Optimization of SSR-PCR system and primers screening of eight species of Stylosanthes

Stylosanthes are good legume forages. The high level of polyphenol and polysaccharides in the blade make DNA extraction difficult. CTAB method for DNA extraction was improved for Stylosanthes in this study. The orthogonal design was employed to optimize SSR-PCR system of Stylosanthes. The optimal system was each 20 μL sample mixture contained 1 μL 50 ng·μL-1 template DNA, 1.6 μL 5 μmol·L-1 primers, 1.6 μL 10 mol·L-1 dNTPs, 1.2 μL 25 mol·L-1 Mg2+, 0.3 μL 5 U·μL-1 Taq DNA polymerase, 2 μL 10× PCR Buffer (Mg2+ free) and 10.7 μL ddH2O. The PCR was performed with 35 cycles with three steps . One hundred and twenty three polymorphic microsatellite loci from seven species of Stylosanthes were amplified in eight another different species of Stylosanthes with the optimization system. Forty four polymorphic microsatellite loci can be effectively amplified in these eight species of Stylosanthes. Twenty six SSR primers with high polymorphism were selected for fingerprinting, genetic diversity analysis, molecular breeding and variety identification of Stylosanthes.