Title: 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 Authors
|Powell, Charles - UNIV OF FLORIDA|
|Sheng, He Lian - AG-FOOD & VETERINARY|
Submitted to: Journal of Economic Entomology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 19, 2008
Publication Date: April 1, 2009
Citation: Shatters, Jr., R.G., Powell, C.A., Boykin, L.M., Sheng, H.L., McKenzie, C.L. 2009. Improved DNA barcoding method for Bemisia tabaci and related Aleyrodidae: Development of universal and Bemisa tabaci biotype-specific mitochondrial cytochrome c oxidase I polymerase chain reaction primers. Journal of Economic Entomology. 102(2):750-758. Interpretive Summary: Whiteflies are one of the major economic pests in crop plants throughout the United States and worldwide. Bemisia tabaci is a species of the whitefly family of insects that is of extreme economic importance with global distribution. Multiple biotypes (distinct variants with different biological characteristics) of B. tabaci occur, of which the B and Q biotypes are aggressive and invasive pests that have spread worldwide from their origin in the Mediterranean area and as a result have created a need to adapt control methods specific to these variants. Monitoring the movement of different biotypes is an important part of the IPM strategies employed to deal with problematic whitefly outbreaks. We present improved methods for rapid characterization of the whitefly species and biotype to support this monitoring effort. These methods allow rapid adaptation of control strategies to address the specific biotypes as they appear and provide a mechanism through which to monitor which biotypes are emerging as problematic threats to crop production.
Technical Abstract: Whitefly is the common name of heteropteran insects that comprise the Aleyrodidae family composed of over 160 genera and 1500 different species. The mitochondrial cytochome c oxidase I (mtCOI) sequence has been used extensively in whitefly phylogenetic comparisons and in biotype identification of the agriculturally important Bemisia tabaci whitefly. Because of the economic importance of several whitefly genera, and the invasive nature of the B and the Q biotypes of Bemisia tabaci, mtCOI sequence data is continually generated from sampled populations worldwide. Routine phylogenetic comparisons and biotype identification is done through amplification and sequencing of an ~800 bp mtCOI DNA fragment. Despite its routine use, published primers for amplification of this region are often inefficient on some B. tabaci and especially across whitefly species. Because this sequence is used as a monitoring tool for rapid species/biotype identification, we have used currently available whitefly mtCOI sequence data to generate a set of PCR amplification primers (Btab-Uni primers) that are more efficient at amplifying ~748 bps of the ~800 bp fragment currently used from multiple genera within the Aleyrodidae. These universal primers amplify a mtCOI fragment from numerous B. tabaci biotypes and whitefly genera using a single amplification profile. Furthermore, mtCOI PCR-primers specific for the B, Q, and New World biotypes of B. tabaci were designed that allow rapid discrimination among these biotypes. These primers produce a 478 bp, 405 bp, and 303 bp, mtCOI fragment for the B, New World, and Q biotypes, respectively. By combining these primers and employing rapid PCR and electrophoretic techniques, biotype determination can be made within three hours for up to 96 samples at a time. When run in conjunction with the universal Bemisia primers, the 795 bp product can be used for sequence analysis when verification is needed.