A PRELIMINARY ANALYSIS ON GENETIC VARIARTION IN POPULATION OF MONOCHORIA VAGINALIS IN DIFFERENT GEOGRAPHICAL AREAS OF COASTAL KERALA Smitha Thomas K.

, Aneesha Devassy, Linu Mathew School of Biosciences, Mahatma Gandhi University, Kottayam OVERVIEW Molecular markers have proved to be valuable tools in the characterization and evaluation of genetic diversity within and between species and populations (Russell J. R., 1997). In this study genetic diversity among different populations of Monochoria vaginalis seen in different geographical locations of coastal Kerala was studied by RAPD marker. For the above study five plants were collected from four geographical locations such as Pathanamthitta , Ernakulam, Kottayam, Thrissur. Five primers from fourteen decamer primers initially screened displayed RAPD profiles with polymorphic bands and many of these bands varied in molecular weight and intensity. The number of polymorphic bands scored and dendrogram was constructed by using SPSS based on the average linkage between groups. The present study shows that the RAPD analysis is useful for genetic diversity analysis.

INTRODUCTION The plant selected for the present study is Monochoria vaginalis. This plant belongs to the family Pontederiaceae. This is a commercially important native e aquatic medicinal plant that has both medicinal and ornamental value (Warrier, 1995). This plant species that was present in abundance in paddy fields of Kerala is considered as threatened species, due to a variety of reasons. To find out the genetic variation among the population of Monochoria is important for conservation. In this study RAPD was used to analyze the population genetic structure of Monochoria and provide valuable molecular evidence for its genetic variation. Figure of Monochoria OBJECTIVES To standardize the DNA isolation protocol for RAPD reaction To optimize the RAPD reaction parameters. To find out the genetic diversity among different populations of Monochoria seen in different geographical locations of coastal Kerala by RAPD.

MATERIALS AND METHOD Plant material- Five plants were collected from 4 different geographical region and one gram of fresh tender leaf was used for DNA isolation.

DNA extraction- DNA isolation was standardized by modification of Doyle and Doyle, 1987 protocol. Amount and purity of DNA- The yield of DNA per gram of leaf tissue extracted was measured using a UV spectrophotometer at 260 nm. The purity of the DNA was determined by calculating the ratio of absorbance at 260 nm to that of 280 nm. DNA concentration and purity was also dertermined by running the samples on 0.8% agarose gel stained with ethidium bromide. (Figure.2) Selection of Primers- Fourteen decamer oligonuleotide with arbitrary sequences were screened and five were selected for RAPD. Serial No. 1 2 3 4 5 Primer Name P2 P3 P4 P5 P8 Primer Sequence CCAGCTTAGG CCGCCCAAAC TCTGTCGAGG CACCTTTCCC GGTTGTACCC Percentage of GC content 60% 70% 60% 60% 60%

Table No.1 Primers and primer sequences used for polymorphism Optimization of RAPD reaction-Optimization of RAPD reaction was conducted in Bio Rad Gradient Thermo cycler by varying the PCR parameters for getting reproducible banding patterns. PCR products were analyzed on 1.2 % agarose gel and stained with Ethidium Bromide and then visualized by Bio- Rad Gel Documentation system. RESULT AND DISCUSSION Optimum PCR condition had an effect on amplification, banding pattern and reproducibility. The optimized conditions for RAPD protocol are given in Table 2 Table.2 PCR parameters (Modified from Padmalatha K, Prasad M. N. V., 2006) PCR parametrs Optimum conditions DNA Concentration 25 ng dNTPs 0.2 mM Primer Concentration 12 Picomoles Taq DNA Polymerase 1.2 U Initial Denaturation Temperature and 940C for 1 minute Time Denaration temperature and time 920C for 1 minute Annealing temperature and time 360C for 1 minute Extension Temperature 720C for 1 minute Final Extension temperature and time 720Cfor 1 minute Reaction Volume 15l Number of Cycles 40 Cycles

DATA ANALYSIS Amplified fragments were scored as Binary data ie Presence as one and absence as zero. Figure.3 of RAPD Profile For the twenty individuals of Monochoria from the four population , five selected RAPD primers produced 24 reproducible and clear amplification bands of which 23 (95.8%) were polymorphic. Primer No. 7 showed highest number of bands and primer number four and five showed the least number of bands. Table No. 3 Polymorphic amplified bands detected with 8 primer for four populations of Monochoria vaginalis(Percentages of polymorphic band ,PPB) Primer Number of No. Of Polymorphic bands Total No. of Nmae amplified Polymorphic PTT EKM KTM TSR bands bands P2 6 6(1) 0(0) 6(1) 1(0.16) 6(1) P3 5 1(0.2) 1(0.2) 0(0) 0(0) 4(0.66) P4 3 3(1) 3(1) 0(0) 2(0.66) 3(1) P5 3 2(0.66) 2(0.66) 1(0.33) 0(0) 2(0.66) P8 7 1(0.14) 1(0.14) 1(0.14) 0(0) 7(1) Average 4.8 2.6(0.6 1.4(0.6 1.6(0.29 0.6(0.16 4.4(0.86) ) ) ) ) PTT-Pathanamthitta, EKM-Ernakulam, KTM- Kottayam, TSR- Thrissur A dendrogram was constructed using SPSS for estimating genetic similarity among populations. Figure of dendrogram
C A S E Label Num Rescaled Distance Cluster Combine 0 5 10 15 20 25 +---------+---------+---------+---------+---------+

19 20 18 16 17 2 5 6 9 8 10 7 4 1 3 11

12 14 15 13

CONCLUSION Analysis of RAPD data shows high genetivc variation among and between populations of Monochoria. Everyone avoided this plant as a weed eventhough the plant has medicinal and heavy metal scavenging properties. The study reveals the importance of this plant to be conserved for its high genetic variation and also the usefulness of RAPD marker for detecting genetic variation. REFERENCES 1. Doyle, J. J. and J. L. Doyle. 1987. A rapid Dna isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19; 11-15. 2. Padmalatha, K and Prasad, M. N. V. 2006 Optimization of DNA and PCR protocol for RAPD analysis of selected medicinal and aromatic plants of conservation concern from peninsular India. African Journal of Biotechnology 5;230-234.