Evaluation of phylogenetic relationship among Ficus palmata forrsk . wild ecotypes from Azad Jammu and Kashmir using inter simple sequence repeats ( ISSR )

Ficus palmata is a very potent medicinal and multipurpose plant which has gained global significance due to medicinal and multipurpose utility. It bears deep violet to black berries which are known to human beings from centuries for their effects on health. The plant is wildly distributed throughout Azad Jammu and Kashmir. The phylogenetic relationship among these natural Ficus palmata ecotypes from Azad Jammu and Kashmir is not established so for using reliable molecular markers. ISSR has been proved to be an effective tool for the determination of phylogenetic relationship among closely related species. To construct a population level genetic profile for investigation and exploitation of genetic diversity of Ficus palmata, 25 ecotypes of Ficus palmata in AJK were analyzed using ISSR (Inter simple sequence repeats) markers. Phylogenetic distance estimated revealed that the ecotypes expressed common heritage for their phylogenetic relationship with a considerable genetic diversity among them as well. Quite a few Ecotypes showed close relationship irrespective of their geographic distances and morphological attributes. The research evolved a significant outcome to start a breeding program from the evolution of Ficus palmata varieties for the mountain areas of Azad Kashmir.


Introduction
The figs (Ficus palmata species, Moraceae) are among the biggest genera of angiosperms with more or less 750 types of trees, epiphytes and bushes in tropical and subtropical areas around the world.Frodin [1] positioned them as the twenty-first biggest class of seed plants.Ficus palmata is a standout amongst the most differing plant genera as to development propensity, with both deciduous and evergreen detached trees, little bushes, creepers, climbers, stranglers, rheophytes and lithophytes [2].The Asian-Australasian locale has the wealthiest and most different fig greenery with more than 500 species.By examination, the extravagance of Ficus in Africa and the Neotropics is lower, with pretty nearly 110 and 130 species, separately.Generally a large portion of the Ficus palmata species are monoecious, and the rest are practically dioecious [3,4].Characterization and evaluation of genetic diversity is the first step in the conservation and utilization of indigenous medicinal plant species.Portrayal and assessment of hereditary differing qualities is the initial phase in the preservation and usage of indigenous therapeutic plant species.Additionally Estimation of hereditary assorted qualities is an essential for enhancing of any species or hereditary material.Molecular markers have been used keeping in mind the end goal to describe therapeutic plants and assessment hereditary differences inside and among restorative plants genotypes utilizing diverse PCR methods in light of atomic and/or mitochondrial genomes [5].The hereditary assorted qualities in regular plant populaces were shockingly at abnormal state in 1970s when isozyme examinations were made.This set off the environmental geneticists to contemplate the relationship between the levels of hereditary variety, natural and life history attributes of numerous plant species Keeping in view the global importance of Ficus palmata and its presence in Azad Kashmir as wild forests in abundance this research was conducted to assess the hereditary differences among chosen Ficus palmata ecotypes utilizing molecular markers and to look at the morphological, biochemical and molecular assorted qualities to be utilized as quality pool for Ficus palmata change in Pakistan and to suggest and set up the most proper ecotypes for delicate inclines of Azad Jammu and Kashmir.

Molecular characterization
The leaves of Ficus palmata Forssk were used in molecular studies.

Plant material collection and storage
Selected plant species were collected from different geographical regions of Azad Jammu and Kashmir including Muzaffarabad, Pallandri, Rawalakot and Kotli.These plants were collected during their flowering season.All plant species were morphologically identified and taxonomically authenticated while using the flora of Pakistan.The plant material was preserved in sealed zipper bags added with the silica gel (Fisher chemical L-12167) at 4°Ctill further use.

Plant material processing
The leaf material was gently excised from the plants using an already sterilized scissor followed by washing with 70 % ethanol and distilled water.The plant material was allowed for drying.0.4 grams of the leaf material was weighted for DNA extraction using CTAB (Cetyl trimethyl ammonium bromide) method with few modifications [22].Genomic DNA confirmation Genomic DNA of the test samples were assessed by gel electrophoresis.The DNA sample (5 µL) was mixed with loading dye bromophenol blue (2 µl) and was run on 1 % agarose gel.Ethidium bromide was used to stain and visualize the gel by taking picture under UV light by Dolphin Doc Plus gel documentation system (Wealtec).

Polymerase chain reaction
PCR reactions were carried out by 05 ISSR primers.The primers UBC807, UBC810, UBC812, UBC 816, UBC817 were used.The amplification reaction mixture of 25µl for each sample contained 1µl of template, 1µl of primer, 10.5 µl of nuclease free water and 12.5µl of Master Mix (MBI Fermentas).Different concentrations of annealing temperature were tested for amplification using gradient PCR and finally a standard method was applied.

Primers dilution
Primers were initially diluted according to prescription by e-Oligos.Concentration of primer used was 25 picomole per µl.

PCR product confirmation
The successful PCR products were confirmed by using 1.5 % agasrose gel, prepared in 0.5 X TAE buffer.Loading dye along with PCR product was loaded in wells of agarose gel.The gel was stained in ethidium bromide and was visualized under UV light in gel documentation system (Wealtec Dolphin Doc plus).The confirmation of size of particular band was done by running 50 kb DNA ladder with PCR products.

Primers data scoring
Primers data was scored as presence or absence of bands for each sample separately and monomorphic and polymorphic bands were calculated.All the visible segments were counted for each primer and presence or absence of bands was calculated as (1) if present and (0) when absent for each sample.

Results and discussion
Isolation of genomic DNA Genomic DNA of twenty five ecotypes of Ficus palamata are collected from different areas of Azad Jammu and Kashmir was extracted by CTAB method [24] with some modifications (Figure 1).The quantity and quality of extracted DNA was checked by running it on 1.5% gel.Concentration of DNA was determined by spectrophotometer at 260 nm. and the purity of genomic DNA was measured by absorbance at 280 nm .

ISSR analysis of all the amplified products by 05 ISSR primers
A total of 29 bands were produced among the ecotypes of Ficus palmata grown in AJK in which 03 were monomorphic and 22 were polymorphic with a polymorphism of 76% (Table 1) Molecular size of amplified fragments ranged from 50bp(UBC812) to 600bp (UBC810).The minimum number of DNA fragments was 05 while maximum number was 07bands .Moreover among all ecotypes only 10% monomorphism was found (Figure 2).

Conclusion
The method described by [34] with some modifications proves to be more appropriate for the fast obtaining a good quality of DNA of Ficus palmta tree for the amplification by PCR.The utilization of microsatellites markers in order to study diversity of different cultivars of Ficus palmata revel the presence of 29 fragments.This study provides evidence that the ISSR procedure is an informative and suitable approach to the examination of the molecular polymorphism and the phylogenic relationships in the Ficus palmata germplasm.The overall results of this investigation demonstrated that all ecotypes studied could provide valuable material for use in breeding programs.Finally more works are necessary to enlarge the number of markers by the use of other molecular technologies in order to have a deeper insight into the molecular polymorphisms and to establish a varietal identification key in this plant.
Molecular data was analyzed by software NTSYS-pc and PAST.A dendrogram based on similarity index was constructed by using Numerical Taxonomy and Multivariate Analysis System by [23].