Intra Specific Analysis of Gloriosa superba ( L . ) through ISSR finger printing and DNA sequencing of ecotypes collected from different accessions of Tamil Nadu State , India

Diversity within and among the population of Gloriosa superba collected from five different location of Tamil Nadu State, India, were explored by using Inter Simple Sequence Repeat (ISSR) DNA sequencing method. A total of 86.27% of polymorphic bands were seen in 94 reproducible bands generated from 12 number of ISSR primer ranging between 200bp to1000bp of band width. ISSR primer produces the bands with an average polymorphism of more than ninety nine percent among all the ecotypes. The dendrograms drawn for the analysis of genetic similarity in all the ecotypes. Our studies reveals that, genetic variations among different accessions of Gloriosa superba(L) was very well identified with the help of ISSR finger printings technic.


INTRODUCTION
Gloriosa superba (L) is an emerging medicinal plant belongs to the family Colchicaceae found widely throughout tropics and extends its occurrence in Africa, India and Southeastern Asia.The plant initially grown for its showy, broad and beautiful flower, locally named as "Senganthal malar" which is the state flower of Tamil Nadu State, India.Later the crop was evidenced to have high medicinal value both in formal and informal medicine.Its utility is pronounced both traditional and in modern medicine because of the high alkaloid accumulation in various parts of the plant.
The tuber portion of the plant contain two important alkaloids namely Colchicine (C 22 H 25 O 6 N) and Colchicoside (C 27 H 33 NO 11 ) [1] which is used for curing cold, ulcers, hemorrhoids [2] but in the recent study which is used for the treatment of bruises and sprains, colic, chronic ulcer, haemorrhoids, cancer, impotence, nocturnal emission, skin diseases, snake bites, intermittent fever, leprosy and also for inducing labour pains and for abortions [3].In modern medicine, thiocolchicoside a semi -synthetic derivative of cholchicoside is used as a muscle relaxant, analgetic and for anti inflammatory actions.Seeds and tubers of Gloriosa superba are used to treat gouts and rheumatism [4].
There is no specific released variety is available in this crop, only ecotypes with variable proportion of chemical constitutions in accumulation are available.This chemical constitution especially the alkaloid content were quantified and seems to be varying along its distribution and found very much influenced by the micro climatic factors of that particular accession.For further breeding and hybridization between natural populations, it is mandatory to known the genetic relationship that exists between the ecotypes of different accession, the identification

Intra Specific Analysis of Gloriosa superba (L.) through ISSR finger printing and DNA sequencing of ecotypes collected from different accessions of Tamil Nadu State, India
Jasmine and Balakrishnan of similarities and difference between the ecotypes taps the novel genes within the ecotypes for future research purpose.
ISSR-PCR has been widely used DNA-based molecular marker technique used to analyze the relationship between the species and ecotypes [7].The characterization of accessions through finger printing can be very well done using ISSR-PCR technique [8] among many types of DNA markers, the Inter simple sequence repeat (ISSR-PCR) can be very well utilized, as the ISSR distribution throughout the genome can be very well addressed by this technique.The marker based on microsatellites simple sequence repeats (SSRS) provides a co-dominate highly reproducible and genetically informative marker system [9].
But the loci of ISSR in the Gloriosa is unknown, this may be replaced by the advanced technique called ISSR-PCR technique, which has been found to be an efficient and reliable technique [10] as this technique need -no information on the DNA sequence prior to amplification, low cost, done with micro samples, perform with simple operations gives stability and produces abundance of genomic information.
Several factors are important including the geographic range which is strongly associated in determining the genetic diversity within the species it is also noted in this study that the general endemic species have lower genetic diversity than the wide spread species [11].So, it is of great need to find a genetic marker for all the five ecotypes collected from different accessions of Tamil Nadu for authentication and standardization of chemical constituents for further clinical, pharmacological and research purpose through highly reliable ISSR-PCR marker Systems.The objective of the present study is to identify specific, effective and reproducible markers for five ecotype of Gloriosa superba (L) collected from different commercially cultivable accessions.

Planting Materials
Ecotypes of Gloriosa superba (L) were taken from five different accessions from different regions such as Sirumalai (GA1), Mulanoor (GA2), Thuraiyur (GA3), Konganapuram (GA4) and Vedaranyam (GA5) for the investigation.The Randomized Block Design (RBD) with three replication was done.The experimental area mixed with red earth, sand, vermicompost and farmyard manure in the ratio of 1:1:1:1 was applied in the agriculture field in order to enhance the micro and macro nutrient of the soil for better plant growth.The plots were irrigated at seven to ten days interval regularly, the recommended agronomic and plant protection methodology were implemented during the cultivation.

Genetic Variability Studies And Separation Of DNA
Genetic variability studies for five ecotypes from 5 different accessions.Samples taken from five different accessions were used for the present studies (The reagents used to amplify the genomic DNA for ISSR analysis is given in the Table -1) and the DNA was extracted from bulk, fresh leaf tissues from each population of Gloriosa superba (L) as per the procedure [12] and the DNA was assessed by UV-vis spectrophotometer (Techomp 8500) in order to find out the purity and the quantity of DNA [13] and the DNA were separated by electrophoresis in agarose gel (1.0%) and was visualized by UV-vis trans illuminator and photographed.DNA pooling was done by pooling equal amount of purified economic DNA from the individuals of each five species and aliquot from those combined sample was used for PCR.

Inter Simple Sequence (ISSR Analysis)
The genetic variability studies were conducted by ISSR analysis using modified method [10].Earlier twenty primers were tested and twelve primers producing reproducible bands were selected for five accessions.Primers representing different random knock out DNA sequences were issued for ISSR-assay.The experiment were repeated three times and conformed for the reproducibility of bands the amplified products were checked by running through 1.5% agarose gel stained with ethidium bromide, the gel was viewed using UV-trans-illuminator and gel was documented using photo documentation system.The banding patterns were analyzed.

Inter Simple Sequence (ISSR Analysis)
The genetic variability studies were conducted by ISSR analysis by using modified method [10].Earlier twenty primers were tested and in which twelve primers producing reproducible bands were selected for the five accessions.Primers representing different random knock out nucleotide sequences were repeated three times for confirmation of the reproducibility of bands.An initial denaturing period of 2min at 94degree was followed by 35 cycles of 30sec at 94degree and then 35 cycles for 45 sec at 42degree followed by 35 cycles of 1min-30 sec at 72 degree for final extension was done (Table 2).The reaction was done with25 µl containing about 15.9 µl of sterile water, 4 µl of dNTPS,1.5µl of Mgcl2.,1µl of Taq polymerized and 0.5 µl of plant DNA.The amplified products were checked by running through 1.5%agrosegel stained with ethidium bromide.The gel was viewed using UV-Tran-illuminator and the gel was documented using photo documentation system.The banding patterns were analyzed for knowing the similarities and difference between the ecotypes.Out of 20 primers, 12 reproduced primers were chosen for running ISSR-PCR analysis (Table 3).

Data Analysis
The genetic distance matrix was obtained and analyzed through cluster method inorder to construct the UPGMA (Unweighted Pair Group method with Arithmetic mean Dendrogram using MEGA 4.0 [14].

ISSR Polymorphism
Earlier twenty primers were taken and screened for the studies.Primer producing clear and reproducible bands of 12 numbers were selected for further amplification with genome of five ecotypes of Gloriosa Superba (L).The oligonucleotide sequence of the primer with the percent content along with the amplification temperature were taken and their appropriate sequences were used for the analysis of ISSR.The twelve primers on amplification produces 7.8 bands per primer.The size range varies between 200bp to 1000 bp and a total of 94 polymorphic fragments were produced for all the five ecotypes of Gloriosa superba (L) which were summarized.

Genetic Diversity
The 5' anchored primers produces total of 94 polymorphic fragments in which the total diversity (Hg) varies between 0.2479 and 0.4880, and diversity within the population (Hs) varies between 0.0924 and 0.1984.The genetic differentiation (gst) of the produced fragment population range within 0.4418 to 0.8436 with a range of 0.2179-0.6387 of gene flow (Nm) (Hr,Hs,Gst and Nm) With the mean average of 1.9647 numbers of alleles,a mean average of 1.6471 numbers of alleles were found to be effective.The mean Nei's gene diversity between GA1-GA5 were 0.0931, 0.1477,0.1285,0.1193and 0.1013 respectively, with an average mean value of 0.4041 as shown (Table 4).
Nei's Unbiased measures of genetic identity (above diagonal) and genetic distance (below diagonal) were shown (Table 5).
Genetic distance of all loci between the population obtained by assessing the ISSR-PCR banding pattern shown in table 6.
The Similarity matrix shows a maximum similarity of (0.9276) that occurs between GA1 and GA5and a minimum similarity of (0.6307) that occurred between GA2 and GA3 which is given in the similarity matrix (Table 7).The number of polymorphic loci in all the five ecotypes varies between 26 (GA1) to 45(GA3), and the percentage of polymorphism varies from 26.36% (GA1) to 40.64 (GA3) which were represented (Table 8).The value of total number of observed alleles(na), Effective number of alleles(ne), Nei's gene diversity (h), Shannan's information index (I) were given as 1.9647, 1.6471, 0.4041, 0.5607 respectively and the over all polymorphic loci and its percentage were given as 92 and 86.27 respectively were given in Table 9.
The genetic relationship between five ecotypes was arrived by closing it into groups by UPGMA analysis based on Nei's genetic diversity within the population.A dendrogram was generated based on the cluster analysis through which the genetic relationship between the ecotypes can very well be studied.The genetically, ecotypes of GA4 and GA5 were closely related and ecotypes of GA3 and GA2 are widely placed, which were shown in dendrogram (Figure 1).

DISCUSSION
It is of urgent need to standardize and authenticate the chemical constituents of Gloriosa superba(L) ecotypes obtained from various accessions of Tamil Nadu as it varies with varying geological and agroclimatic conditions.Earlier work on Gloriosa superba (L) was done with ISSR-PCR system to evaluate genetic fidelity of micro propogated Gloriosa superba(L) using ISSR marker [15] was seems to be single work regarding genetic studies of the plant.In the present study reveals that the ISSR-PCR system was taken as tool for analyzing the genetic diversity (Figure 2).Twelve number of 5' sequence anchored primer were selected as it produces clear and reproducible fragments, about the 20 primers were taken initially and they were amplified.The ISSR marker technique produces much greater number of total polymorphic and discrimitant fragments then RAPDS [16] and lower cost than AFLPS [17].Simpler to use than SSR technique [18].Molecular fingerprints of four different species of Curcuma amada, C.caesia, C.longa and C.zedoa [19].Similar results are also reported in three different DNA based technique such as RAPD, ISSR and AFLP were used for fingerprinting Dactylis glomerata genotype and for detecting genetic variation between the three subspecies [20].Various species of genus Saccharina are economically important brown algae cultivated in China.