18S rRNA Approach for Identification of Chara L . Species

The study focused on the molecular phylogenetics for the identification of genus Chara species through 18S rRNA genes. Genus Chara sample is collected in the freshwater streams of Talakona region, Chittoor district, Andhra Pradesh and DNA was isolated. The 18S rRNA, gene sequence was used to carry out BLAST with the ‘nr’ database of NCBI GenBank database. First ten sequences were selected based on maximum identity score using multiple alignment software program Clustal W. Ribosomal Database Project was used for generating distance matrix and Molecular Evolutionary Genetics Analysis 6 Software was used for constructing the phylogenetic tree. Based on the maximum identifying score Chara species was identified as Chara foetida.

Charophyceae is macroscopic algae, habit in both fresh and brackish water, upright stem like structure with branches, in addition to whorl of secondary branches 15 .These members are in close relation to higher plants due to the presence of similar pigmentation and reproductive structures 4 .They are used as research models in biotechnology, genetic engineering, biochemistry, cell cycle and physiology 5 .Algae have the great importance in ecological and economical context.Hence the availability of systematic details, diversity and distribution is essential for future research work.Systematics deals with identification of plant and evolutionary relationships 3 .They are studied by the classical and molecular methods.In classical method the plants are identified based on the morphological characteristics and inmolecular method plant are identified with the DNA sequence 1 .Identification of organism through molecular phylogenetic study can be done in short gene from a uniform locality on a genome.Each organism has different type of DNA barcode, so it has the great importance in the taxonomy, evolution and phylogenetical arrangement 2 .The first step of the Molecular phylogenetic studies is to select the suitable primers, amplified the barcode region PCR.In plant species which show similar morphology, differences can be identified through molecular phylogenetic study 12 .Sometimes it is difficult to identify the Chara speciesin which do not bear sex organs 15 .In such conditions the Molecular phylogenetic study helps in the identification of Organism.Anyone who has the Basic knowledge on DNA technology is adequate to identify the organism.Molecular phylogenetic study is not the only solution for all taxonomical problems, but it provides the effective nomenclature 10 and identification for the unknown or un recognized species.The organisms which are not identified in the classical methods can be identified by the molecular phylogenetic study.

Collection and Culture of Chara species
Chara sample was collected from the fresh water streams of Talakona region, Chittoor district, Andhra Pradesh.The sample was isolated from other associated algae, cleaned thoroughly with the water 19 .Chara sample was cultured in in vivo condition and named as CH9.Molecular Phylogenetic DNA was isolated from the samples with DNeasy plant Mini Kit(Quiagen, Hilden, Germany).1.0 % agarose gel was used for quality assessment, a high-molecular weight single band DNA has been identified.
CDMFP and CDMRP primers were used for amplifying the fragments of 18S rRNA gene.700 bp a single discrete PCR amplicon band identified in the Sample when resolved in agarose gel.CDMFP and CDMRP primers are carried out the DNA sequencing reaction.Forward and reverse PCR amplicon was used in BDT v3.1 Cycle (sequencing kit on ABI 3730xl)on Genetic analyzer for eliminating contaminants.
Aligner software used for generating Consensus sequence of 18S rRNA from forward and reverse sequence data.The 18S rRNA gene sequence was used to carry out BLAST with the 'nr' database of NCBI GenBank database.Ten sequences are selected based on the Maximum identity score and multiple alignment software program Clustal W was used for alignment.
Ribosomal Database Project was used for generating distance matrix.Molecular Evolutionary Genetics Analysis 6 software was used for constructing the phylogenetic tree.

Morphological Characters
Plant is 30 cm long, 620 µm wide and 6-9 whorls branch lets,incurved, terminal cell with 3 projections.Collection number: Talakona surrounding areas.Distribution: First report from Andhra Pradesh 6

Molecular Phylogenetic
Charaspecies shows 91.16% homology to the Charafoeitda.So, Chara sample was identified as Chara foeitda   Maximum Likelihood method (Tamura-Neimodel) are used for the evolutionary history 16,17 CH9 sample tree shows highest log likelihood at (-1309.77).11 nucleotide sequences are involvedin analysis and Codon positions included were 1st+2nd+3rd+Noncoding.In Chara foetida total of 737 positions in the final data set.Evolutionary analyses were conducted in MEGA 6.

Table 1 .
Sequences producing significant alignment