Identification of genetic marker by RAPD to differentiate normal and viral disease tolerance black tiger prawns Penaeus monodon

Abstract

Genetic differentiation between normal and white spot viral tolerance shrimps was examined using Randomly Amplified Polymorphic DNA analysis (RAPD). Forty-two RAPD primers were screened and 26 primers which gave resolvable and reproducible RAPD patterns were selected. Two primers, OPA-04 and OPB-20, of 26 selected arbitrary primers showed different DNA bands which found only in normal shrimps but not in viral tolerance shrimps. For OPA-04 primer, DNA band with size about 800 bp was found in all normal samples. Amplification using OPB-20 showed a DNA band with size about 1,250 bp which existed only in 12 of 19 normal shrimps (63.2%). Amplification of other geographically separated P. monodon using OPA-04 primer, showed only a faint band of 800 bp in samples from Satun-Trang collected later in 1997 but not in other normal groups. This fragment was further analyzed by cloning and sequencing. The 800 bp fragment was cloned and transformed into E. coli DH5alpha. Clone no. 28 containing the desired 800 bp fragment was isolated and partially sequenced by the ABI-PRISM automated sequencer. Four hundred and sixty nine bases (58.6%) were sequenced and then aligned to other genes in the GenBank using BLAST program. The sequence comparisons, nucleotides and amino acids, showed no similarity to any known genes or proteins. To test specificity of this fragment using PCR technique, the specific primers were designed from nucleotides of the 800 bp fragment and PCR amplification yielded a 173 bp product. The presence of a 173 bp fragment was found 80% and 15% in normal samples and Satun samples (collected in 1997), respectively. In viral tolerance samples and other normal groups did not showed this PCR product