Optimization of PCR Conditions to Amplify SSR Markers in Sweet Cherry Cultivars

Abstract

In all Romanian national fruit collections, all accessions holding a phenotypic, biochemical and genetic evaluation, but less a molecular level assessment of their….. For introduction of this material (accessions) into the breeding programs is necessary and important to know the level of characterization. Considering this fact, in the present study was optimization of the PCR protocol, specially annealing temperature of fifteen SSR primer pairs to characterize and study the genetic similarity and to determine the polymorphism between 36 sweet cherry accessions from I.C.D.P. Iasi, Romania. In the optimization process of annealing temperature for primers, was started with 5oC less them the temperature recommendated by the manufacturer, gradually ascending with 1-2oC/PCR cycle, up to 10-11oC, above the recommended value. For every primers pairs it was 6-7 steps for optimization of annealing temperature, obtaining satisfactory results to the following primers pairs: UCDCH17, UDP96-001 and PceGA25 at 65oC, UCDCH21: 64oC, UCDCH31: 66oC,:UPD97-402: 60oC, PMS3: 68oC. The primer pairs UDP96-008 obtained at 65oC, monomorphic bands, so it was excluded from the analysis. The other reagents used in PCR and temperature regimes (denaturation and extension temperature) were kept constant. From these results we conclude the transferability and applicability of SSR markers for genotyping and phylogenetic studies in the genus Prunus.