MOLECULAR ENTOMOLOGY Improved DNA Barcoding Method for Bemisia tabaci and Related Aleyrodidae: Development of Universal and Bemisia tabaci Biotype-Specific Mitochondrial Cytochrome c Oxidase I Polymerase Chain Reaction Primers

Whiteßies, heteropterans in the family Aleyrodidae, are globally distributed and severe agricultural pests. The mitochondrial cytochrome c oxidase I (mtCOI) sequence has been used extensively in whiteßy phylogenetic comparisons and in biotype identiÞcation of the agriculturally important Bemisia tabaci (Gennadius) whiteßy. Because of the economic importance of several whiteßygenera, and the invasivenatureof theBand theQbiotypes ofBemisia tabaci,mtCOI sequence data are continually generated from sampled populations worldwide. Routine phylogenetic comparisons and biotype identiÞcation is done through ampliÞcation and sequencing of an 800-bp mtCOI DNA fragment. Despite its routine use, published primers for ampliÞcation of this region are often inefÞcient for some B. tabaci biotypes and especially across whiteßy species. Through new sequence generation and comparison to available whiteßy mtCOI sequence data, a set of polymerase chain reaction (PCR) ampliÞcation primers (Btab-Uni primers) were identiÞed that are more efÞcient at amplifying 748 bp of the 800-bp fragment currently used. These universal primers amplify an mtCOI fragment from numerous B. tabaci biotypes andwhiteßy genera by using a single ampliÞcation proÞle. Furthermore,mtCOI PCR primers speciÞc for the B, Q, and NewWorld biotypes of B. tabaci were designed that allow rapid discrimination among these biotypes. These primers produce a 478-, 405-, and 303-bpmtCOI fragment for the B, NewWorld, and Q biotypes, respectively. By combining these primers and using rapid PCR and electrophoretic techniques, biotype determination can be made within 3 h for up to 96 samples at a time.