Introduction

Black Pepper (Piper nigrum. L) is one of the oldest spice know to the world P.nigrum belongs to the family piperacea.  The tropical ever green forests  of Western Ghats are considered as the center of origin of Black Pepper (1).  The major center of diversity of the genus piper is central & Northern South America, where 60% of the species are distributed.  More than 1000 Spps are included in the genus of which 115 are of Indian origin.  Black pepper, the most popular spice is cultivated in about 21 countries including India, Indonesia, Brazil, Malaysia, Srilanka, Vietnam, Thailand, China, Mexico, Guatemala.  In India, Black pepper is grown predominantly in the states  of Kerala, Karnataka and Tamil Nadu & to a certain extent in Maharashtra, AndraPradesh, Andaman and Nicobar Islands and North Eastern states viz, Assam, Meghalaya, Manipur and Arunachal Pradesh.

Morphological characters have been used extensively to study diversity of different form  in the past.  In recent years, attempts   to study biodiversity at molecular level have gained importance. Among DNA based approaches for crop-improvement, the first step of molecular  is the use of molecular markers as a tool to detect the extent and structure of Genetic variation(3,4) Providing insight into the diversity of crop- varieties  and potential contributions  represented by their wild relatives.

Microsatellite markers have become the DNA markers of choice for a wide range of application in genetic mapping and genome analysis genotype identification of Variety protection (5)) seed purity evaluation and germ plasm conservation, diversity studies (6, paternity determination and pedigree analysis (7)give and quantitative trait locus analysis (8) and Markers assisted breeding(9)  In measuring genetic diversity assigning  lines to heterotic groups and genetic fingerprinting , Microsatellite  provides  Power of discrimination equal to greater than that  RFLP in an more cost effective manner and case of study the objective of this study were to assess the extent of genetic diversity and relationship among 20 Piper nigrum genotypes.

Materials  and  Methods

Plant  Materials and Dna Extraction

Five Land Races & 13 advanced cultivars & 2 wild accessions  of Piper nigrum collected from IISR Experimental farm, Peruvannamuzhi were used in the present study  (TABLE-1) DNA was extracted according to the CTAB method with Minor modifications(2).  Leaf tissue of 2g was ground to fine powder in liquid N₂, followed by incubation  in 15 ml of preheated extraction buffer (4% W/V CTAB, 1.4 M Nacl, 100 mm Tris-HCL  PH 800, 20mm EDTA, 1.4m Nacl, 100mm Tris-HCL, PH 8-0, 20 mm EDTA, 2% PVP & 0.1 % V/V & Mercaptoethanol) for 2hr at 55⁰C.  The homogenate was extracted once with chloroform: Iso- amyl alcohol (24:1) and centrifuged at 15000 rpm at 25⁰C.  Nucleic acids were precipitated in 0.6 volume of isopropanol and collected by centrifugation (15,000rpm, 15 min, 25⁰C).  The precipitate was dissolved in TE- Buffer (100Mm Tris HCL PH : 8.0, 1 mm EDTA).  DNA was treated with bovine pancreatic  RNase and extracted with phenol: chloroform (1:1) and chloroform: iso-amyl alcohol (24:1) in succession.  DNA was quantified in a Fluorometer and dissolved to appropriate dilution in TE Buffer

Ssr and Rapd Primers

15 Rapd Primers And 9 Ssr Primers Were Used In The Study Are Mentioned In The Table2(A,B) As Follows. The Primers Were Obtained From Sigma –Aldrich,Usa.Pcr Reactions Were Carried Out In A Eppendorf Thermocycler

Table 1: Piper Accessoins used in the Present Investigation.

 Table 2: Operon Primers Which Showed Polymorphism For Developing RAPD Profiles .

 Table 3: List of SSR Primers used in the Study.

source

Menezes., et al

Pcr  Amplification For Rapd The PCR was performed in a 20µL reaction mixture comprising  50 ng of template DNA, 2µL 10X assay Buffer, 25 Mm Mgcl₂ (1µL), 100 mm each DNTP’s (dATP, dGTP, DCTP and dTTP), .85 U Taq DNA polymerase 10 pmolar  primer, PCR reaction were carried out on a perkin Elmer 9600 DNA thermal cycler, After a predenaturation step of 4min at 94⁰C, amplification reaction were cycled 40 times at 94⁰C for 1 min, 36⁰C for 1 min and 72⁰C for 2 min followed by 5 min at 72⁰C, was followed by completion of the primer extension on an Eppendorf thermal cycler.

Pcr  Amplification for Ssr

The PCR was performed in a 20µL reaction mixture comprising  20  ng of template DNA, 2µL 10X assay Buffer, 25 Mm Mgcl₂ (1µL), 100 mm each DNTP’s (dATP, dGTP, DCTP and dTTP), .85 U Taq DNA polymerase 10 pmolar  primer, PCR reaction were carried out on a perkin Elmer 9600 DNA thermal cycle.Temperature DNA was initially denatured at 940C for 5 min ,followed by 35 cycles of 940 C for 1 min denaturation ,primer annealing temperature between 570C to 680C FOR 1 min and 2 min primer extension at 720C.Final 5 min incubation at 720C was followed for completionof primer extension on an Eppendorf thermal cycler.

Gel Scoring and Data Analysis

Amplified DNA samples were analyzed by electrophoresis on 1.4% and 3%agarose gels in 1XTBE Buffer.  Clear and well resolved bands were scored for presence (1or absence (O).(Fig:1)  Statistical analysis was carried out using STATISTICA Package.  The program used was treeing with raw input data.  The main parameter, which guided the joining process, is unweighed pair group method with Arithmetic mean (UPGMA) & Euclidean distance was computed.  The relationship among 20 Black Pepper accessions was portrayed graphically in the form of a Dendogram.

Figure 1   Click here to View figure

Figure 2   Click here to View figure

Results and Discussion

Molecular profiles were developed with 15 RAPD Primers and 9 SSR primers Good polymorphism was observed between the genotypes for most of the primers studied. A total of 130 markers were obtained with 15 RAPD Primers .Out of which 71 markers were polymorphic and 51 were monomorphic. RAPD primer OPE-20 produced maximum numberof markers(13).Out of which 10 were polymorphic and 2 were monomorphic.  Among the 9 SSR primers studied ,5 were Polymorphic(PNF1,PND10,PNA5,PNG11 and PNH8),rest of the primers produced Monomorphic(PNB5,PNE3,PNH4,PNB9,) bands.The PCR products were run on 4% Polyacrylamide gel electrophoretic system to check the extent of polymorphism.   These polymorphic loci amplified approximately of size between 120-210bp.The maximum number of alleles amplified were at an average of 2 bands for each polymorphic primer pairs.

Conclusion

Grouping of individuals based on RAPD and SSR indicate a similarity in code region of   the  genome and hence common morphological traits. The molecular markers identified using RAPD and SSR techniques in the present study could be used in the identification of pepper genotypes quickly.  Among this the SSR markers were found to the useful in discrimination and identification of the genotypes and it gave more genotypic specific bands. The genetic diversity obtained in this study might be useful in future strategies for evaluation of desired genotypes.Such molecular data would be useful for detecting DNA patterns unique for a given accession or a set of accessions . This will through some light on the pepper crop improvement and boost our domestic needs  as well as the earnings of foreign exchange.

Acknowledgement

Deeply acknowledged to the Director IISR experimental form Peruvanamuzhi Calicut, Kerala for providing the plant materials for study.

This paper forms a part of PhD thesis ( Plant biology & Biotechnology submitted by Miss Remmia Raghavan to University of Madras,  Nov 2010.

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