Design and validation of SSR microsatellite primers for castor bean

Abstract

The objective of this work was to design, validate, and optimize pairs of SSR microsatellite primers for castor bean. The design of the primer pairs was done by means of the Websat application from sequences deposited in the GenBank of the National Center for Biotechnology Information (GenBank/NCBI), and its quality was assessed using the NetPrimer web application. Different concentrations of DNA, magnesium chloride, primer pairs, dNTPs, and annealing temperatures were used for the optimization of PCR conditions. A total of 30 primer pairs were designed, synthesized, and optimized. Agarose gel was used for detection of the amplified products, and denaturing polyacrylamide gel for the optimization of PCR conditions and the identification of polymorphism. The primer pairs presented average guanine/cytosine (GC) percentage of 47.29% and amplified fragment sizes varying from 128 to 381 bp. Twenty‑nine pairs of SSR primers (96.7%) were validated, of which nine were polymorphic (23.3%). The concentrations optimized for amplification are: DNA, 25 ng; magnesium chloride, 1.2 mmol L^‑1; Forward and Reverse primers, 0.4 mmol L^‑1; dNTPs, 0.1 mmol L^‑1; and annealing temperature, 62 to 64°C. The bioinformatic tools Websat and Net Primer can be used to develop quality microsatellite primers for castor bean from sequences deposited at the GenBank/NCBI.