GENETIC ANALYSIS ON HIBISCUS SPECIES BY USING RAPD MARKERS

This study was undertaken for genetic analysis within the 7 varieties of Hibiscus rosa-sinensis by using RAPD markers the method involves a extraction of DNA from leaves of Hibiscus rosa-sinensis and yield of DNA ranged from 158-200μg and purity (ratio) was between 1.3-1.6 indicating minimum level of contaminating metabolites. Genetic analysis and relationship among seven Hibiscus rosasinensis germplasm were analyzed using Random Amplified Polymorphic DNA (RAPD). Total nine primers were used as, RPI-07, RPI-08, RPI-10, RPI-12, RPI-18, RPI-19, RPI-21, RPI-22 and RPI-23. No PCR product was generated through the primer RPI-23 indicating that no binding sites for this primer. In all the primers RPI-07, RPI-08, RPI-10, RPI-19 and RPI-21 are more suitable for the genetic variation analysis in Hibiscus species as it has shown maximum polymorphic bands. Primers RPI-12, RPI-18 and RPI-22 are weak in showing the polymorphic banding pattern for the all species of Hibiscus rosa-sinensis. By using PAST and MEGA programme Consensus Phylogenetic tree generated based on genetic distances segregated the seven Hibiscus germplasm into two different clusters. 75% times of species Hibiscus rosa-sinensis Red S.P. and D.P. (Single petal, Scarlet and Double petal, Rubroplenus) clustered in a single cluster. 87% times the cluster of Hibiscus rosasinensis Pink varieties clustered with Hibiscus rosa-sinensis White varieties. The results of the present study indicated that the RAPD analysis could be utilized by breeders for further improvement of Hibiscus rosa-sinensis species.


Introduction
Hibiscus rosa-sinensis can endure extreme heat and cold, poor soil environment, and its flowers are modest in size and color. H. rosa-sinensis, the tropical hibiscus, has glossy heavy foliage with large, brilliant and spectacular flowers 1 . There are numerous varieties of H. rosa sinensis obtained either by hybridization or mutation breeding 2 . The initial activities of DNA extraction and quantification can be used in a variety of settings with a wide range of organisms as the source of the DNA 2 . Advances in molecular techniques increased the availability of different DNA-based markers, which have become efficient tools 3 in conservation genetic studies 4 . Random amplified polymorphic DNA (RAPD) is a simple and straightforward PCR-based technique, which uses arbitrary primers for amplification of discrete regions of genome 5 .
Medicinal applications • Root is demulcent and used for Cough.
• A decoction of root is used for venereal diseases and fevers.
• Fresh root juice is given for gonorrhoea and powdered root for menorrhagia. • Leaves are emollient, aperient, anodyne and laxative. • Leaves and Stembark are used for abortion. • Staminal column is diuretic used for Kidney trouble. • Flowers are astringent, demulcent, emollient, refrigerant, constipating, hypoglycaemic, aphrodisiac, emmenagogue and used for treating aloparia, burning sensation in the body, diabetes, and menstrual. • Disorders.
• Buds are used in treatment of vaginal and uterine discharges. • Leaves and flowers are good for healing ulcers and for promoting growth and color of hair 6 . And our Present work is on the genetic analysis in this mainly focused on the isolation of good quality of DNA from selected Hibiscus species, PCR amplification of selected Hibiscus species for RAPD and Analysis of all Hibiscus species for genetic variability. Electrophoresis was conducted at 50V for 2-3 hours and the gel was observed and photographed under UV-Transilluminator 8 .

DNA Analysis:
Bands were manually scored 1 for presence and 0 for the absence and the binary data were used for statistical analysis by using the software "PAST". The sizes of the fragments (molecular weight in base pairs) were estimated by using 100 bp ladder markers, which was run along with the amplified products. A genetic dissimilarity matrix was calculated according to Squared Euclidean Distances which estimated all pair-wise differences in the amplification product and cluster analysis was done by wards method using variance algorithm 9 . PCR (polymerase chain reaction) technology had led to the development of several novel genetic assays based on selective DNA amplification. Protocol is also relatively quick and easy to perform. Because the RAPD technique is amplification based assay, only nanogram quantities of DNA are required. One of the strengths of this new assay is that they are more amenable to automation than conventional techniques. It is to perform and is preferable to experiments where the genotypes of large number of individual are to be determined at a few genetic loci. With this idea the experiment was undertaken to evaluate genetic variation and relationship of some Hibiscus cultivars by RAPD technique.

Results
In order to perform RAPD, the Hibiscus leaves of 7 different varieties were collected and used for the good quality of genomic DNA had been isolated. Extraction of DNA from leaves of Hibiscus rosa-sinensis and then DNA quantified through spectrophotometer then DNA was amplified by using PCR technique. The PCR product produced were run on 2% agarose gel and observed under UV-transilluminator and photographs were taken. PAST can read and export files in the NEXUS format. Here tree were generated by PAST program and visualization using MEGA tool with some editing of tree file to convert it to Newick Trees format. Table-1 shows the Jaccards similarity coefficient matrix and Fig-1 is the dendogram generated using Jaccard's similarity coefficient for the gel banding patterns generated by Primer RPI-07. Fig.-

Conclusion
RAPD marker is an efficient tool which permits to obtain information on genetic similarity among Hibiscus plants. The main objective of this investigation is to isolate a good quality genomic DNA of four Hibiscus cultivars, standardization of PCR amplification protocol for RAPD and analysis of four Hibiscus species for genetic variability. The genomic DNA was isolated using CTAB method. Isolated DNA was quantified and provided optimized conditions for PCR amplification appropriate reaction mixture using 9 RAPD primers, the PCR product then run on agarose gel electrophoresis, the bands are manually scored from amplification profile on gel. In the Consensus Phylogenetic tree which is based on Phylogenetic tree generated from the banding pattern by different primers RPI-07, RPI-08, RPI-10, RAPD conditions may serve as an efficient tool for further molecular studies. The genetic distance was very close within the varieties and also among the species. Thus, these RAPD markers have the potential for the identification of species and varieties and characterization of genetic variation within the varieties. This is also helpful in Hibiscus breeding programs and provides a major input into conservation biology.

Acknowledgement
The authors are grateful to the School of Biotechnology, Rajiv Gandhi Proudyogiki Vishwavidyalaya, for their continuous support and technical assistance and the authors wish to thank for valuable advice on data analysis.  DNA Template (10ng/µl) 1.0µl 6.