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

Abstract

DNA markers were identified for the molecular detection of the Asian long-horned beetle (ALB), Anoplophora glabripennis (Mot.), based on sequence characterized amplified regions (SCARs) derived from random amplified polymorphic DNA (RAPD) fragments. A 2,740-bp DNA fragment that was present only in ALB and not in other Cerambycids was identified after screening 230 random primers in a PCR-based assay system. Three pairs of nested 22-mer oligonucleotide primers were designed on the basis of the sequence of this fragment and were used to perform diagnostic PCR. The first pair of primers (SCAR1) amplified a single 745-bp fragment of ALB DNA, but this did not differentiate ALB from other species. The other two pairs of SCAR primers (SCAR2 and SCAR3) amplified bands of 1,237- and 2,720-bp, respectively, that were capable of differentiating ALB from other closely related non-native and native Cerambycids, such as A. chinensis (Forster), A. malasiaca (Thomson), A. nobilis (Ganglbauer), Monochamus scutellatus (Say), Plectrodera scalator (Fab), Saperda tridentata (Olivier), and Graphisurus fasciatus (Degeer). The latter two SCAR markers could be amplified using DNA extracted from body parts of ALB such as the wing, the leg, and the antennae as well as tissues from all the developmental stages including the egg, larva, pupa, and adult. These markers were also capable of identifying ALB using the DNA extracted from frass. Our results demonstrate that the SCAR markers we have identified can be used for unambiguously identifying ALB from other closely related Cerambycids using a simple PCR procedure.

Pine wilt disease in Yunnan, China: Evidence of non‐local pine sawyer Monochamus alternatus (Coleoptera: Cerambycidae) populations revealed by mitochondrial DNA

“…Molecular techniques using genetic data are effective in intra‐specific and phylogenetic research (Boge et al ., 1994; Hidayat et al ., 1996; Brown et al ., 1997; Miller et al ., 1999; Kethidi et al ., 2003). The COII gene is a coding sequence for cytochrome oxidase subunit II located in mitochondrial DNA.…”

Molecular authentication of the medicinal herb Ruta graveolens (Rutaceae) and an adulterant using nuclear and chloroplast DNA markers

“…It is a monolocus dominant marker and uses specific primers, which was designed from dissimilar sequences of nrDNA-ITS. The development of this marker is easy, fast and reliable, less sensitive to reaction conditions, and easy to perform in any laboratory (Kethidi et al, 2003). Therefore, results are more reliable with this marker as compared to other markers.…”

Genetic variation among species, races, forms and inbred lines of lac insects belonging to the genus Kerria (Homoptera, Tachardiidae)

“…RAPD-PCR is widely used for identifying cultivars, clones, natural populations, etc. Despite the limitation posed by reproducibility, unless reaction conditions are stringent (Baruffi et al , 1995 and Bertin et al , 2007), this technique offers the advantages of simplicity, independence from prior DNA sequence information, and the evaluation of a large number of loci across the genome (Hadrys et al , 1992; Lynch and Milligan, 1994; Weising et al , 2005), besides providing the basis for developing more reliable SCAR (sequence characterized amplified regions) markers (Kethidi et al , 2003). The technique has already been widely employed for assessing the genetic diversity of other insect populations (Reyes and Ochando, 1998; Castiglioni and Bicudo, 2005; Dvorak et al , 2006; Lopes-Da-Silva and Vieira, 2007; Martins et al , 2007; Magaña et al , 2007; Karam et al , 2007; Sosa-Gomez et al , 2008; Sharma et al , 2009).…”