Development of sequence amplified characterized region (SCAR) markers of Helicoverpa armigera: a new polymerase chain reaction-based technique for predator gut analysis

A method is described for the development of DNA markers for detection of H elicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) in predator gut analy sis, based on sequence characterized amplified regions (SCARs) derived from a randomly amplified polymorphic DNA (RAPD) band. A 1200-bp DNA fragment o f H. armigera, absent in the predator band pattern and in other closely rel ated prey species, was identified by RAPD analysis. This fragment was clone d and its extremes sequenced to design extended strand-specific 20-mer olig onucleotide primers. Three pairs of SCAR primers, which amplified three dif ferent DNA fragments, were used to study the effect of fragment length on d etection of prey in the predator gut. Using the pair of primers that amplif ied the longest fragment of H. armigera DNA, a single band of 1100 bp was o btained, but its detection was not possible in the predator gut. Detection of the ingested prey was possible with the other two pairs of SCAR primers, obtaining bands of 600 and 254 bp, respectively. Detection of H. armigera DNA in the gut of the predator Dicyphus tamaninii was evaluated immediately after ingestion (t = 0) and after 4 h. Detection of H. armigera DNA after 4 h was only possible using the pair of primers that amplified the shortest fragment (254 bp). The test for specificity, using these last pair of prim ers, showed that H. armigera was the only species detected. The detection t hreshold was defined at a 1:8192 dilution of a H. armigera whole egg in all samples.