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AMPLIFICATION OF FUNGAL rDNA-ITS REGIONS FROM NON-FERTILE SPECIMENS OF THE LICHEN-FORMING GENUSPARMELIA
Lichenologist 29(3): 275–282 (1997)
AMPLIFICATION OF FUNGAL rDNA-ITS REGIONS FROM NON-FERTILE SPECIMENS OF THE LICHEN-FORMING GENUS PARMELIA Ana CRESPO*, Paul D. BRIDGE‡ and David L. HAWKSWORTH‡
Abstract: A method is described for the selective amplification of single mycobiont rDNA sequences from vegetative, non-ascomatal material of the lichenized thalli of Parmelia sulcata and P. saxatilis. The method includes a simple DNA extraction procedure employing rhizines, which avoids the use of liquid nitrogen disruption methods and phenol based extractions. The resulting amplified DNA is suitable for restriction enzyme analysis, and can be used to provide material for investigating population distribution and species concepts. ? 1997 The British Lichen Society
Introduction The molecular analysis of DNA is making a major contribution to the understanding of fungal biology and relationships (Hawksworth & Mouchacca 1994) and there is considerable potential for the application of molecular methods to the examination of phylogenetic relationships and population biology within the lichen-forming fungi. A number of different methods have recently been reviewed for the extraction and amplification of DNA from lichen mycobionts (Ahmadjian et al. 1987; Armaleo & Clerc 1991, 1995; DePriest 1994; Grube et al. 1995). DNA extraction methods for lichens need to be able to purify the DNA from both small samples of freshly collected environmental material or preserved material, which can be of considerable age. A further complication to the extraction of DNA from lichens is that initial samples often contain high levels of polysaccharides and other complex molecules, which can reduce both the efficiency of the DNA extraction and the final purity of the DNA extract. A significant problem with amplification of mycobiont DNA from lichens is the common presence of more than one ribosomal DNA type in a single specimen (Gargas & Taylor 1992; Fasshelt 1996) in addition to DNA derived form the photobiont. A further complication is that both the upper and lower surfaces of mature foliose macrolichens can harbour a wide range of other fungi and other microorganisms, bryophytes, invertebrates, and invertebrates’ eggs. This has been demonstrated by DePriest (1994) where polymerase chain reaction (PCR) primers used to amplify parts of the rDNA repeat unit from the fruticose lichen Cladonia chlorophaea were also found to amplify DNA from the photobiont Trebouxia erici and Saccharomyces cerevisiae used as a carrier DNA in the extraction procedure. *Departamento de Biologı´a Vegetal II, Universidad Complutense de Madrid, 28040 Madrid, Spain. ‡International Mycological Institute, Bakeham Lane, Egham, Surrey TW20 9TY, UK. 0024–2829/97/030275+08 $25.00/0/li960071
? 1997 The British Lichen Society
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The specificity of PCR primers is therefore of considerable importance if algal DNA products are to be avoided. Universal primers, such as ITS1 and ITS4 described for the rDNA internally transcribed spacer regions by White et al. (1990) may preferentially amplify fungal DNA in some plant-fungal interactions (Gardes et al. 1991), but in other cases both plant and fungal DNA may be amplified (Gardes & Bruns 1993). The sensitivity of PCR reactions is such that contamination with algal DNA cannot be excluded even from apparently algal-free tissue such as the medulla, lower cortex, and rhizines. Recently Grube et al. (1995) published a protocol that allowed for the extraction of DNA from lichen ascomata, but this is of limited use in lichenized species where ascomata are rarely or never produced, and a reliable method is required for the extraction of purified DNA in those cases. A possible complication to the use of ascomata is that several kinds of ascomata may also contain photobiont material or lichenicolous fungi. Ascomata also contain ascospores derived from the thallus mycobiont through meiosis, therefore a molecular analysis of lichens based on these structures will be an analysis of a mixture of the parental material in the ascomatal and hamathecial tissues and the progeny of the individuals, and so may be based on mixed genotypes. Without extensive sampling of local and dispersed populations it is not possible to predict which genotypes will persist in a particular environment, and some comparison to the genotype of the vegetatively reproducing thallus material will be necessary in any study of populations or extant relationships. In this paper we report the extraction of DNA from vegetatively produced lichen rhizines through a simple procedure avoiding the use of phenol and liquid nitrogen extractions. This DNA was amplified with fungal specific rDNA ITS primers, and size and restriction site analysis of the PCR product showed differences between specimens of Parmelia sulcata and P. saxatilis. The PCR amplification from rhizine and thallus samples of Parmelia sulcata was compared between two different sets of rDNA ITS primers. Materials and Methods Lichens Four specimens comprising isolated single thalli of Parmelia sulcata Taylor and P. saxatilis (L.) Ach. were collected from Spain (Madrid: El Escorial, Silla de Felip II, July 1995, A. Crespo, MAF & IMI). Specimens were air-dried and stored at room temperature. Preliminary procedure The air-dried specimens were washed in a continual stream of tap water and dried at room temperature for a minimum of 24 h. Specimens were then washed in 95% ethanol. Tissue samples of rhizines and thallus material were dissected with ethanol-sterilized razor blades and tweezers with the aid of a dissecting microscope, avoiding any other evident living material. Samples were stored in microcentrifuge tubes at "20)C until required. DNA extraction DNA extraction was based on the general CTAB method detailed in Paterson & Bridge (1994). Lichen samples (30-40 mg) were suspended with 15 mg carborundum in 100ìl of 2% CTAB
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buffer (700 mM NaCl; 50 mM Trizma-HCl, pH 8; 10 mM EDTA; 2% cetyltrimethyl ammonium bromide). The lichen material was disrupted by grinding this mixture in a microcentrifuge tube with a close-fitting micro-homogenizer attached to a small electric motor. The samples were each ground for three separate periods of 2 min alternated with 2 min chilling. The resulting slurry was then diluted by the addition of a further 400 ìl of the CTAB buffer with 1% â-mercaptoethanol. The microcentrifuge tubes were than incubated at 60)C for 30 min to inactivate nucleases, after which an equal volume (500 ìl) of chloroform-isoamyl alcohol (24:1 v/v) was added to each tube. The tubes were briefly mixed and then centrifuged at 10 000 # g for 10 min to separate aqueous and solvent layers. The upper aqueous layers were collected in clean microcentrifuge tubes with a wide-bore micropipette tip and precipitated by the addition of 0·54 vol isopropyl alcohol. The precipitate was collected by a brief centrifugation, drained and redissolved in 700 ìl of TE buffer (10 mM Trizma-HCl, pH 8; 1 mM EDTA). After redissolving, 20 units of ribonuclease A were added and the tubes were incubated at 37)C for 30 min. The DNA solutions were further purified by a second treatment with an equal volume of chloroform-isoamyl alcohol and the upper aqueous layer was again collected with a wide-bore micropipette tip. The aqueous layer was adjusted with 7·5 M ammonium acetate to give a final concentration of 1·5 M ammonium acetate (1/4 total volume added) and DNA was precipitated by adding 95% ethanol to give a final volume of 1·5 ml. The precipitate was briefly centrifuged and drained before redissolving in 500 ìl of 200 mM ammonium acetate. The final purification step was to re-precipitate the DNA with a double volume of 95% ethanol as before and the pellet was vacuum dried and dissolved in an minimum volume of TE buffer (5–10 ìl). PCR methods Amplification of the internally transcribed spacer region of the rDNA gene cluster was undertaken with the primer pairs SR6R 5*AAGTAGGTCGTAACAAGG3* and LR1 5*GGTT GGTTTCTTTTCCT3* used by DePriest (1994), and ITS1-F (5*CTTGGTCATTTAGAG GAAGTAA3*) described by Gardes & Bruns (1993) and ITS4 (5*TCCTCCGCTTATTGA TATGC3*) described by White et al. (1990). ITS1-F was designed to be specific for fungal sequences at the 3* end of the small subunit gene of the rDNA and overlaps with ITS5, whereas ITS4 has been described as a ‘ universal ’ primer and hybridizes at the 5* end of the large subunit gene (White et al. 1990; Gardes & Bruns 1993). Amplification was carried out in 50 ìl volumes consisting of 5 ìl of 10 # Tth buffer (HT Biotechnology, Cambridge), 1 ìl of a 10 "1 dilution of stock DNA, 3 ìl of 25 mM MgCl2, 4 ìl of a dinucleotide triphosphate (dNTP) mixture containing each base (Pharmacia) at 2·5 mM, 2·5 ìl of each primer (all at 200 ìM except ITS1-F at 100 ìM) and 0·25 ìl of a 1 in 5 dilution of Tth DNA polymerase (HT Biotechnology, Cambridge). Amplification was performed in Techne PHC3 thermal cycler set at 1 min at 94)C, 45 s at 50)C, and 2 min at 72)C for 40 cycles, followed by a final cycle of 5 min at 72)C. DNA products were separated by electrophoresis in 1·3% NuSieve 3:1 agarose (FMC Ltd) in tris-acetate-EDTA buffer (TAE; Sambrook et al. 1989). DNA was visualized under UV light after staining for 1 h in ethidium bromide (0.5 ìg ml "1). DNA digestion PCR products (5 ìl) were digested for 16 h with 30U EcoRI or HaeIII (Life Technology Ltd) in a 20 ìl reaction of diluted restriction enzyme buffer. Restriction products were separated by electrophoresis as described above. Reproducibility Each extraction and amplification was carried out at least twice on different occasions.
Results and Discussion DNA was successfully extracted from both rhizine and thallus material. DNA concentrations were in general low, with typical yields of 25–100 pg purified DNA from 30 mg rhizines. This may be in part due to the nature of the rhizines, which were often very vacuolated.
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T 1. Amplification products from Parmelia sulcata and P. saxatilis P. sulcata Location
18062
18064
P. saxatilis
26072
26074
18061
18063
26071
26073
na 2b
na 2b
na 2b
1b 1b
3b 3b
1b 1b
Primer pair SR6R/LR1 Rhizinae Thallus
1b* 2b
1b 2b
na 2b
Rhizinae Thallus
1b 1b
1b 1b
1b 1b
1b 1b
1b 1b
Primer pair ITS1-F/ITS4 1b 1b
na 1b
*b=number of bands seen, na=not available.
Samples of DNA from rhizine and thallus material were amplified with the primer pair described by DePriest (1994). This procedure generally gave either one or two amplification products of about 500–1000 base pair (bp) length (Table 1; Fig. 1). The number of products varied both between samples and with the storage of the sample. In general rhizine material either failed to amplify or gave one band and thallus material gave multiple bands. The multiple PCR products may indicate amplification from both the mycobiont and the photobiont, the presence of more than one mycobiont or multiple rDNA types. DePriest (1994) reported amplification of rDNA from both Trebouxia erici and Saccharomyces cerevisiae with the same primer pair. The thallus material will undoubtedly contain both the photo- and mycobionts, whereas rhizine material will be largely from the mycobiont although there may also be some photobiont contamination of rhizines. The rhizine material usually showed fewer bands than the thallus material, and it would seem most likely that the second fainter band is due to photobiont contamination. The variation in intensity of bands with the condition of the sample has also been described by DePriest (1994), who reported greater intensities from Trebouxia-derived fragments in dilute and degraded DNA stocks. Amplification of rhizine-derived DNA from the same Parmelia specimens with the primer pair ITS1-F and ITS4 gave a single PCR product of about 650 bp in the P. sulcata material and 600 bp from P. saxatilis. Amplification of thallus material of one specimen (26071; Table 1) gave between 1 and 3 bands, and this specimen has since been found to contain the parasitic fungus Abrothallus parmeliarum, in addition to the lichen symbionts. The primer pair ITS1-F and ITS4 has been investigated by Gardes & Bruns (1993) for amplifying fungal ITS regions in comparison to plant DNA. This pair of primers was reported to specifically amplify fungal DNA, although they found that attempts at amplification of some plant DNA also gave faint bands. As the reported increased specificity of these primers has excluded the previously seen fragments from the rhizine samples, it would seem likely that the multiple bands amplified with these primers from thallus material may indicate the presence of multiple rDNA types, further mycobionts or other associated fungi (e.g. lichenicolous fungi).
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Parmelia fungal and DNA sequences—Crespo et al. (A)
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F. 1. 1A & B. A, Example of PCR products from Parmelia saxatilis and P. sulcata. Lanes 1 & 10=Kb markers, lanes 2 & 3 and 6 & 7=paired rhizinae and thallus material from P. saxatilis, lanes 4 & 5 and 8 & 9=the same for P. sulcata. Both amplified with primers described by DePriest (1994). B, As A but amplified with the ITS1-F/ITS4 primer pair.
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2 3
4 5 6
7 8
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9 10 11
F. 2. Example of digestion of PCR products. Lanes 1, 6 & 11=Kb markers, lanes 2, 3, 4 & 5=P. sulcata PCR products: (2) digested with EcoRI, (3) undigested, (4) digested with HaeIII or (5) HinfI. Lanes 7, 8, 9 & 10=P. saxatilis PCR products (7): digested with EcoRI, (8) undigested, (9) digested with HaeIII or (10) HinfI.
The single 650 and 600 bp bands obtained from the rhizine material were digested with EcoRI, HaeIII and HinfI in order to verify singularity. The result of the EcoRI restriction suggested a single site near the centre of the ITS region, as the restriction products from P. sulcata and P. saxatilis were two bands of 355 and 295 bp and 330 and 270 bp, respectively (Fig.2; lanes 2 & 7). The sums of these fragments correspond to the relative sizes of the single ITS fragments. The HaeIII digestion resulted in a reduction of about half in the size of the ITS fragments from the two species (Fig. 2, lanes 4 & 9), which may be due to either multiple HaeIII sites at the end of the fragment, or a single site giving rise to a small single fragment. The P. sulcata samples gave a broad band at below 100 bp that may suggest multiple small fragments (see Fig. 2). The HinfI digestion gave 3 fragments from the P. sulcata material and 2 from P. saxatilis (Fig. 2; lanes 5 & 10). The results presented here demonstrate that a simple DNA extraction technique can be combined with specific PCR primers to reliably isolate mycobiont DNA. As the starting material is existing vegetative structures from non-fertile specimens, the genotypes examined are those of the original
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specimen, and not meiotically derived progeny or mixtures. This technique therefore has particular value in the determination of relationships within and between existing populations and the examination of the extent to which several mycobionts are involved in a single thallus. The DNA extraction reported is simple and inexpensive and avoids the use of phenol and liquid nitrogen. Subsequent work has shown that the mercaptoethanol may be omitted from the initial disruption buffer without significantly reducing the efficiency of the extraction from freshly isolated specimens, however the mercaptoethanol has been found necessary when dealing with older herbarium specimens. Although there is the possibility of co-purification of non-DNA contaminating material, in our experience contamination can be nullified by dilution of the DNA sample of up to 1:20 without loss of PCR product. The PCR products in this case are the systematically important ITS regions of the rDNA gene cluster and this extraction and amplification procedure is suitable for the production of material for sequencing, in addition to restriction digestion analysis. The primer ITS1-F overlaps the primer ITS5 and is located before the ‘ universal ’ ITS1 primer (Gardes & Bruns 1993). This method therefore also offers some potential in lichens where pure mycobiont DNA is not easily available, for example because of the absence of rhizines. In those cases a modification of the above procedure with the ITS-1F and ITS-4 primers and subsequent amplification of either the two single spacer regions with ‘ universal ’ primers in a nested-PCR, may provide sufficient specificity. This work was carried out at the International Mycological Institute with the support of projects Ant 94-0905 from the Secretaria General del Plan Nacional de I+D, and APC-0020 from Dirección General de Investigación Cientifica y Técnica, both from the Ministerio de Educación y Ciencia, Spain. R Ahmadjian, V., Chadegani, M., Koriem, A. M. & Paracer, S. (1987) DNA and protoplast isolation from lichens and lichen symbionts. Lichen Physiology and Biochemistry 2: 1–11. Armaleo, D. & Clerc, P. (1991) Lichen chimeras: DNA analysis suggests that one fungus forms two morphotypes. Experimental Mycology 15: 1–10. Armaleo, D. & Clerc, P. (1995) A rapid and inexpensive method for the purification of DNA from lichens and their symbionts. Lichenologist 27: 207–213. DePriest, P. T. (1994) Variation in the Cladonia chlorophaea complex II: Ribosomal DNA variation in a southern Appalachian population. Bryologist 97: 117–126. Fahselt, D. (1996) Individuals, populations and population ecology. In Lichen Biology (T. H. Nash , ed.): 181–198. Cambridge: Cambridge University Press Gardes, M. & Bruns, T. D. (1993) ITS primers with enhanced specificity for basidiomycetes— application to the identification of mycorrhizae and rusts. Molecular Ecology 2: 113–118. Gardes, M., White, T. J., Fortin, J. A., Bruns, T. D. & Taylor, J. W. (1991) Identification of indigenous and introduced symbiotic fungi in ectomycorrhizae by amplification of nuclear and mitochondrial ribosomal DNA. Canadian Journal of Botany 69: 180–190. Gargas, A. & Taylor, J. W. (1992) Polymerase chain reaction (PCR) primers for amplifying and sequencing nuclear 18s rDNA from lichenized fungi. Myocologia 84: 589–592. Grube, M., DePriest, P. T., Gargas, A. & Hafellner, J. (1995) DNA isolation from lichen ascomata. Mycological Research 99: 1321–1324. Hawksworth, D. L. & Mouchacca, J. (1994) Ascomycete systematics in the nineties. In Ascomycete Systematics: Problems and Perspectives in the Nineties (D. L. Hawksworth, ed.): 3–11. New York: Plenum Press.
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Paterson, R. R. M. & Bridge, P. D. (1994) Biochemical Techniques for Filamentous Fungi. [IMI Technical Handbooks No. 1.] Wallingford: CAB International. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd Edn. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. White, T. J., Bruns, T. D., Lee, S. & Taylor, J. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols (M. A. Innis, D. H. Gelfand, J. J. Sninsky, & T. J. White, eds). New York: Academic Press. Accepted for publication 5 September 1996
AMPLIFICATION OF FUNGAL rDNA-ITS REGIONS FROM NON-FERTILE SPECIMENS OF THE LICHEN-FORMING GENUS PARMELIA Ana CRESPO*, Paul D. BRIDGE‡ and David L. HAWKSWORTH‡
Abstract: A method is described for the selective amplification of single mycobiont rDNA sequences from vegetative, non-ascomatal material of the lichenized thalli of Parmelia sulcata and P. saxatilis. The method includes a simple DNA extraction procedure employing rhizines, which avoids the use of liquid nitrogen disruption methods and phenol based extractions. The resulting amplified DNA is suitable for restriction enzyme analysis, and can be used to provide material for investigating population distribution and species concepts. ? 1997 The British Lichen Society
Introduction The molecular analysis of DNA is making a major contribution to the understanding of fungal biology and relationships (Hawksworth & Mouchacca 1994) and there is considerable potential for the application of molecular methods to the examination of phylogenetic relationships and population biology within the lichen-forming fungi. A number of different methods have recently been reviewed for the extraction and amplification of DNA from lichen mycobionts (Ahmadjian et al. 1987; Armaleo & Clerc 1991, 1995; DePriest 1994; Grube et al. 1995). DNA extraction methods for lichens need to be able to purify the DNA from both small samples of freshly collected environmental material or preserved material, which can be of considerable age. A further complication to the extraction of DNA from lichens is that initial samples often contain high levels of polysaccharides and other complex molecules, which can reduce both the efficiency of the DNA extraction and the final purity of the DNA extract. A significant problem with amplification of mycobiont DNA from lichens is the common presence of more than one ribosomal DNA type in a single specimen (Gargas & Taylor 1992; Fasshelt 1996) in addition to DNA derived form the photobiont. A further complication is that both the upper and lower surfaces of mature foliose macrolichens can harbour a wide range of other fungi and other microorganisms, bryophytes, invertebrates, and invertebrates’ eggs. This has been demonstrated by DePriest (1994) where polymerase chain reaction (PCR) primers used to amplify parts of the rDNA repeat unit from the fruticose lichen Cladonia chlorophaea were also found to amplify DNA from the photobiont Trebouxia erici and Saccharomyces cerevisiae used as a carrier DNA in the extraction procedure. *Departamento de Biologı´a Vegetal II, Universidad Complutense de Madrid, 28040 Madrid, Spain. ‡International Mycological Institute, Bakeham Lane, Egham, Surrey TW20 9TY, UK. 0024–2829/97/030275+08 $25.00/0/li960071
? 1997 The British Lichen Society
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The specificity of PCR primers is therefore of considerable importance if algal DNA products are to be avoided. Universal primers, such as ITS1 and ITS4 described for the rDNA internally transcribed spacer regions by White et al. (1990) may preferentially amplify fungal DNA in some plant-fungal interactions (Gardes et al. 1991), but in other cases both plant and fungal DNA may be amplified (Gardes & Bruns 1993). The sensitivity of PCR reactions is such that contamination with algal DNA cannot be excluded even from apparently algal-free tissue such as the medulla, lower cortex, and rhizines. Recently Grube et al. (1995) published a protocol that allowed for the extraction of DNA from lichen ascomata, but this is of limited use in lichenized species where ascomata are rarely or never produced, and a reliable method is required for the extraction of purified DNA in those cases. A possible complication to the use of ascomata is that several kinds of ascomata may also contain photobiont material or lichenicolous fungi. Ascomata also contain ascospores derived from the thallus mycobiont through meiosis, therefore a molecular analysis of lichens based on these structures will be an analysis of a mixture of the parental material in the ascomatal and hamathecial tissues and the progeny of the individuals, and so may be based on mixed genotypes. Without extensive sampling of local and dispersed populations it is not possible to predict which genotypes will persist in a particular environment, and some comparison to the genotype of the vegetatively reproducing thallus material will be necessary in any study of populations or extant relationships. In this paper we report the extraction of DNA from vegetatively produced lichen rhizines through a simple procedure avoiding the use of phenol and liquid nitrogen extractions. This DNA was amplified with fungal specific rDNA ITS primers, and size and restriction site analysis of the PCR product showed differences between specimens of Parmelia sulcata and P. saxatilis. The PCR amplification from rhizine and thallus samples of Parmelia sulcata was compared between two different sets of rDNA ITS primers. Materials and Methods Lichens Four specimens comprising isolated single thalli of Parmelia sulcata Taylor and P. saxatilis (L.) Ach. were collected from Spain (Madrid: El Escorial, Silla de Felip II, July 1995, A. Crespo, MAF & IMI). Specimens were air-dried and stored at room temperature. Preliminary procedure The air-dried specimens were washed in a continual stream of tap water and dried at room temperature for a minimum of 24 h. Specimens were then washed in 95% ethanol. Tissue samples of rhizines and thallus material were dissected with ethanol-sterilized razor blades and tweezers with the aid of a dissecting microscope, avoiding any other evident living material. Samples were stored in microcentrifuge tubes at "20)C until required. DNA extraction DNA extraction was based on the general CTAB method detailed in Paterson & Bridge (1994). Lichen samples (30-40 mg) were suspended with 15 mg carborundum in 100ìl of 2% CTAB
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buffer (700 mM NaCl; 50 mM Trizma-HCl, pH 8; 10 mM EDTA; 2% cetyltrimethyl ammonium bromide). The lichen material was disrupted by grinding this mixture in a microcentrifuge tube with a close-fitting micro-homogenizer attached to a small electric motor. The samples were each ground for three separate periods of 2 min alternated with 2 min chilling. The resulting slurry was then diluted by the addition of a further 400 ìl of the CTAB buffer with 1% â-mercaptoethanol. The microcentrifuge tubes were than incubated at 60)C for 30 min to inactivate nucleases, after which an equal volume (500 ìl) of chloroform-isoamyl alcohol (24:1 v/v) was added to each tube. The tubes were briefly mixed and then centrifuged at 10 000 # g for 10 min to separate aqueous and solvent layers. The upper aqueous layers were collected in clean microcentrifuge tubes with a wide-bore micropipette tip and precipitated by the addition of 0·54 vol isopropyl alcohol. The precipitate was collected by a brief centrifugation, drained and redissolved in 700 ìl of TE buffer (10 mM Trizma-HCl, pH 8; 1 mM EDTA). After redissolving, 20 units of ribonuclease A were added and the tubes were incubated at 37)C for 30 min. The DNA solutions were further purified by a second treatment with an equal volume of chloroform-isoamyl alcohol and the upper aqueous layer was again collected with a wide-bore micropipette tip. The aqueous layer was adjusted with 7·5 M ammonium acetate to give a final concentration of 1·5 M ammonium acetate (1/4 total volume added) and DNA was precipitated by adding 95% ethanol to give a final volume of 1·5 ml. The precipitate was briefly centrifuged and drained before redissolving in 500 ìl of 200 mM ammonium acetate. The final purification step was to re-precipitate the DNA with a double volume of 95% ethanol as before and the pellet was vacuum dried and dissolved in an minimum volume of TE buffer (5–10 ìl). PCR methods Amplification of the internally transcribed spacer region of the rDNA gene cluster was undertaken with the primer pairs SR6R 5*AAGTAGGTCGTAACAAGG3* and LR1 5*GGTT GGTTTCTTTTCCT3* used by DePriest (1994), and ITS1-F (5*CTTGGTCATTTAGAG GAAGTAA3*) described by Gardes & Bruns (1993) and ITS4 (5*TCCTCCGCTTATTGA TATGC3*) described by White et al. (1990). ITS1-F was designed to be specific for fungal sequences at the 3* end of the small subunit gene of the rDNA and overlaps with ITS5, whereas ITS4 has been described as a ‘ universal ’ primer and hybridizes at the 5* end of the large subunit gene (White et al. 1990; Gardes & Bruns 1993). Amplification was carried out in 50 ìl volumes consisting of 5 ìl of 10 # Tth buffer (HT Biotechnology, Cambridge), 1 ìl of a 10 "1 dilution of stock DNA, 3 ìl of 25 mM MgCl2, 4 ìl of a dinucleotide triphosphate (dNTP) mixture containing each base (Pharmacia) at 2·5 mM, 2·5 ìl of each primer (all at 200 ìM except ITS1-F at 100 ìM) and 0·25 ìl of a 1 in 5 dilution of Tth DNA polymerase (HT Biotechnology, Cambridge). Amplification was performed in Techne PHC3 thermal cycler set at 1 min at 94)C, 45 s at 50)C, and 2 min at 72)C for 40 cycles, followed by a final cycle of 5 min at 72)C. DNA products were separated by electrophoresis in 1·3% NuSieve 3:1 agarose (FMC Ltd) in tris-acetate-EDTA buffer (TAE; Sambrook et al. 1989). DNA was visualized under UV light after staining for 1 h in ethidium bromide (0.5 ìg ml "1). DNA digestion PCR products (5 ìl) were digested for 16 h with 30U EcoRI or HaeIII (Life Technology Ltd) in a 20 ìl reaction of diluted restriction enzyme buffer. Restriction products were separated by electrophoresis as described above. Reproducibility Each extraction and amplification was carried out at least twice on different occasions.
Results and Discussion DNA was successfully extracted from both rhizine and thallus material. DNA concentrations were in general low, with typical yields of 25–100 pg purified DNA from 30 mg rhizines. This may be in part due to the nature of the rhizines, which were often very vacuolated.
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T 1. Amplification products from Parmelia sulcata and P. saxatilis P. sulcata Location
18062
18064
P. saxatilis
26072
26074
18061
18063
26071
26073
na 2b
na 2b
na 2b
1b 1b
3b 3b
1b 1b
Primer pair SR6R/LR1 Rhizinae Thallus
1b* 2b
1b 2b
na 2b
Rhizinae Thallus
1b 1b
1b 1b
1b 1b
1b 1b
1b 1b
Primer pair ITS1-F/ITS4 1b 1b
na 1b
*b=number of bands seen, na=not available.
Samples of DNA from rhizine and thallus material were amplified with the primer pair described by DePriest (1994). This procedure generally gave either one or two amplification products of about 500–1000 base pair (bp) length (Table 1; Fig. 1). The number of products varied both between samples and with the storage of the sample. In general rhizine material either failed to amplify or gave one band and thallus material gave multiple bands. The multiple PCR products may indicate amplification from both the mycobiont and the photobiont, the presence of more than one mycobiont or multiple rDNA types. DePriest (1994) reported amplification of rDNA from both Trebouxia erici and Saccharomyces cerevisiae with the same primer pair. The thallus material will undoubtedly contain both the photo- and mycobionts, whereas rhizine material will be largely from the mycobiont although there may also be some photobiont contamination of rhizines. The rhizine material usually showed fewer bands than the thallus material, and it would seem most likely that the second fainter band is due to photobiont contamination. The variation in intensity of bands with the condition of the sample has also been described by DePriest (1994), who reported greater intensities from Trebouxia-derived fragments in dilute and degraded DNA stocks. Amplification of rhizine-derived DNA from the same Parmelia specimens with the primer pair ITS1-F and ITS4 gave a single PCR product of about 650 bp in the P. sulcata material and 600 bp from P. saxatilis. Amplification of thallus material of one specimen (26071; Table 1) gave between 1 and 3 bands, and this specimen has since been found to contain the parasitic fungus Abrothallus parmeliarum, in addition to the lichen symbionts. The primer pair ITS1-F and ITS4 has been investigated by Gardes & Bruns (1993) for amplifying fungal ITS regions in comparison to plant DNA. This pair of primers was reported to specifically amplify fungal DNA, although they found that attempts at amplification of some plant DNA also gave faint bands. As the reported increased specificity of these primers has excluded the previously seen fragments from the rhizine samples, it would seem likely that the multiple bands amplified with these primers from thallus material may indicate the presence of multiple rDNA types, further mycobionts or other associated fungi (e.g. lichenicolous fungi).
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F. 1. 1A & B. A, Example of PCR products from Parmelia saxatilis and P. sulcata. Lanes 1 & 10=Kb markers, lanes 2 & 3 and 6 & 7=paired rhizinae and thallus material from P. saxatilis, lanes 4 & 5 and 8 & 9=the same for P. sulcata. Both amplified with primers described by DePriest (1994). B, As A but amplified with the ITS1-F/ITS4 primer pair.
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F. 2. Example of digestion of PCR products. Lanes 1, 6 & 11=Kb markers, lanes 2, 3, 4 & 5=P. sulcata PCR products: (2) digested with EcoRI, (3) undigested, (4) digested with HaeIII or (5) HinfI. Lanes 7, 8, 9 & 10=P. saxatilis PCR products (7): digested with EcoRI, (8) undigested, (9) digested with HaeIII or (10) HinfI.
The single 650 and 600 bp bands obtained from the rhizine material were digested with EcoRI, HaeIII and HinfI in order to verify singularity. The result of the EcoRI restriction suggested a single site near the centre of the ITS region, as the restriction products from P. sulcata and P. saxatilis were two bands of 355 and 295 bp and 330 and 270 bp, respectively (Fig.2; lanes 2 & 7). The sums of these fragments correspond to the relative sizes of the single ITS fragments. The HaeIII digestion resulted in a reduction of about half in the size of the ITS fragments from the two species (Fig. 2, lanes 4 & 9), which may be due to either multiple HaeIII sites at the end of the fragment, or a single site giving rise to a small single fragment. The P. sulcata samples gave a broad band at below 100 bp that may suggest multiple small fragments (see Fig. 2). The HinfI digestion gave 3 fragments from the P. sulcata material and 2 from P. saxatilis (Fig. 2; lanes 5 & 10). The results presented here demonstrate that a simple DNA extraction technique can be combined with specific PCR primers to reliably isolate mycobiont DNA. As the starting material is existing vegetative structures from non-fertile specimens, the genotypes examined are those of the original
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specimen, and not meiotically derived progeny or mixtures. This technique therefore has particular value in the determination of relationships within and between existing populations and the examination of the extent to which several mycobionts are involved in a single thallus. The DNA extraction reported is simple and inexpensive and avoids the use of phenol and liquid nitrogen. Subsequent work has shown that the mercaptoethanol may be omitted from the initial disruption buffer without significantly reducing the efficiency of the extraction from freshly isolated specimens, however the mercaptoethanol has been found necessary when dealing with older herbarium specimens. Although there is the possibility of co-purification of non-DNA contaminating material, in our experience contamination can be nullified by dilution of the DNA sample of up to 1:20 without loss of PCR product. The PCR products in this case are the systematically important ITS regions of the rDNA gene cluster and this extraction and amplification procedure is suitable for the production of material for sequencing, in addition to restriction digestion analysis. The primer ITS1-F overlaps the primer ITS5 and is located before the ‘ universal ’ ITS1 primer (Gardes & Bruns 1993). This method therefore also offers some potential in lichens where pure mycobiont DNA is not easily available, for example because of the absence of rhizines. In those cases a modification of the above procedure with the ITS-1F and ITS-4 primers and subsequent amplification of either the two single spacer regions with ‘ universal ’ primers in a nested-PCR, may provide sufficient specificity. This work was carried out at the International Mycological Institute with the support of projects Ant 94-0905 from the Secretaria General del Plan Nacional de I+D, and APC-0020 from Dirección General de Investigación Cientifica y Técnica, both from the Ministerio de Educación y Ciencia, Spain. R Ahmadjian, V., Chadegani, M., Koriem, A. M. & Paracer, S. (1987) DNA and protoplast isolation from lichens and lichen symbionts. Lichen Physiology and Biochemistry 2: 1–11. Armaleo, D. & Clerc, P. (1991) Lichen chimeras: DNA analysis suggests that one fungus forms two morphotypes. Experimental Mycology 15: 1–10. Armaleo, D. & Clerc, P. (1995) A rapid and inexpensive method for the purification of DNA from lichens and their symbionts. Lichenologist 27: 207–213. DePriest, P. T. (1994) Variation in the Cladonia chlorophaea complex II: Ribosomal DNA variation in a southern Appalachian population. Bryologist 97: 117–126. Fahselt, D. (1996) Individuals, populations and population ecology. In Lichen Biology (T. H. Nash , ed.): 181–198. Cambridge: Cambridge University Press Gardes, M. & Bruns, T. D. (1993) ITS primers with enhanced specificity for basidiomycetes— application to the identification of mycorrhizae and rusts. Molecular Ecology 2: 113–118. Gardes, M., White, T. J., Fortin, J. A., Bruns, T. D. & Taylor, J. W. (1991) Identification of indigenous and introduced symbiotic fungi in ectomycorrhizae by amplification of nuclear and mitochondrial ribosomal DNA. Canadian Journal of Botany 69: 180–190. Gargas, A. & Taylor, J. W. (1992) Polymerase chain reaction (PCR) primers for amplifying and sequencing nuclear 18s rDNA from lichenized fungi. Myocologia 84: 589–592. Grube, M., DePriest, P. T., Gargas, A. & Hafellner, J. (1995) DNA isolation from lichen ascomata. Mycological Research 99: 1321–1324. Hawksworth, D. L. & Mouchacca, J. (1994) Ascomycete systematics in the nineties. In Ascomycete Systematics: Problems and Perspectives in the Nineties (D. L. Hawksworth, ed.): 3–11. New York: Plenum Press.
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Paterson, R. R. M. & Bridge, P. D. (1994) Biochemical Techniques for Filamentous Fungi. [IMI Technical Handbooks No. 1.] Wallingford: CAB International. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd Edn. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. White, T. J., Bruns, T. D., Lee, S. & Taylor, J. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols (M. A. Innis, D. H. Gelfand, J. J. Sninsky, & T. J. White, eds). New York: Academic Press. Accepted for publication 5 September 1996