Microsatellites, often abbreviated as simple sequence repeats (SSRs), are 1 to 5 bp repeat mutation prone motifs. Due to mutation prone specific and high rate of polymorphism, microsatellites (SSR) have become a modern genetic resource tool for the genetic mapping studies, developing and establishing new patterns of molecular breeding and in inferring the phylogenetic and comparative genome analysis. These repetitive stretches are distributed in the coding and non-coding region with a bias towards the noncoding regions. The variation of SSR is controlled by a complex two-level system allowing changes in the number of repeat units and changes in the repeat sequences by point mutations (Sharopova Natalya, 2008). Simple Sequence Repeats have attracted relatively more attention because of their abundance in plants genome, reproducibility, high level of polymorphism, co-dominant inheritance and hypervariability. The ‘hypervariability’ can be documented and supported by illustrating the fact that the SSR differs in the number of repeats from genotype to genotype making them as the best suitable model genetic markers. The linkage maps of nuclear genomes are constructed through the analysis of di-, tri- or tetra-nucleotide SSRs. Single nucleotide repeats have been used in the population genetic analyses of chloroplast genomes (Powell et al., 1995). SSR are also finding applications in the field of comparative genomics where SSR of one species are finding the utilization in the (genetic mapping of the other species. Cordeiro et al., 2001; Peakall et al., 1998; Rallo etal., 2003). The frequency of the SSR reported is 1 SSR /6 kb in plant genomes (Cardle et al., 2000). Recent investigation and analysis on several plant genomes have also demonstrated that the frequencies of SSRs were significantly higher in ESTs than in genomic DNA (Morgante et al., 2002). Bioinformatics is currently acting as a resource tool for the development of SSR with the availability of the genomic sequences and Expressed Sequence Tags (ESTs) as molecular databases. Expressed Sequence Tags (ESTs) are currently the most widely sequenced nucleotide commodity in the terms of number of sequences and total nucleotide count. These provide a robust sequence resource that can be used for genome annotation, gene discovery and comparative genomics (Rudd, 2003). The concept of using cDNA as a route to expedited gene discovery was first discovered in early 1980s (Putney et. al., 1983). (Brenner 1990) proposed that an obvious method for characterizing the important part of human genome will involve looking at messengers from the expressed genes thus advocating the application of highthroughput methods for transcriptome sampling. Mark Adams (1991) first used the term EST in relation to gene discovery and the Human Genome Project. The first step in deriving a gene index from an EST set is to remove redundancy by clustering ESTs representing the same native transcripts (Kalyanaraman et al., 2003). This strategy is implicit in DNA assembly tools such as PHRAP (http://www.phrap.org/), TIGR Assembler (Sutton et al., 1996) or CAP3 (Huang and Madan, 1999) that are widely used for EST clustering. The Nucleotide Redundancy Index is calculated at the whole organism level, and represents the ratio of the total length of all clusters for that organism to the total length of all ESTs for that organism; at the extreme case, a value of 1 would indicate that every cluster was a singleton containing one EST. This measure should provide some indication of when newly sequenced ESTs are not finding new gene products. In this study we screened SSRs based upon ESTs that were publicly available for Prosopis species. We evaluated different motifs of mono, di-, tri-, tetra-, and pentanucleotide SSRs for variation in length and location, relative abundance, distribution for further usage as molecular markers. We have also analyzed the distribution of the SNP and the frequency of the SNP distribution.

All the Prosopis ESTs (1467) used are downloaded from the public database NCBI dbEST division (http://www.ncbi.nlm.nih.gov/dbEST/index.html) in May of 2008. ESTs downloaded from dbEST division (http://www.ncbi.nlm.nih.gov/dbEST/index.html) of NCBI were analyzed for the nucleotide redundancy index and for putative gene mining. The ESTs of Prosopis used were directly downloaded from the NCBI dbEST database. Cluster analysis was employed to define groups of overlapping EST sequences and by so doing eliminate EST redundancy while simultaneously improving the length of sequences in the local database. This type of clustering also assists in the estimation of genome coverage and SSR frequency per genome. ESTs were respectively clustered using PHRAP obtained under an academic License. The putative unigenes were saved as dual resource databases in FASTA-formatted files for SSR detection. The number of sequences per cluster varied widely. All unigene databases were used to identify and characterize SSRs using a Perl Script for SSR identification. The minimum repeat unit was defined as ten for mononucleotides, six for dinucleotides, and five for all the higher order motifs including tri-, tetra-, penta-, and hexanucleotides The FASTA-formatted sequence file was allowed to search each sequence for all possible combination of mononucleotide, dinucleotide, trinucleotide, tetranucleotide, and pentanucleotide repeats. The SNP were identified using the alignment with the consensus sequence and in house perl script was used to validate it.

This work is done to implement the clustering algorithm on the identification of the SSR in the Prosopis juliflora spp. The work described the identification of the SSR and the removal of the Poly A tail in the identification of true mononucleotide tracts. The work also helps in the identification of SNP as a resource tool for further usage as a molecular marker.