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

Abstract

A method is described for the development of DNA markers for detection of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) in predator gut analysis, based on sequence characterized amplified regions (SCARs) derived from a randomly amplified polymorphic DNA (RAPD) band. A 1200-bp DNA fragment of H. armigera, absent in the predator band pattern and in other closely related prey species, was identified by RAPD analysis. This fragment was cloned and its extremes sequenced to design extended strand-specific 20-mer oligonucleotide primers. Three pairs of SCAR primers, which amplified three different DNA fragments, were used to study the effect of fragment length on detection of prey in the predator gut. Using the pair of primers that amplified the longest fragment of H. armigera DNA, a single band of 1100 bp was obtained, 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 primers, showed that H. armigera was the only species detected. The detection threshold was defined at a 1:8192 dilution of a H. armigera whole egg in all samples.

Development of scar markers for the dna‐based detection of the Asian long‐horned beetle, Anoplophora glabripennis (Motschulsky)

“…In the present work, we have shown that the SCAR primers developed can amplify the corresponding markers using a concentration as low as 1:24,576 dilution of the genomic DNA isolated from egg samples. A similar sensitivity was reported with the SCAR marker developed for H. armigera (Agusti et al, 1999). The accuracy rate for the SCAR markers in correctly identifying ALB samples was 100% for the 20 insects collected in North America.…”

“…To develop reliable and specific markers, we followed an approach previously described by Paran and Michelmore (1993) and Agusti et al (1999) to convert this RAPD marker into SCAR markers. Three pairs of SCAR primers (22 bp in length) were designed according to the sequence of the ALBspecific 2,740-bp fragment and used in diagnostic PCR (Fig.…”

“…The SCAR technique was first applied to the identification of the downy mildew resistance genes in lettuce (Paran and Michelmore, 1993), and more recently to the biological control agents Gliocladium catenulatum (Paavanen-Huhtala et al, 2000) and Trichoderma atroviride (Rosa-Hermosa et al, 2001). SCAR markers have been successfully developed for the detection of gut contents from the tobacco budworm, Helicoverpa armigera (Agusti et al, 1999) and the whitefly, Trialeurodes vaporariorum (Agusti et al, 2000). SCAR markers have also been used to differentiate between different strains of the Asian rice gall midge, Orseolia oryzae (Behura et al, 1999), and to distinguish the Asian from the North American gypsy moth, Lymantria dispar (Garner and Slavicek, 1996).…”

Genetic Variability of Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) Populations from Latin America Is Associated with Variations in Susceptibility to Bacillus thuringiensis Cry Toxins

“…Randomly selected fourthinstar S. frugiperda larvae were collected from each population and maintained in 100% ethanol at Ϫ20°C. DNA was extracted according to the procedure of Agusti et al (1). The selected larvae were placed individually in Eppendorf tubes, homogenized with 500 l extraction buffer (10 mM Tris-HCl [pH 8], 1 mM EDTA, 0.3% Triton X-100, and 60 g/ml DNase-free proteinase K), incubated for 30 min at 65°C, and centrifuged for 10 min at 10,000 ϫ g. The lysate was extracted twice with phenol-chloroform (1:1, vol/vol).…”

Mitochondrial markers to distinguish Spodoptera frugiperda populations associated with corn and cotton crops

“…All these rearing strains were established with insects collected from corn crops in each country, and the insects were reared in artificial diet under laboratory conditions, at 28±2°C and 65±5% relative humidity, under a 12:12 (light-dark) photoperiod (Monnerat et al, 2006). DNA was extracted from insect samples according to the procedure in Agusti et al (1999). For RAPD molecular analysis, approximately ten immature stages (second or third instar) of S. frugiperda were collected from different corn and cotton field crops.…”