Journalof Bryology (2002) 24: 127±131

Directampli® cation and NaOH extraction: two rapid and simple methodsfor preparing bryophyte DNA forpolymerase chain reaction(PCR)

OLAF WERNER, ROSA MARI Â AROS andJUAN GUERRA

University ofMurcia, Spain

SUMMARY

PCR(polymerase chain reaction) has become one of the most important techniques used in molecular systematics.Generally, the methods applied to isolate DNA forPCR ampli® cation depend on multiple stepsto isolate and clean the ® nalproduct. This involves considerable e€ ort and time when many sampleshave to be processed and poisonous organic solvents are often needed (phenol, chloroform etc.).In this contribution, two very rapid and simple techniques intended for use in higher plants are shownto be useful in bryophyte molecular biology: direct ampli® cation from plant tissues and the NaOHextraction method. In 15 of the 17 investigated bryophytes (two hepatics and 15 mosses) the

trnLUAA intronof the chloroplast DNA wassuccessfully ampli® ed by the direct approach, while the NaOHextraction method gave ampli® able DNA inall 17 species. DNA ampli®ed by both methods wassuccessfully used in cycle sequencing.

KEYWORDS:direct PCR, NaOH DNA isolation, trnLUAA intron,bryophyte DNA extraction.

INTRODUCTION whenlarge numbers of samples have to be processed, for examplein population genetic investigations or when PCR(polymerase chain reaction)-based techniques are extremelysmall bryophytes are studied. Therefore, we becomingincreasingly popular for studying relatedness of comparedboth methods with several small moss and bryophytetaxa. The sequencing of PCR-ampli® ed DNA hepaticspecies. fragmentsand RAPD (randomampli® ed polymorphic DNA)aretwo frequently used techniques. The methods thatare generally used for DNA extractionfrom bryo- MATERIALSAND METHODS phytesare based on those described by Doyle & Doyle Plantmaterial (1987,1990) using CTAB assolvent (cf. Ashton et al., 1994;Boisselier-Dubayle et al.,1995;Capesius & Bopp, Plants of Metzgeriafurcata (L.) Dum., Lophocoleabiden- 1997;Meiû ner et al.,1998;Patterson et al.,1998;De Luna tata (L.)Dum. and Fissidenstaxifolius Hedw. were et al.,1999;Stenù ien, 1999) although other methods, such collectedfrom S.W. Germanyin January 2000. Didymodon asthe SDS extractionmethod of Edwards, Johnstone & sicculus M.J.Cano,Ros, Garcõ Âa-Zamora & J.Guerra, Ptery- Thompson(1991) are sometimes used (cf. Cox & Hedder- goneurumlamellatum (Lindb.)Jur., Bryumargenteum son,1999). All these methods require a considerable Hedw.,and one sample of Aloinaaloides (Schultz)Kindb. investmentin both time and the chemicals needed. Two werecollected during February 2000 from S.E. Spain. methodshave been published for the very rapid prepara- Othersamples of Aloinaaloides wereherbarium specimens tionof plant material for PCR, although so far no studies ofSpanish origin sampled in the years 1999, 1997, 1993 havebeen published that have applied these techniques to and 1990. Grimmiatrichophylla Grev., Brachytheciumvelu- bryophytes:the direct ampli® cation of DNA fromplant tinum (Hedw.)Schimp., Pterogoniumgracile (Hedw.) Sm, tissues(Berthomieu & Meyer,1991; Rogers & Parkes, Homalotheciumlutescens (Hedw.)Robins., Hypnum 1999)and the alkaline isolation of total DNA (Wang,Qi cupressiforme Hedw., Rhynchostegiummegapolitanum &Cutler,1993). Such techniques could be very useful (H.F.Weber& D.Mohr)Bruch, Schimp. Eurhynchium

# BritishBryological Society 2002 Received 20 November 2000. Revision accepted 4May 2001 DOI:10.1179/ 037366802125000980 128 O.WERNER,R. M.ROSAND J. GUERRA meridionale (Bruch,Schimp. & W.Gu È mbel)De Not. and membranewas made in two steps using 50 l loftheelution Homalotheciumaureum (Spruce)Robins. were collected bu€er supplied by the manufacturer. The eluates were duringSeptember 2000 from Sierra Espun Äa,S.E. Spain. thencombined to give a ®nalvolume of 100 l l. NaOH- Additionalsamples of Homalotheciumaureum were extractedor Qiagen-extracted DNA (1 l l)that was then herbariumspecimens collected in the years 1999, 1997, usedin the PCR reactions. At least three independent sets 1994,1991, 1986 and 1981. Tortulamuralis Hedw. and ofreplicate experiments were run for all the extraction Tortularevolvens (Schimp.)G.Roth. were collected methods. duringMarch 2001 from Murcia, S.E. Spain. All the ThePCR reactions were performed in 0.2 ml reaction plantswere stored dry until direct PCR orDNA-extrac- tubesusing 1 UTaqDNA polymerase(Appligene Oncor), tionwas carried out. Herbarium vouchers are deposited 0.2mM dNTPs(Appligene Oncor), 10 mM Tris±HCl, pH at MUB. 8.3(RT), 50 mM KCl and1.5 mM MgCl 2 in 25 l l ®nal volume.The primers were designed to amplify the

trnLUAA intronof cp DNA. 10pmol of primers C andD Preparationof plantmaterial for direct PCR (Taberlet et al.,1991)were used with the modi® cations introducedby Meiû ner et al.(1998)to improve annealing Inthe case of terricolous mosses, the lower part of the inbryophytes. An Eppendorf Mastercycler gradient was plantswas cut o€ to remove the soil particles adhering to programmed(2 min 94 ¯C,35cycles with 15 sec 94 ¯C, 30 therhizoids. All plants were rinsed with double-distilled sec 50¯C, 1 min 72¯Canda ®nalextension step of 2 min water(DDW) to remove dirt and biological contaminants at 72¯)toperform the ampli® cation reactions. The ampli® - asfar as possible.The bryophytes were then transferred to cationproducts were separated on 9% PAA gelsand 1.5ml Eppendorf tubes with sterile DDW andtreated in DNAbandswere visualized using the silver staining anultrasonic cleaner to remove any remaining foreign methodof Dean & Milligan(1998). Gels were scanned material.Such ultrasonic cleaning of small bryophyte usinga TWAINcompatible scanner (Agfa SnapScan 600) samplesin plastic tubes works well andis routinely witha transilluminationmodule and stored in digital appliedin our laboratory for the preparation of material format. forSEM studies.Clean leaves or thallus fragments of PCRproductswere puri® ed using the QIAquick Puri® - ca 0.2 mm2 wereremoved with sterile forceps and trans- cationKit (Qiagen). Sequencing reactions were carried out ferredto thin-walled 0.2 ml Eppendorf tubes, to which usingthe Biocycle Sequencing Kit of GATC (Konstanz,

PCRcomponents were added. In order to investigate the Germany)in an Eppendorf Mastercycler (2 min 95 ¯C, 35 dependanceof the PCR process on the amount of plant cycles30 s95 ¯C, 30 s 55¯C, 1 min 72¯C)andprimers C/ D material,varying amounts of Bryumargenteum plant ofTaberlet. Sequencing reactions were separated in a materialwere used. GATC-1500-systemand transferred to Nylon membranes. Basically,NaOH extraction followed the protocol of Thebiotin-modi® ed ddNTPs of the sequencing kit allowed Wang et al. (1993)with minor modi® cations. One whole thevisualization of the DNA-bands by the application of plantwas put in an Eppendorf tube and 5 l l of 0.5 M astandardprotocol with StreptavidinAlkaline Phospha- NaOHwere added. The plant was then ground with a tase(GATC) and BCIP/ NBT (Aldrich). stainlesssteel needle, which had been cleaned with 0.5 M HCl, untilno larger fragments were visible. An additional 15 l l0.5M NaOHwere then added and grinding was RESULTS AND DISCUSSION continuedfor 1 or2min.The samples were centrifuged at 13,000rpm for 2 minand the supernatant was diluted 1:10 Fig.1 showsan example of the direct ampli® cation of the in100 mM Tris±HCl, pH8.3. When necessary, this dilu- trnLUAA intronof chloroplast DNA. Ampli®cation tionwas used for the preparation of further dilutions with productswere obtained for all species studied with the 100mM Tris±HCl, pH8.3. exceptionof Lophocoleabidentata and Rhynchostegium Forcomparison purposes, DNA wasextracted with a megapolitanum .Insome species ( Grimmiatrichophylla , commerciallyavailable kit, which had previously given Brachytheciumvelutinum , Homalotheciumlutescens , and goodresults in our laboratory with higher plants (DNeasy Eurhynchiummeridionale )notall replicates resulted in PlantMini Kit,Qiagen GmbH, Hilden, Germany). This detectableampli® cation products. Rogers & Parkes(1999) kituses silica-gel membranes to bind DNA selectively, observedthat an excess of tissue rather than too little is whichcan then be washed with an appropriate bu€ er and morelikely to cause problems in direct PCR of higher latereluted. In a modi®cation of the manufacturer’s proto- plants,attributing these problems to leaf matrix compo- col,one plant was ground in 5 l lbu€er AP1 in an Eppen- nentssuch as proteins, carbohydrates, chlorophyll, etc., dorftube at room temperature. When no large fragments thatmay act as Taq polymerase inhibitors. Therefore an werevisible, 195 l lofAP1 were added and the Qiagen attemptwas made to reduce the amount of plant material protocolwas followed but with all the bu€ er volumes, of Lophocoleabidentata tothe minimum that could be withthe exception of the washing bu€ er, reduced by half. easilyhandled ( ca 25 25 l m)but no ampli® cation was £ The® nalelution of the DNA fromthe silica-gel observedfor any L.bidentata directPCR reaction. DIRECTAMPLIFICATION AND NaOHEXTRACTION OF BRYOPHYTEDNA 129

Figure 1. Directampli® cation ofthe trnLUAA intronusing bryophyte leaves orthallus fragments. Lane M 50bpladder; 1 Fissidens ˆ ˆ taxifolius; 2 Metzgeria furcata ; 3 Lophocolea bidentata ; ˆ ˆ 4 Didymodon sicculus ; 5 Pterygoneurum lamellatum ; 6 Bryum ˆ ˆ ˆ argenteum; 7 Aloina aloides ,collected 2000; 8 Aloina aloides , 1999; ˆ ˆ 9 Aloina aloides , 1997; 10 Aloina aloides , 1993; 11 Aloina aloides , Figure 3. Inhibitorye€ ect oflarge amountsof plant material on ˆ ˆ ˆ 1990. One leaf ora thallusfragment ofcorresponding size was used in direct PCR ampli®cation. Increasing quantitiesof Bryum argenteum a 25 l lreaction volume. were added tothe PCR assays. Lane 1 ‰leaf;lane 2 1 leaf; lane ˆ ˆ 3 2leaves; lane 4 4leaves; lane 5 ‰gametophore; lane 6 1 ˆ ˆ ˆ ˆ gametophore; lane 7 2gametophores; M=50 bpladder. The ˆ However,the fact that the DNA fragmentwas successfully reaction volumein all cases was 25 l l. ampli®ed starting with template DNA extractedfrom L. bidentata usingthe Qiagen DNeasy Plant Mini Kit,indi- catesthat the primers used were suitable for amplifying clearlyinhibited the reaction and the optimal length of theintron in this taxon (Fig. 2). fragmentswas found to be below 0.5 mm. Although these Testswith other species showed that an excess of plant resultsseem to suggest that hepatics are more resistant to materialdoes indeed have an inhibitory e€ ect on direct directPCR than most mosses, perhaps due to the high PCRampli® cation (Fig. 3). In the case of Bryumargen- numberof secondary substances that are produced and teum,halfa stemor more of plant material completely storedin the oil bodies of these plants, more species must inhibitedthe reaction, although the strength of the inhibi- betestedbefore this can be con®rmed. torye€ ect depended on the species. In Metzgeriafurcata , Whenthe NaOH extraction method was tried in an forexample, thallus fragments more than 1 mmin length attemptto overcome the problems that we foundin the ampli®cation of Lophocolea and Rhynchostegium , a 1:10 dilutionof the extract gave positive results in all bryophyte speciestested, with the exception of Lophocoleabidentata . However,higher dilutions worked with Lophocolea (Fig.4), which suggests that a lackof DNA isnot the problem,but rather the presence of inhibitory substances inthe plants. Inmolecular taxonomic studies, investigators frequently dependon herbarium material as a sourceof DNA. For thisreason, we triedto amplify DNA fromherbarium specimensof Aloinaaloides upto ten years old. Fig. 1

showsthat the direct PCR ampli®cation of the trnLUAA intronwas possible with all tested samples. Also DNA extractedusing the NaOH method was capable of ampli®- cationin the case of herbarium samples of Aloina stored forup to 10 years (Fig. 5). The same is true for specimens of Homalotheciumaureum storedunder normal herbarium conditionsfor up to 19 years (data not shown). However, wheninterpreting this result, one should keep in mind that both Aloinaaloides and Homalotheciumaureum are well Figure 2. Ampli®cation ofthe trnLUAA intronusing DNAextracted adaptedto tolerating desiccation and that DNA mightbe withthe DNeasy PlantMini Kit (Qiagen). The DNAofall stabilizedby special cell components under herbarium investigated bryophytes was ampli®ed. Lane 1 Fissidens taxifolius ; ˆ 2 Metzgeria furcata ; 3 Lophocolea bidentata ; 4 Didymodon conditions.Some bryophytes are known to survive up to ˆ ˆ ˆ sicculus; 5 Pterygoneurum lamellatum ; 6 Bryum argenteum ; 23years in an ahydrobiotic state (Breuil-Se Âe,1993, 1994). ˆ ˆ 7 Aloina aloides ,collected 2000; M=50 bpladder. Herbariumspecimens of species that are less tolerant to ˆ 130 O.WERNER,R. M.ROSAND J. GUERRA

Figure 4. Ampli®cation ofthe trnLUAA intronusing NaOH- extracted DNAas template.All dilutions are 1:10 unless otherwise indicated.Lane 1 Fissidens taxifolius ; lane 2 Metzgeria furcata ; ˆ ˆ lane 3 Lophocolea bidentata ; lane 4 L.bidentata, diluted1:20; lane ˆ ˆ 5 L.bidentata ,1:40; lane 6 L.bidentata ,1:80; lane 7 Didymodon ˆ ˆ ˆ sicculus; lane 8 Pterygoneurum lamellatum ; lane 9 Bryum ˆ ˆ argenteum; lane 10 Aloina aloides ,collected 2000; M=50 bpladder. ˆ Notethat no ampli® cation was observed with Lophocolea bidentata at dilutionsof 1:10 and 1:20, butat dilutionsof 1:40 and 1:80 the ampli®cation was successful.

Figure 5. Ampli®cation ofthe trnLUAA intron in Aloina aloides stored dry forup to 10 years inthe herbarium.The DNAwas NaOH- desiccationare generally more resistant to PCR ampli® ca- extracted fromsingle plants,diluted 1:10 and then1 l lofthe diluted tion(M. Stech,personal communication). Therefore, when extract was added tothe PCR reaction (25 l l®nal volume).Lane collectingplant material for DNA studieswhen the DNA- 1 collectionyear 2000; lane 2 year 1999; lane 3 year 1997; lane ˆ ˆ ˆ 4=year 1993; lane 5 year 1990; M 50 bpladder. extractionis not possible immediately, a rapiddrying ˆ ˆ (usingsilica gel or CaSO 4)isrecommended (Sytsma et al., 1993).Nevertheless, all samples collected during 2000 were theampli® ed DNA priorto its use in downstreamapplica- capableof ampli®cation using the direct PCR approach or tions(sequencing reaction etc.). However, this is more theNaOH extraction method, when the experiments were time-consumingthan the application of spin columns in repeatedin April 2001. NaOH extracted DNA canbe thecase of single bands. storedat 20ë C inthe dilution bu€ er for up to 1 yearand Themost important downstream-reaction of ampli® ed successfullyused in ampli® cation reactions. DNAisDNA-sequencing. In order to ascertain that the Bothextraction methods were used to amplify DNA productsof NaOH-extraction and direct ampli® cation are withshort random primers (RAPD) (Welsh& McClel- usable,we sequencedthe trnLUAA intron of Tortulamura- land,1990; Williams et al.,1990).A highproportion of lis obtainedby direct ampli® cation and of Tortularevol- theampli® ed bands were not reproducible (data not vens usingNaOH extracted DNA template.In both cases shown).This may be due to the low concentrationof theDNA sequencingreactions were successful. DNAinthe reaction mix. Williams et al.(1993)reported, thatin the case of soybean, Glycine max,aconcentration of 1.5 ng/25l lreactionwas necessary to guarantee the CONCLUSIONS reproducibilityof the bands. This corresponds to the DNAcontentof 850haploid genomes. It is probable, that Ourconclusion is that direct PCR ampli® cation from theamount of available DNA inthe reaction mix is well bryophytefragments or NaOH extraction of DNA could belowthis level. In the case of direct DNA ampli®cation bean interesting alternative to the extraction of DNA by theaccess to the DNA mightbe partially restricted by the othermethods, which, although more sophisticated and chromosomalDNA structureand the proteins associated possiblyyielding cleaner DNA, aremore complicated. No withthe DNA. DNAextractedwith NaOH is denatured organicsolvents or any other treatment (heating, protei- andalthough such denatured DNA issuitable for PCR- naseK etc.)are necessary and the time needed is drasti- basedampli® cation it may not be for some other down- callyreduced, since manipulation is minimized,which is of streamapplications such as restriction enzyme digestion. specialimportance when large numbers of specimens are Insome of our gels more than one band could be seen. In beingstudied. The reduced number of steps may also sucha casegel extraction methods are necessary to clean decreasethe risk of cross contamination. This is especially DIRECTAMPLIFICATION AND NaOHEXTRACTION OF BRYOPHYTEDNA 131 truein the case of direct PCR, since it is not necessary to Capesius I,Bopp M.1997. Newclassi® cation ofliverworts based on handleDNA-containing liquids. These may form aerosols molecular and morphologicaldata. 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OLAF WERNER, ROSA MARIÂ A ROS and JUAN GUERRA,Departamentode Biologõ ÂaVegetal(Bota Ânica),Facultad de BiologõÂa, Universidadde Murcia,E-30100 Espinardo, Spain. Author for correspondence:[email protected]