HortFlora Research Spectrum www.hortflorajournal.com Vol 6, Issue 3; 159-162 (September 2017) NAAS Rating : 3.78 ISSN: 2250-2823 STANDARDIZATION OF PHENOL FREE GENOMIC DNA EXTRACTION OF POMEGRANATE GENOTYPES FOR DIVERSITY ANALYSIS Dimpy Raina 1* and A S Sundouri KVK Ferozepur, Punjab Agricultural University, Ludhiana-141 004, India Faculty of Horticulture, Division of Fruit Science, SKUAST, Shalimar, Kashmir *Corresponding Author’s E-mail: dimpy.raina@gmail.com ABSTRACT : The isolation of intact high-molecular-mass genomic and good quality deoxyribonucleic acid (DNA) is the pre-requisite for many molecular biology applications including long polymerase chain reaction (PCR), endonuclease restriction digestion, southern blot analysis, and genomic library construction The presence of high concentrations of polysaccharides, polyphenols, proteins, and other secondary metabolites in pomegranate leaves poses problem in getting good quality DNA The study aimed to determine a reliable and modified protocol based on the cetyltrimethylammonium bromide (CTAB) method for DNA extraction from pomegranate leaves Easy purification method was added to modify CTAB method using Tris-saturated phenol: chloroform (1:1) and 3M sodium acetate Polyvinylpyrrolidone (PVP) and β-mercaptoethanol were employed to manage phenolic compounds Extended chloroform-isoamyl alcohol treatment followed by RNase treatment Efficient yields of high-quality amplifiable DNA (200-1200 ng) was produced rapidly with modified CTAB method Quantity of obtained DNA from this extraction method was controlled in terms of absorbance at wavelength of 260, 230 and 280 nm The absorbance ratio of A260/A280 indicates presence of dense protein Spectrophotometric analysis at A260/A280 revealed ratio range of 1.77–1.94 The purified DNA which has excellent spectral quality was efficiently amplified by 48 SSR primers and was suitable for long-fragment PCR amplification Keywords : Polyphenolic compounds, genomic DNA, Tris-saturated phenol, chloroform, genetic diversity Pomegranate (Punica granatum L.), belongs to the family Punicaceae, is one of the important and oldest edible fruit, cultivated in Mediterranean countries extensively in Iran, India, Pakistan, Afghanistan, Saudi Arabia and in the sub-tropical areas of South America (Elyatem and Kader, 3) In India, the major pomegranate growing states are Maharashtra, Karnataka, Gujarat, Andhra Pradesh and Rajasthan In recent years, pomegranate fruit is in demand worldwide because of its superior pharmacological and therapeutic properties The arils and husk of pomegranate fruit bear properties such as antioxidant, anti-inflammatory, and anti-atherosclerotic against some diseases (osteoarthritis, prostate cancer, heart disease, HIV-1) (Malik et al., 7; Neurath et al., 10; Sumner et al., 17) Due to its multipurpose medicinal uses it is also known as “Super fruit” in the global functional food industry (Martins et al., 8) The germplasm of pomegranate needed to be evaluated and conserved for its valuable properties The morphological characterization provided the inefficient information about the germplasm so genomic based Article’s History: Received : 12-08-2017 Accepted : 02-09-2017 approaches have been taken to assess the intrinsic knowledge about the germplasm Genomic DNA extraction is pre-requisite for the molecular applications The presence of high concentrations of polysaccharides, polyphenols, proteins, and other secondary metabolites in pomegranate leaves poses problem in getting good quality DNA which is almost insolvable in water or TE buffer, and inhibits enzyme reactions (Reski, 13) and are also unstable for long term storage (Sharma et al., 15).The present study aimed to determine a reliable and modified protocol based on the cetyltrimethylammonium bromide (CTAB) method for DNA extraction from pomegranate leaves with the addition of purification method Tris-saturated phenol: chloroform (1 : 1) and 3M sodium acetate MATERIALS AND METHODS Ten pomegranate genotypes viz Kandhari, Moga local, Assam local, Russian seedling, G-137, P-26, Khug, Jhodpur white, Panipat selection, Kandhari Kabuli were used as plant material for genomic DNA extraction The leaf samples of genotypes were collected from germplasm maintain at New Orchard of PAU, Ludhiana, India 160 Raina and Sundouri HortFlora Res Spectrum, 6(3) : September 2017 Chemicals Used Purification of Genomic DNA An extraction buffer solution (pH 8.0) consisting of 1.5% CTAB (w/v), NaCl (1.4 M), Tris HCl pH 8.0 (100 mM) and EDTA pH 8.0 (20 mM), PVP (2 %), â-mercaptoethanol % The other chemicals were chloroform:Isoamylalcohol (24:1) v/v/v), iso-propanol, Tris saturated phenol, phenol: chloroform: isoamyl alcohol (25:24:1),Sodium acetate (3M) solution (pH 8.0),70% ethanol, RNase (10mg/ml of RNase in buffer, 10 mM Tris HCl, 15 mM NaCl pH 7.5) and TE buffer (Tris HCl, 10 Mm pH 8.0, 1mM EDTA pH 8.0) As the freshly isolated DNA contained certain impurities like, polyphenolic, proteins, polypeptides etc it was necessary to purify the DNA before PCR analysis The DNA samples were thawed to room temperature and an equal volume of Tris-saturated phenol: chloroform (1:1) was added The mixture was then mixed thoroughly and centrifuged for about minutes at 12000 rpm The aqueous phase was pipette out in a fresh tube and two chloroform: isoamyl alcohol (24:1) extractions were performed as before Both the times, the mixture was centrifuged at 10,000 rpm for 10 minutes The upper phase was again pipette out and 2.5 times the total volume of chilled ethanol were added to it The contents were mixed gently and the precipitated DNA was spooled out The extra salts were removed by two washings with 70per cent ethanol at 10,000 rpm for minutes and the pellet was dried at room temperature The pellet was dissolved in appropriate volume of 1X TE The DNA samples were dissolved at room temperature and stored at -20° C until used Quantity of obtained DNA from this extraction method was determined by spectrophotometer analysis at wavelength of 260, 230 and 280 nm The absorbance ratio of A260/A280 indicates presence of dense protein DNA concentration and purity was also determined by running the samples on 0.8 % agarose gel (Fig.1) Isolation of Genomic DNA Plant DNA was isolated using CTAB (Cetyl Trimethyl Ammonium Bromide) (Saghai-Maroof et al., 14) method with some modifications Young leaves from 10-12 years old trees were harvested, placed in glassine bags and stored at –80°C until use The leaves were ground to fine powder using liquid nitrogen to make leaves brittle as well as to stop DNase activity The powder was transferred immediately to a 50 ml autoclaved Oakridge tube containing 20 ml of pre-warmed (60°C) CTAB extraction buffer The powder was suspended in the buffer by inverting and rotating the tubes gently The tubes were incubated at 60°C for one hour in a water bath and were mixed occasionally After incubation, 15ml of chloroform: isoamyl alcohol (24:1) was added and tubes were swirled, till it made a dark green emulsion The tubes were placed on a rotary shaker for 30 minutes and then centrifuged at 10,000 rpm for 10 minutes at room temperature Following centrifugation, the upper aqueous phase was transferred to a clean sterile 50 ml Falcon tube and 30 µl of sodium acetate (3M) was added and contents were mixed thoroughly and centrifuged at 12000 rpm for about minutes About 10 ml of chilled isopropyl alcohol was added to precipitate the DNA and a good quality DNA floated at top was hooked out using a sterile hooked Pasteur pipette The DNA was transferred into a clean sterile 2.0 ml microfuge tubes rinsed with 70 per cent ethanol for five minutes so as to remove any residual salts followed by re-centrifugation Pellet was collected and was allowed to air dry (at room temperature) for one hour Then 300-500µl volume of 1X TE was added and left for few hours at room temperature The dissolved DNA was added with 10 µl of RNAse to each sample and incubated at 370C in water bath for 45 minutes The samples containing DNA were stored at -20°C until used PCR Analysis A set of 25 SSR molecular markers ((Hasnaoui et al., and Pirseyedi et al., 11) was used for PCR amplification In vitro amplification using polymerase chain reaction (PCR) was performed using 40 ng of genomic DNA of each genotype in a final volume of 20µl per reaction containing x PCR Buffer, 1.5mM MgCl , 200 µM dNTP mix, 1U Go Taq polymerase (Promega), 0.5 µM of each single primer Amplification reactions were allowed to perform in a DNA thermocycler (MJ Research) for 35 cycles after an initial denaturation at 94°C for minute In each cycle denaturation for minute at 94°C, annealing for 1minute at (50-55°C) and elongation at 72°C for minutes was performed with a final extension step at 72°C for minutes Negative control was used initially to check the fidelity of the PCR reaction The amplified products were separated by electrophoresis on a 3% agarose gel (0.03g/ml) ethidium bromide Wells were loaded with 20 µL of reaction volume Electrophoresis was conducted approximately 3hours at 90 volts, and at the end, the gels were visualized and photographed on an ultraviolet light transluminator Standardization of Phenol Free Genomic DNA Extraction of Pomegranate Genotypes for Diversity Analysis RESULTS AND DISCUSSION Molecular marker analysis in genome studies greatly enhances the speed and efficiency of crop improvement The extraction of good quality and quantity of DNA suitably meet the various molecular based techniques for genome analysis In the present study DNA extraction was improved by modifying some of the steps in the original CTAB DNA isolation protocol given by Saghai-Maroof et al (14) This procedure resulted in extracting high quality, low-polysaccharide genomic DNA from mature leaves of pomegranate Large quantities of polysaccharides are known to interfere in many analytical applications and therefore, lead to wrong interpretations (Singh et al, 16) CTAB found to prevent the polysaccharide co-precipitations (Dellaporta et al., 2).The presence of polyphenols can reduce the yield and purity by binding covalently with the extracted DNA, the mixing of PVP along with CTAB might bind to the polyphenolic compounds by forming a complex with hydrogen bonds and help in removal of impurities to some extent Similar residual phenols and polysaccharides were removed and DNA was precipitated selectively in the presence of high salts in some woody plants (Fang et al., 4) Tannins, terpenes and resins considered as secondary metabolites are also difficult to separate from DNA (Mazid et al., 9) Sodium acetate treatment fixed and removed tannins and the extra secondary metabolites The additional step of purification with Tris Saturated Phenol : Choloform (pH 8.0) followed by chloroform: isoamyl (24:1) removed excess impurities of proteins, polysaccharides and phenols DNA isolation procedure also yields large amounts of RNA, especially 18S and 25S rRNA (Joshi et al., 6) Large amounts of RNA in the sample can chelate Mg 2+ and reduce the yield of the PCR A prolonged RNase treatment degraded RNA into small ribonucleosides that would not contaminate the DNA preparation and yielded RNA-free pure DNA Additional precipitation steps removed large amounts of precipitates by centrifugation and modified speed and time Several plant DNA extraction protocols have been reported in fruit crops like mango (Mangifera indica L.) citrus (Citrus spp.), litchi (Litchi chinensis S.), custard apple (Annona squasoma L.), guava (Pisidium guajava L.) and banana (Musa spp.) (Porebski et al., 12).The spectrophotometer analysis showed good quality DNA (A 260 A 280 1.77–1.94) (Table 1) Cheng et al (1) also reported spectrophotometer analysis of DNA extracted from old frosted citrus spp leaves (A 260/A280 1.5 -1.87) The present modified protocol was able to quantify high amount of DNA (200-1200 ng) on 0.8% agarose (Table and Fig.1) DNA isolated 161 by this method yielded strong and reliable amplification products showing its compatibility for SSR-PCR For SSR almost all the tested parameters like the primer, Taq polymerase, dNTPs, magnesium chloride, concentration of template DNA and temperature and time intervals during denaturation , annealing and elongation were optimized which also had an effect on amplification, reproducibility and banding patterns (Fig 2.) The optimized DNA isolation and SSR technique may serve as an efficient tool for further genetic studies Table : Qualitative and quantitative differences in genomic DNA of 10 pomegranate genotypes extracted from mature leaves S.No Genotypes DNA concentration (ng/2 µl DNA) A 260/A280 Kandhari 300 1.92 Moga Local 200 1.94 Assam Local 300 1.89 Russian seedling 1200 1.77 G-137 1200 1.79 P-26 1200 1.77 Khug 1000 1.87 Jhodpur white 500 1.84 Panipat selection 1000 1.87 Kandhari Kabuli 1200 1.78 10 Fig : Electrophorotic separation of genomic DNA of 10 pomegranate genotypes Numbers at the top of each line are the genotypes as presented in Table Fig 2: In vitro amplification profile of 10 pomegranate genotypes for SSR marker Numbers at the top of each line are the genotypes as presented in Table 162 Raina and Sundouri CONCLUSION The results proved the reproducibility, reliability and practicality of the modified protocol which yielded high quality DNA on isolation and found accessible for PCR analysis It provides the opportunity to collect good quality DNA from mature leaves from other species high in polysaccharides and polyphenols Acknowledgements Thanks to Inspire Fellowship Scheme (Department of Science and Technology, Govt of India) for financial assistance and Molecular laboratory of School of Agricultural Biotechnology, PAU for technical advice 10 11 REFERENCES Cheng J., Guo W.W and Deng X.X (2003) Molecular charcterization of cytoplasmic and nuclear genomes in phenotypicaly abnormal Valencia orange (Citrus sinensis) + Meiwa kumquat (Fortunella crassifolia) intergeneric somatic hybrids Plant Cell Rep., 21 : 445-451 Dellaporta S.L and Wood J., Hicks J.B (1983) A plant DNA minipreparation: Version II Plant Mol Biol Rep., : 19-21 Elyatem S.M, Kader A (1984) Post harvest physiology and storage behaviour of pomegranate fruits Scientia Hort., 24 : 284-98 Fang G., Bammar S and Grumnet R (1992) A quick and inexpensive method for removing polysaccharides from plant genomic DNA Biofeed back, 13 : 52-54 Hasnaoui N., Mars M., Chibani J and Trifi M (2010) Molecular polymorphism in Tunisian pomegranate (Punica granatum L.) as revealed by SSR fingerprints Diversity, : 107-114 Joshi N., Rawat R.A, Subramanian B and Rao K.S (2010) A method for small scale genomic DNA isolation from chickpea (Cicer arietinum L.) suitable for molecular marker analysis Indian J Sci Technol., : 1214-1217 Malik A., Afaq F., Sarfaraz S., Adhami V.M., Syed D.N and Mukhtar H (2005) Pomegranate fruit juice for chemotherapy of prostate cancer PNAS, 102 : 14813-18 Martins P.S.U., Jilma S.P., Rios J., Hingorani L and Derendorf M (2006) Absorption, metabolism 12 13 14 15 16 17 HortFlora Res Spectrum, 6(3) : September 2017 and antioxidant effect of pomegranate (Punica granatum L.) poly-phenol after ingestion of standardized extract in healthy human volunteers J Agric Food Chem., 54 : 8956-61 Mazid M., Khan T.A and Mohammad F (2011) Role of secondary metabolites in defense mechanisms of plants Biol Med., : 232-224 Neurath A.R., Strick N., Li Y and Debnath A.K (2005) Punica granatum (pomegranate) juice provide an HIV-1 entry inhibitor and candidate to icalmicrobicide Ann N Y Acad Sci., 1056 : 311-27 Pirseyedi S.M., Valizadehghan S., Mardi M., Ghaffari M.R., Mahmoodi P., Zahravi M., Zeinalabedini M and Nekoui S.M.K (2010) Isolation and characterisation of novel microsatellite markers in pomegranate Int J Mol Sci., 11 : 10-16 Porebski S., Bailey L.G and Baum B.R (1997) Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and ployphenol components Plant Mol Biol Rep., 15 : 8-15 Reski R (2002) Preparing high-quality DNA from Moss (Physcomitrella patens) Plant Mol Biol Rep., 20 : 423-442 Saghai-Maroof M.A., Soliman K.M., Jorgensen R.A and Allard R.W (1984) Ribosomal DNA sepacer- length polymorphism in barley: Mendelian inheritance, chromosalmal localtionk, and population dynamic Proc Natl Acad Sci USA, 81 : 8014-8019 Sharma A.D., Gill P.K and Singh P (2002) DNA isolation from dry and fresh samples of polysaccharide-rich plants Plant Mol Biol Rep., 20 : 415a- 415f Singh B., Yadav R., Singh H., Singh G and Punia A (2010) Studies on effect of PCR-RAPD conditions for molecular analysis in asparagus (Satawari) and Aloe vera medicinal plants Aust J Basic Appl Sci., : 6570-6574 Sumner M D., Raisin C.J and Ornish D (2005) Effect of pomegranate juice consumption on myocardial perfusion in patients with coronary heart disease J Cardiol., 96 : 810-814 q Citation : Raina D and Sundouri A.S (2017) Standardization of phenol free genomic DNA extraction of pomegranate genotypes for diversity analysis HortFlora Res Spectrum, 6(3) : 159-162