Genetic Diversity Analysis of Couroupita guianensis Aubl . based on RAPD Marker

Couroupita guianensis Aubl. Commonly known as cannon ball tree, belongs to family Lecythidaceae and widely used in Chinese medicine. C. guianensis is being threatened due to habitat destruction in its native range for human settlement and other development related activities. Native populations of C. guianensis were collected from different locations of the India states and characterized through. To study the genetic diversity of C. guianensis populations we performed RAPD – PCR with several primers. The results were revealed in this study.

Couroupita guianensis Aubl.commonly known as cannon ball tree, belongs to family Lecythidaceae and widely used in Chinese medicine (Han, 1994).C. guianensis is native of South Ameria, specially to Amazonian basin.It is grown as ornamental plant in various countries and in India is generally grown in Lord Shiva temple because of its special featured flowers which look like hood of Naga (snake) protecting the shivalinga, hence is called as Nagalinga Pushpa (Kumar, 2011).
Couroupita guianensis widely used for its medicinal properties and is extensively studied for its pharmacological properties by various researchers from different countries.In its native range, fruit pulp is used to treat the canine and other animal diseases.Leaves used for the cure of odontalgia and various skin infections including protozon diseases in humans.Flowers are used for treatment of mood disorders, tumors and inflammation.Flowers are used for used for treatment of mood disorders, tumors and inflammation.Antibacterial activity of fruit pulp extracts Oil extracts was demonstrated against gram positive and gram negative bacteria.Ethyl acetate fractions of aqueous extracts of flowers proved effective in DPPH and superoxide radical scavenging (Azimi, 2012).Antianxity and other neuropharmacological activities of flower extracts have been demonstrated in mice models.With immense traditional uses and demonstrated pharmacological activities, C. guianensis is being threatened due to habitat destruction in its native range for human settlement and other development related activities (Bafna, 2011).Although its extensively planted in Thailand, India and United States as ornamental tree, there is an urgent need of conservation strategies for the species.Considering the medicinal, ecological and economical importance of this tree species, the genetic diversity needs to be studied for genetic improvement and germplasm conservation in the wake of deforestation and adaptatability in its exotic habitats.The DNA based molecular markers have been successfully implemented due to its numerous merits over morphological markers against geographic and environmental variations (Williams, 1990).Among variety of molecular markers available to evaluate the genetic diversity of medicinal plants, Random Amplified Polymorphic DNA (RAPD) markers proved most simple, rapid, handy, inexpensive where small sample is involved and reliable when prior genetic information of population under study is lacking.

Sample collection
Plant materials (young leaves, clean and disese free) from different populations of Gujarat, Maharashtra, Karnataka, Goa, Kerala and Tamilnadu were collected in sterile polythene bags and transported to the laboratory.On arrival to the laboratory, leaf samples were sterilized with 70% alcohol and airdried.Then the samples were processed for DNA isolation.Sample collection location details and accession codes are given in table 1.

Primers
The OPA set of random decamer primers were purchased from Eurofins Genomics India.The primers were reconstituted in molecular biology grade water to 100pm/µl as stock solution.For working solution 5pm/ µl is diluted from stock solution in molecular biology grade water and stored at -20ºC for further use.

PCR amplification
The PCR amplification was done using gradient thermal cycler (Master cycler, Eppendorf).The reaction mixture was containing og 50ng genomic DNA, 5pm primer, 200µm dNTP mix (Bangalore Genie), 1.5 M MgCl2, 1X taf buffer and 1U Taq DNA polymerase(New England Biolabs).The final volume of the reaction mixture is made up to 20µl using molecular biology grade water.The reaction conditions were, initial for 5 min at 95ºC followed by 35 cycles of cycle denaturation for 1min at 95ºC, primer annealing for 1 min at 38ºC and primer extension for 2 min at 68ºC and final extension for 30 min at 68ºC.The primers which are giving scorable polymorphic bands were selected for final amplification.The bands amplified were documentaed in gel documentation unit.

Scoring and Statistical Analysis
The each band amplified was considered as unit character and were scored as absent (0) or present(1).Based on the binary scoring data similarity matrix was constructed in accordance to Jaccard Similarty Coefficient.Dendrogram based on similar was constructed by UPGMA method using NTSys PC Version 2.0.

Results and Discussion
A dendrogram was constructed for population using Jaccard's similarity index values using the NTSYS-pc ver 2.02.In Sequential Agglomerative Hierarchical Non overlapping (SAHN) UPGMA were used to generate dendrogram.Based on the Dendrogram, the 11 population formed four clusters at similarity index of 0.69 for Operon H9 primer.It was observed that for H9 primer four clusters were formed in which Cluster l comprises of CBE 1 TP Pm 1 and KKM 1 CT Pm 2 were grouped closely (Figure 1).Along with these the remaining clusters such as II, III and IV had close association with KGR 1 PA Pm 2 ; TVM 1 TP Pm 1 , KKM 1 TP Pm 1 , KGR 1 TP Pm 3, KKM 1 JA Pm 3, KGR 1 HS Pm 1 ; MDU 1 PA Pm 2 , MDU 1 HB Pm 1, TRY 1 TP Pm 1 resppectively.In addition to the above observation for the primer H16 primer two clusters were formed.

Table 1 .
Sample collection and accession code details