Random Amplified Polymorphic DNA Analysis For Several Cyprinoidei Fishes

In this article we applied the Random Amplified Polymorphic DNA technique to analyse eight Cyprinoidei fishes included Myxocyprinus asiaticus, Spinibarbus sinensis, Spinibarbus caldwelli, Sckizothorax prenanti, Tinca tinca, Mesocyprinus multitaeniata, Procypris rabaudi and a hybrid(Procypris rabaudi). At last, we reconstructed eight Cyprinoidei fishes' genetic relationship, distinguished the hybrid from the pure breed (Procypris rabaudi), and selected the RAPD molecular markers of the seven Cyprinoidei fishes.In the experiments we compare four different fish genomic DNA extraction methods included genomic blood DNA purification kit, genomic tissue DNA purification kit, the manual isolation of genomic blood DNA and the manual isolation of genomic tissue DNA. At last, we concluded that the manual isolation of genomic blood DNA is the best method to get the fish genomic DNA, and we use this method to get the eight Cyprinoidei fishes' genomic DNA. According to RAPD's traits, the experimental parameters were optimized, and the optimal experiment conditions were ascertained (1.5mmol/L Mg2+, 30ng template, anneal at 36 ℃). Under the optimal experiment conditions, total 60 random 10-mer oligonucleotide primers of OPA , OPC and OPM groups were applied to theRandom Amplified Polymorphic DNA Analysis of eight Cyprinoidei fishes. After we studied the RAPD's results, 35 primers were selected to amplify repeatedly. We got the statistic results of the RAPD's products of the 35 primers included total amplified bands of single primer, total amplified bands of single fish, et al. Amplified bands of 35 primers were selected to calculate similarity index (S=2Nab/Na+Nb) and genetic distances (D=1-S) . According to genetic distances (D) , cluster analysis based on the RAPD results was performed by UPGMA method and the genetic relationship of the eight Cyprinoidei Fishes was reconstructed. The result was in good agreement with the result of the traditional analysis and distinguished the hybrid (Procypris rabaudi) from the pure breed (Procypris rabaudi). The experiment also selected the RAPD molecular markers of the seven Cyprinoidei fishes and discussed the feasibility of using RAPD marker to identify the fishes.