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Rapid isolation of DNA from Dioscoreaspecies suitable for PCR, restrictiondigestion and pathogen screeningMithun Raja, Vishnu S. Natha, M. Senthil @ Sankara, M.L. Jeevaa &Vinayaka Hegdea

a Central Tuber Crops Research Institute (CTCRI),Thiruvananthapuram, India.Published online: 01 Aug 2013.

To cite this article: Mithun Raj, Vishnu S. Nath, M. Senthil @ Sankar, M.L. Jeeva & VinayakaHegde (2014) Rapid isolation of DNA from Dioscorea species suitable for PCR, restriction digestionand pathogen screening, Archives Of Phytopathology And Plant Protection, 47:6, 753-760, DOI:10.1080/03235408.2013.821753

To link to this article: http://dx.doi.org/10.1080/03235408.2013.821753

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Rapid isolation of DNA from Dioscorea species suitable for PCR,restriction digestion and pathogen screening

Mithun Raj, Vishnu S. Nath, M. Senthil @ Sankar, M.L. Jeeva and Vinayaka Hegde*

Central Tuber Crops Research Institute (CTCRI), Thiruvananthapuram, India

(Received 17 June 2013; final version received 29 June 2013)

The methods employed for DNA extraction from many plants is difficult because ofthe metabolites that interfere with DNA isolation procedures. We have developed areliable and efficient method for isolating genomic DNA free from polysaccharide,polyphenols and protein contaminants from Dioscorea spp. The method involvesinactivation of contaminant proteins by using CTAB/Proteinase K and precipitationof polysaccharides in the presence of high concentration of salt. The purity ofgenomic DNA was confirmed by A260/280 and A260/230 ratios calculated from thespectrophotometric readings and further by restriction analysis of the isolated DNAusing restriction enzymes Eco RI. The total genomic DNA extracted by the newprotocol was used for polymerase chain reaction amplification, RAPD analysis,restriction digestion and pathogen screening. The new protocol can be successfullyused for both small- and large-scale preparation of genomic DNA from different tis-sues of Dioscorea spp. The quarantine of seed tubers and use of pathogen-freetubers for planting is a prerequisite for integrated disease management strategy. Theprotocol can be used for the isolation of genomic DNA from other crop plants too.

Keywords: greater yam; RAPD; ITS; rDNA; molecular detection; restriction enzymedigestion; Dieback; Anthracnose

Introduction

Yam (Dioscorea spp.) is the fourth most important root crop, with a worldwide productionestimated at about 3600million tonnes (Mt) in 1999 (FAO 2002). The most widely culti-vated yam species include the greater yam or water yam (Dioscorea alata), the white yam(Dioscorea rotundata) and the lesser yam (Dioscorea esculenta). The air potato or potatoyam (Dioscorea bulbifera) is a medically important yam species (Figure 1). Yams are avaluable source of carbohydrate to the people of the tropical and subtropical Africa,Central and South America, parts of Asia, the Caribbean and Pacific Islands (Coursey1967; Adelusi & Lawanson 1987). Dioscorea spp. constitutes a staple food in the tropics(Han et al. 1987). The development of molecular markers as a tool for tuber crops germ-plasm characterization and early progeny selection is highly desirable for developing anefficient breeding programme to speed the integration of new genetic material into elitegermplasm. In addition, germplasm characterisation of tropical tuber crops usingmolecular markers will contribute to knowledge of genetic relationships betweenaccessions of the wild and cultivated gene pool and hence facilitate the breeding ofimproved genotypes in tuber crops to satisfy market needs and also helps develop

*Corresponding author. Email: ctcritvm@yahoo.com

Archives of Phytopathology and Plant Protection, 2014Vol. 47, No. 6, 753–760, http://dx.doi.org/10.1080/03235408.2013.821753

� 2013 Taylor & Francis

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genotypes possessing tolerance against diverse biotic and abiotic challenges (Figure 2). Aprerequisite for successful implementation of these crops improvement strategies is theability to isolate good amount of high-quality genomic DNA, which can be used forSouthern blot analysis, polymerase chain reaction (PCR) amplification, restriction frag-ment-length polymorphisms, arbitrary primed DNA amplification (RAPD, AP-PCR,DAF) and genomic library construction (Clark 1997). To fulfil this criteria, a rapid, simpleand reliable DNA isolation method is highly solicited. Problems encountered in the

Figure 1. Different species of Dioscorea. (A) D. alata, (B) D. esculenta, (C) D. rotundata,(D) D. bulbifera.

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isolation and purification of high molecular weight DNA from plant species include: (1)degradation of DNA due to endonucleases and co-isolation of highly viscous polysaccha-rides, and (2) inhibitor compounds like polyphenols and other secondary metaboliteswhich directly or indirectly interfere with the enzymatic reactions (Weishing et al. 1995).The presence of polyphenols, which are powerful oxidising agents present in many plantspecies, can reduce the yield and purity of extracted DNA (Loomis 1974; Porebski et al.1997). Plants species belonging to the same or related genera can exhibit enormous vari-ability in the complexity of pathways of dispensable functions. Thus, the biochemicalcomposition in plant tissues of different species is expected to vary considerably. Thechemotypic heterogeneity among species may not permit optimal DNA yields from oneisolation protocol, and perhaps even closely related species may require different isolationprotocols (Weishing et al. 1995). Polysaccharides are also problematic (Scott & Playford1996) as acidic polysaccharides inhibit the digestion of lambda DNA by certain endonu-cleases like Hind III (Do & Adams 1991) and classical 2-primer PCR amplification (Dem-ke & Adams 1992) by inhibiting Taq DNA polymerase activity (Fang et al. 1992). Inaddition, polysaccharides can cause anomalous reassociation kinetics of DNA sample(Merlo & Kemp 1976). They coprecipitate with DNA during alcohol precipitation to formhighly viscous solution (Do & Adams 1991) making DNA unsuitable for digestion byrestriction enzymes and southern blot hybridisation and the DNA tends to stick to thewells of the gel during electrophoretic analysis. NaCl at a concentration of more than0.5M together with CTAB is known to remove polysaccharides (Murray & Thompson1980). The problem of polyphenols is exacerbated if green, overmatured tissue is takenrather than etiolated leaves (Sharma et al. 2008). The method we developed for extractingDNA is specially designed to isolate genomic DNA from leaves of Dioscorea spp. withina short period of time using small amounts of plant tissues and yielding a high quality ofpurified DNA suitable for restriction digestion and PCR-based analysis (Figure 3).

Materials and methods

All the experiments were done in triplicates and values shown are the mean of the threeexperiments.

Figure 2. RAPD analysis.

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Plant materials

Young leaf tissues of D. alata, D. esculenta, D. rotundata and D. bulbifera were col-lected during early hours in the morning from Central Tuber Crops Research Institute,Thiruvananthapuram (India). Fresh leaf materials were kept in moist tissue paper in aplastic bag and kept away from sunlight. The leaves were destarched by keeping themat 4 °C overnight in a refrigerator before extraction. Plant tissues for pathogen screeningwas collected on the same day and processed immediately (Figure 4).

Reagents and chemicals

Extraction buffer: 100mM Tris–Cl (pH 8.0), 25mM EDTA, 1.5M NaCl, 2.5% CTAB,0.2% β-mercaptoethanol (v/v) (added just before use) and 1% PVP (w/v) (added justbefore use).

Chloroform: isoamyl alcohol (24:1), 2M NaCl with 4% PEG solution, Washsolution [15mM ammonium acetate in 75% (v/v) of ethanol] and a TE buffer (10mMtris-HCl (pH 8) and 1mM EDTA (pH 8).

DNA extraction protocol

Destarched leaf tissues (200–250mg) were ground to a fine powder using liquid nitro-gen. Prewarmed extraction buffer (1ml) was added to the samples and it was groundonce more. The samples were transferred to 2.0ml Eppendorf tubes and 10 μlProteinase K (10mg/ml) was added. The tube was incubated in 37 °C for 30min andthen at 65 °C for another 30min with frequent swirling. Samples were centrifuged at12,000 rpm for 15min at RT and supernatant was transferred to fresh eppendorf tube.Equal volume of Chloroform: isoamyl alcohol (24:1) were added and mixed by gentleinversion for 30–40 times. The samples were centrifuged at 12,000 g for 10min at RT

Figure 3. Restriction digestion. M: Moleular weight Ladder, Da: D. alata, De: D. esculenta,Dr: D. rotundata, Db: D. bulbifera.

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and the supernatant was transferred to a fresh tube. The above step was repeated againto remove any further proteins present (Figure 5). To the supernatant collected in afresh tube, 150 μL of 2M NaCl solutions containing 4% PEG was added. The sampleswere centrifuged at 12,000 g for 10min at RT. The supernatant was transferred to afresh tube and precipitated with 200 μl of ethanol. The nucleic acids was precipitatedand collected by centrifuging at 12,000 rpm for 10min. The nucleic acid pellet was

Figure 4. PCR using infected plant parts. M: Molecular weight ladder, 1: Naturally infectedwine, 2: Artificially infected leaf, 3: Naturally infected leaf, 4: Naturally infected tuber.

Figure 5. Anthracnose-infected leaf.

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washed twice with wash solution, air-dried until the ethanol was removed and dissolvedin appropriate amount of TE buffer (100–150 μl). The nucleic acid dissolved in TE buf-fer were treated with ribonuclease (RNase, 10mg/ml), incubated at 37 °C for 30minand stored at �20 °C until use.

DNA isolation from other plant parts

The above protocol was used for DNA isolation from soft wine, hard wine, tuber skinand tuber flesh.

Measurement of amount and purity of DNA

The quality of the isolated DNA was analysed by using 0.8% agarose gel containing0.1 μgml�1 ethidium bromide. DNA quantity was determined by calculating the absor-bance ratio A260/280 and A260/230 (Quant-iT™ assay, Invitrogen, USA). DNA puritywas further confirmed by digestion with Eco RI, incubating reaction mixture at 37 °Covernight and followed by 0.8% agarose gel electrophoresis.

RAPD analysis

Suitability of the isolated DNA for downstream analysis was assessed by a RAPDdecamer primer OPT-15 (Operon Technologies). The PCR reaction mixtures wereheated at an initial step of 94 °C for 2min and then subjected to 35 cycles of {94 °Cfor 30 s, 48 °C for 2min, 72 °C for 1min 45 s}. After the last cycle, the temperaturewas maintained at 72 °C for 8min and final hold at 4 °C. The amplified products wereresolved on a 2% agarose gel containing 0.5mgmL�1 ethidium bromide and the imagewas visualised through Gel Doc System (Alpha imager).

Comparative study

For comparison, the genomic DNA of the same leaf sample was extracted using theexisting CTAB protocol (Doyle & Doyle 1987), GenElute™ Plant Genomic DNAMiniprep Kit, GeNeiTM Ultrapure Plant Genomic DNA Prep Kit and further DNAanalysis was performed using the same conditions and reagents as described.

DNA isolation from Dieback-infected plant leaves

Anthracnose/Dieback-infected plant leaves were collected from yam fields of CTCRI,Trivandrum, India, and used for DNA isolation using the protocol. The extracted DNAwas used for PCR with species-specific PCR assay suing previously described methods(Raj et al. 2012).

Results and discussion

The method was standardised for the four species of Dioscorea. The standardisedextraction method yielded good quantity of pure, high molecular weight DNA fromplant parts including the seldom used plant parts such as wines and tuber parts. TheA260/280 ratio ranged from 1.80 to 2.20 and A260/280 ratio ranged from 1.21 to 1.21,showing that DNA was of high purity. The purity of DNA was further confirmed with

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restriction digestion analysis using the enzymes EcoRI and monitoring the bandingprofile of the completely digested genomic DNA. The results indicated that the isolatedDNA was suitable for further downstream applications. Successful amplification andvariation among species was obtained by using primer OPT15 in RAPD analysis, whentested with genomic DNA isolated using this new protocol from different Dioscoreaspecies revealed its suitability for PCR-based analysis and further downstream process-ing. The DNA extraction using the new protocol was comparable to that isolated fromthe commercially available kits. This shows the effectiveness of this protocol to replacecommercially costly nucleic acid purification system. Plants are sources of natural prod-ucts or bioactive substances that also produce large amounts of secondary metabolitesand substances of medicinal or industrial importance. Thus, while working with a vari-ety of plants, it is common to encounter problems arising from the presence of essentialoils, polysaccharides, polyphenols and other secondary metabolites in the lysate and theDNA preparations (Khanuja et al. 1999). The PCR analysis for Colletotrichumgloeosporioides using species-specific PCR assay using infected leaf yielded ampliconof � 300 bp in all the infected plant parts. This highlights the efficiency of this protocolin isolation of total genomic DNA and its use in pathogen detection. The aim of thiswork was to develop a highly reliable, simple and efficient method for the isolation ofgenomic DNA from the Dioscorea spp. The DNA extraction protocol described here israpid and technically easy for preparing high molecular weight DNA without any ultra-centrifugation or column purification steps. Another advantage of this protocol is that isdoes not involve the use of phenol. It can serve as a low-cost and effective alternativeto kit-based DNA extraction methods or high-end protocols. Since this protocol wassuccessfully used to isolate DNA from four species of Dioscorea, it can be furtherapplied for other plant species. The use of PCR-based diagnostics is getting more valuethese years; the current protocol can be applied for the detection of more pathogensaffecting yams, including the viral pathogens.

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