MOLECULAR CHARACTERIZATION USING RAPD …

1 , (4)2017: 658-666 ISSN2278 9103658 MOLECULAR CHARACTERIZATION USING RAPD AND SSR MARKERIN DIVERSE POMEGRANATE [PUNICA GRANATUM(L.)] GERMPLASMS aroj, , Parmar, ** Scholar, Department of Plant MOLECULAR Biology and Biotechnology,2**Advisor and Associate Professor, Collage of Agriculture,Tharad,Principal, Sardarkrushinagar Dantiwada Agricultural University, Saradrkrushinagar, Gujarat, Author (Punica granatumL.) belongs to the family Punicaceae exhibiting enormous morphological diversity is animportant horticultural crop of region of western India. We have inferred evolutionary relationship of 50 diverse genotypesusing RAPD and SSR markers. We have design new 14 SSR markers of which 3 were successful to depict polymorphicnature. Overall, 29 RAPD primers and eleven microsatellite (SSR) employed or MOLECULAR CHARACTERIZATION and screeningproduced a total 80/99 and 54/66 DNA fragments RAPD and SSR marker respectively.

2 Wider polymorphism becomeapparent with Average number of polymorphic bands per primer was found to be (for RAPD) and (for SSR). InSSR highest polymorphism ( ) was exhibited by PG5 while the lowest polymorphism ( ) was evinced with PG2,PG7 and PG31. The average polymorphism detected by the SSR loci in the present investigation was %. UPGMA andNJ based analysis of the genotypes was performed, USING Jaccard's similarity coefficient, PCA and PCO analysis. Thehighest and lowest similarities detected between genotypes were and , respectively. Dendrogram in showed twomajor clusters with coefficient value r = showing the goodness of it of the dendrogram of RAPD. In SSRdendrogram showed two major clusters with co-efficient value r = RAPD and SSR markers showed to be autilitarian instrument or analyzing the genetic variety of WORDS:(RAPD) random amplified polymorphic DNA and (SSR) simple sequence repeats (Punica granatumL.)

3 Belongs to the familyPunicaceae comprises single genus Punica and two sub-speciesP. protopunicaBalf. (2n=2X=16) (Jalikop, 2010) (Smith, 1976,Stover and Mercure,2007). It is the imperative delicious fruit crop of thetropical, subtropical region and commercially grown fromEurope, Africa, Mediterranean region to the Himalayasterrain (north India) (Stover and Mercure,2007,Ozgen,etal.,2008,Morton and Miami, 1987) due to their nutrientrich pool of vitamin A, C & E with 15-19% sugar andother antioxidant (Chauhan, and Kanwar, 2012). India is asecond largest producer million tonnes (Chandra,etal.,2010) after Iran. Out of this, nearly 80000 hectare areais covered in Maharashtra, which produces fruits of over 1lakh metric tons (about 85% of the total production).

4 InGujarat thousand hectare area is under cultivation withproduction thousand tons fruit and productivity per hectare in 2010-11(Anonymous, 2011). In Indiamajor states that cultivate pomegranate are Maharashtra,Karnataka, Gujarat, and Andhra Pradesh and to a smallerextent in Rajasthan, Tamil Nadu and Himachal is often cross-pollinated crop (Jalikop andKumar,1990) and exhibits five to six branchhermaphrodite flowers in different cultivars and rangedfrom to 49% (Mir,et al.,2012) therefore it isevident with enormous morphological diversity. More than250 germplasm lines are available in India. Total 187(both exotic and indigenous) germplasm are available innational yield gene bank of National Research Centre onPomegranate, Solapur. In the last 50 years, tenpomegranate cultivars have been recommended or onlycommercial cultivation.

5 Bhagawa, Ganesh and Sinduri arepopular varieties among farmers (Chandra,et al.,2010).Pomegranate varietiesviz., Ganesh, Bhagwa, Ruby, Araktaand Mridula are being cultivated in Maharashtra andGujarat. So, current research was planned out tosystematically analyze economically important varietywith wider morphological variation at MOLECULAR level tounrevealed their MaterialsFor diversity study more than 2 years old, 50 accessions ofPomegranate were selected from Horticulture arm andAICRP Project on Arid Horticulture, S. D. AgriculturalUniversity. Includes variety Achikdana, Ahor Seedless,Bedana Suri, Bedana Sedana, Bassein Seedless, Damini, GR Pink, Kerala Collection, Jallore Seedless, Jodhapur Red,Jodhapur collection, Jyoti, Maha, Nimali, Sirin Anar,Saharanpur, Spendanadar, Surat Anar, Utkal, IC 318705,IC 318703, IC 318779, IC 318790, IC 318753, IC 318718, A K Anar, Bhagawa, Chawala, China Orange, of genomic DNAG enomic DNA isolated, by talking about one g of freshyoung disease free leaf materials, were ground to make afine powder in liquid nitrogen and DNA was isolated byfollowing the Cetyl trimethyl ammonium bromide(CTAB) method as described by Doyle and Doyle (DoyleRAPD and SSR marker in diverse pomegranate germplasm659and Doyle, 1987) with some modification.

6 The quality andconcentration of the extracted DNA were estimated byspectrophotometer (Johnson,et al.,1955) and the sampleswere diluted to make a final concentration of 30-50 ng and SSR Analysis and PCR amplification:The 29 primers of RAPD(Table )and 24 SSR primers(Table )obtained from Bangalore Genie, Bangalore,India, was screened or the fifty pomegranates genomicDNA extracted or polymorphism RAPD,Polymerase chain reaction reactions were carried out in a25 l reaction volume containing l ofPCR Buffer B(10X), lMgCl2(25 mM),1 l of each of dATP,dCTP, dGTP and dTTP, M of primer, 1 unit of TaqDNA polymerase, 50 ng of tempelate were performed in a DNA thermocycler(Eppendorf, Hamburg, Germany), programmed or PCR,initial denaturation or min at 94 C, followed by 45consecutive cycles of 36 s at 94 C, min at 36 C and72 C or min and final extension at 72 C or 5 inSSRreactions except primers (forward andreverse)

7 Were used M of each, others were used insame conc. And the programmed as or initial denaturationor min at 94 C, followed by 45 consecutive cycles of36 s at 94 C, 2 min at 35-37 C and 72 C or 2 min andfinal extension at 72 C or 7 min (Bedafet al.,2011). Theannealing temperatures of the cycling parameter werereadjusted or each microsatellite primers according to theircalculated Tm based on the sequence composition: Tm=4 (G+C) + 2 (A+T)-3 C. The amplified products weresubjected to electrophoresis in % agarose gelin buffer[Tris borate (EDTA)] buffer running at 50 Vor gel was stained with (4 g/100 ml)Ethidium Bromide and viewed under UV scoringData was scored or computer analysis on the basis of thepresence or absenceof the PCR products.

8 The presence ofthe product / band was scored as 1 while the absence wasdesignated as 0 . The data were maintained in thespreadsheet format or further analysis. PolymorphicInformation Content (PIC) was calculated based on thefrequency of alleles of each 1 P2ij,Where,Pijis the frequency of the jthallele or the ithmarker locusand summation extends over n alleles. The polymorphismpercentage was calculated as per lowing method: (Blairetal.,1999). Polymorphism (%) = {(Total number of bands-Number of monomorphic bands)/Total number of bands}x cluster analysis and Neighbor Joining (NJ) clusteringwas done based on different similarity matrices usingPAST version software (PAleontological STatistics)(Hammeret al.,2001) and analyzed by the multivar toolwith Jaccard s similarity coefficient(Jaccard, P.)

9 ,1908), aswell as or ordination plot based on Principal ComponentAnalysis (PCA) and (PCO) Principal coordinate analysiswere also carried out . Cophenetic correlation wasdetermined to check the fitness of dendrograms was constructedusing UPGMA (UnweightedPair-Group Method with Arithmetic Averages).RESULTSG enetic diversity was studied on the basis of the bandingpattern thus obtained by both RAPD and SSR primersclearly distinguished cultivars into different clustersshowing sufficient diversity. The data collected fromrandom amplification of polymorphic DNA with 14arbitrary oligonucleotide primers produced a total 99 DNAfragments, among which 80 fragments were found to bepolymorphic. As such the mean number of polymorphicbands per was found to be The size of PCR amplifiedDNA fragment varied from to bp.

10 Thehighest amplified band (12) was exhibited by primerOPYH18 and the lowest amplified band (4) was exhibitedby primer OPAI18. The highest polymorphism ( )was exhibited by primer OPY6 out of 8 band, all the 8were found polymorphic while the lowest polymorphism( %) was evinced with OPAJ14. The averagepolymorphism detected by the RAPD loci in the presentinvestigation was (Table ). The polymorphicinformation content (PIC) values ranged from (average ), a reflection of allele diversityand frequency among the germplasm , were uniformlyhigher or all the RAPD loci eleven microsatellite (SSR) produced a total 66 DNAfragments out of 24 primers, among which 54 fragmentswere found to be polymorphic. As such, the mean numberof polymorphic bands per primer among fifty pomegranategenotypes was found to be The size of PCR amplifiedDNA fragment varied to (Table ).