Exploitation of Walnut (Juglans regia L.) Expressed Sequence Tags for Development of SSR Markers After In Silico Analysis

The walnut (Juglans regia) has been extensively characterized for expressed sequence tags (EST) sequences and currently 6169492 ESTs are available in National Center for Biotechnology Information. Although this is a valuable resource for marker development, the redundancy in sequences makes the mining out of unique candidates for designing markers cumbersome. Keeping this in view, the present study was undertaken with the aim to remove the data redundancy in walnut ESTs and then to develop simple sequence repeats (SSRs) markers. The EST sequences were assembled into a non-redundant set of 85 contigs and 1584 singletons (total sequences 1699), indicating 16.55% reduction in data redundancy. These 1699 sequences were then used to mine out SSR motifs. 132 EST-SSRs were detected, with dinucleotide repeats being predominant (70.45%), followed by trinucleotide repeats (27.27%) and very less frequent hexanucleotides (2.27%). These markers were validated by designing primer pairs. 15 of these designed primers were tested on a group of 37 walnut genotypes. Out of which 7 markers gave robust amplification, generating polymorphism. These findings indicate the usefulness of EST-SSRs in genome analysis. This study further emphasizes the importance of assembly of the vast amounts of data submitted in public databases. Our results have generated a set of non redundant walnut ESTs which is of prime importance for development of marker systems without any repetition or overlapping.