POLYMERASE CHAIN REACTION-RESTRICTION FRAGMENT LENGTH POLYMORPHISMS IDENTIFY MTDNA HAPLOTYPES OF GREENBUG (HEMIPTERA: APHIDIDAE)

Authors: SHUFRAN, KEVIN

Submitted to: Journal of Kansas Entomological Society
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/3/2003
Publication Date: 9/25/2003
Citation: Shufran, K.A. 2003. Polymerase chain reaction-restriction fragment length polymorphisms identify mtDNA haplotypes of greenbug (Hempitera: Aphididae). Journal of Kansas Entomological Society. 76(4):551-556.

Interpretive Summary: DNA sequencing of a mtDNA gene has previously established the existence of three genetically distinct races of the greenbug, Schizaphis graminum. DNA sequencing, while very accurate, unfortunately is time consuming and expensive, and does not lend itself as an ideal tool for population studies where hundreds of individuals are involved. To further aid in population studies of the greenbug, a PCR (polymerase chain reaction) assay was developed that correctly identifies single greenbugs to their proper race. A portion of a mtDNA gene was PCR amplified, and the PCR products then digested (cut) with a combination of two enzymes. Electrophoresis of the digested products revealed diagnostic RFLP (restriction fragment length polymorphism) patterns, which is analogous to a DNA fingerprint. The technique is faster (results can be achieved in an 8 h work day) and ultimately cheaper in materials and labor, thus making large population genetic studies of the greenbug more feasible.

Technical Abstract: DNA sequencing of the mtDNA cytochrome oxidase I (COI) gene has previously established the existence of three distinct sub-specific clades within populations of the greenbug, Schizaphis graminum. Each clade roughly corresponds to a specific host use. DNA sequencing, while very accurate, unfortunately is time consuming and expensive, and does not lend itself as an ideal tool for population studies where hundreds of individuals are involved. To further aid in studies of phylogeography and host adapted races in the greenbug, a PCR-RFLP assay was developed that correctly identifies single greenbugs to their proper sub-specific clade. A 525 bp portion of the COI gene was amplified, and the PCR products digested with a combination of two restriction enzymes (DraI + XmnI, or BclI + XmnI). Electrophoresis of the digested products revealed diagnostic RFLP patterns. The technique is faster (results can be achieved in an 8 h work day) and ultimately cheaper in materials and labor, thus making large population genetic studies of the greenbug more feasible.