In-Silico Mining and Characterization of EST-SSRs for the Genetic Diversity Analysis of Lemon

Abstract

One of the economically important plants of the family of Rutaceae, Citrus limon L. Osbeck also known as lemon exists as different varieties of which many are indigenous to our country. Therefore, to assess the diversity or the genetic variations among the lemon varieties, the development of molecular markers is necessitated. Expressed Sequence Tags (ESTs) are small region of expressed DNA sequences of a genome which are highly conserved across species and are co-dominant in nature by having Simple Sequence Repeats (SSRs) unlike RAPD and AFLP which are dominant. Hence, this study was taken up for mining and characterizing SSR markers from the publically available EST data. The ESTs downloaded from NCBI database were mined for SSRs using MISA followed by primer designing using Primer3. Functional domains and GO analysis were performed on Blast2GO and primer-BLAST was done to check the suitability of the primers flanking the SSR regions. Total 1505 sequences were retrieved from NCBI database which after initial clean up filtered 1406 sequences containing 59 contigs and 1347 singletons. Total 80 SSRs were identified with frequency of 1SSR/Kb to be 8.27 of the total sequences examined. The 76 SSR containing EST sequences were functionally annotated, which resulted in 20 annotated ESTs and 22 ESTs having IPR (PFAM) domains. Successful primer pairs were designed for these EST-SSRs and out of these, 33 sequences having >200bp products were sorted of which 26 were optimised further and finally 12 primer pairs gave successful primer-BLAST results without any mismatch with two other citrus species. Thus, the findings gave an insight on the distribution and frequency of SSR in the ESTs of lemon. In the future, the usage of these functional molecular markers may further assist to explore the genetic diversity analysis of different lemon varieties.