams, J. G. K., A. R. Kubelik, K. J. Livak, J. A. Rafalski, and S. V.

Tingey.1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18:6531–6535.

57. Woodbury, W., A. K. Spencer, and M. A. Stanmann. 1971. An improved procedure using ferricyanide for detecting catalase isozymes. Anal. Biochem.

44:301–305.

on May 15, 2020 by guest

http://jcm.asm.org/

Figure

TABLE 1. A. fumigatus strains examined in our study

TABLE 1.

A. fumigatus strains examined in our study p.2
TABLE 2. Enzymes examined in the study

TABLE 2.

Enzymes examined in the study p.3
FIG. 1. Isoenzyme patterns of the A. fumigatuspseudofischeri strains. For the abbreviations used and for the strains showing these patterns, see Table 2
FIG. 1. Isoenzyme patterns of the A. fumigatuspseudofischeri strains. For the abbreviations used and for the strains showing these patterns, see Table 2 p.4
FIG. 2. EST patterns of some A. fumigatusstrain numbers above the lanes, see Table 1. Lane A,lane a, strains
FIG. 2. EST patterns of some A. fumigatusstrain numbers above the lanes, see Table 1. Lane A,lane a, strains p.5
FIG. 3. UPGMA dendrogram of the A. fumigatusliaedendrogram was based on their isoenzyme patterns
FIG. 3. UPGMA dendrogram of the A. fumigatusliaedendrogram was based on their isoenzyme patterns p.5
FIG. 4. RAPD patterns of some of the A. fumigatusLane M, strains with OPC-10 used as the primer
FIG. 4. RAPD patterns of some of the A. fumigatusLane M, strains with OPC-10 used as the primer p.6
FIG. 5. UPGMA dendrogram of the A. fumigatusdendrogram was based on their RAPD patterns
FIG. 5. UPGMA dendrogram of the A. fumigatusdendrogram was based on their RAPD patterns p.6

References