Extraction of Total DNA from Locusts and Optimization of Reaction Conditions for RAPD Analysis

In this study,we had developed a fast and easy method to extract total DNA from migratory locusts.We could extract about 50 μg and 100 μg DNA from single male and female individual respectively.The extracted total DNA was about 45 kb in length with OD260/OD280 between 1.5 and 2.2,which could satisfy the requirements of RAPD and PCR on DNA template.In order to obtain high-molecular-weight DNA from migratory locusts and repeatable RAPD results,we used autoclaved tips to collect precipitated DNA after being extracted with phenol/chloroform instead of collecting by centrifugation.We tested experimental conditions including different PCR amplifiers,tubes and concentrations of template DNA,Taq DNA polymerase,dNTP and primer that might affect RAPD results.Within some limits,the concentrations of template DNA,Taq DNA polymerase,dNTP and primer did not greatly affect RAPD profiles.The optimized reaction conditions of RAPD for three Chinese locust subspecies in 25 μL reaction volume were as follows:20 ng template DNA,0.1 mol/L dNTP,0.2 μmol/L primer,1 U Taq polymerase.Some advice was also given,especially all individuals of all populations should be amplified with every primer at the same reaction conditions.With the optimized reaction conditions for RAPD,we analyzed the genetic relationship among three locust subspecies in China.The genetic distances generated by RAPDDIST between populations belonging to the same subspecies were less than those to different subspecies.In the UPGMA Phenogram of three locust subspecies,the two populations of L.m.manilensis and L.m.tibetensis were clustered together with a bootstrap of 100%,respectively.The Bameng population of L.m.migratoria was clustered with two populations of L.m.manilensis with a bootstrap of 66%,meaning that the Bameng population of L.m.migratoria could also clustered with L.m.tibetensis.In the UPGMA Phenogram of all individuals of three subspecies,all individuals of one subspecies were clustered together to form a branch,so three subspecies were significantly different in DNA polymorphism.Thus,RAPD method could be used to separate the three subspecies.There was a gene flow between the populations of L.m.tibetensis and L.m.manilensis.In order to get the true relationships among the three subspecies,more samples and primers should be used in the analysis on the three locust subspecies with RAPD,and other molecular techniques should be applied to test the results of RAPD.