The aim of the experiment was to look for natural genetic variation in different natural strains of Sordaria fimicola isolated from natural and stressed ecological conditions. It was challenging because there was no sequence information at the time, and we planned to design a set of PCR primers that would reliably amplify in S. fimicola and hoped that the amplified products were polymorphic. Hence we decided to target ribosomal protein genes, putting primers in the exonic parts to amplify the intron(s) they flank. We used S. macrospora sequence information to strategically place the "exon-primed, intron-crossing" primers in a couple of Rp genes (RpS29, RpS23, RpS26, RpL17, and RpL30) and to determine nucleotide variations in fifty strains of S. fimicola by sequencing PCR amplicons. As compared to the strains isolated from natural environment, strains isolated from stressed environment exhibited point mutation on three positions C (122) A; G (127) A and T (137) G for RpS29 gene, at two positions G (257) T; T (327) G for RpS23 gene and one base substitution A (152) T in case of RpS26 gene and polymorphisms on four nucleotides were observed for RpL30 gene while 100% homology was found for RpL17 ribosomal proteins. In this study, we have also explored the phosphorylation status of ribosomal protein S29 and predicted the phosphorylation sites using NetPhos3.1 server. Serine-phosphorylation on two residues Ser9 and Ser18 out of 56 amino acids was found evolutionarily conserved in five model organisms while Ser18 modification is not present in Sordaria species.
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- Genetic diversity
- High resolution melt analysis
- Real-time PCR