Polymerase chain reaction-restriction fragment length polymorphisms identify mtDNA haplotypes of greenbug (Hemiptera: Aphididae)

Phylogenetic studies of the greenbug, Schizapwhis graminum (Rondani) (Hemiptera: Aphididae), based on DNA sequences of the mtDNA cytochrome oxidase I (COI) gene, have revealed the presence of three sub-specific clades. Each clade has been found to roughly correspond to a host ecotype, i.e., crops or wild grasses. An alternative method to DNA sequencing followed by maximum parsimony (and other phylogenetic analyses) to classify individual greenbugs to a specific clade was developed. Using published DNA sequences a 525-bp portion of the COI gene was found to contain endonuclease restriction sights that were diagnostic for each clade. Greenbug DNA template was PCR amplified and the resulting products subjected to digestion with Dral plus XmnI, or Bc/I plus XmnI. The resulting RFLP patterns identified COI haplotypes I, II and III, which directly corresponded to clades 1, 2, and 3, respectively. Furthermore, the Dral plus XmnI digest was diagnostic for biotype H. The PCR-RFLP technique is very efficient and will facilitate genetic studies of greenbug populations that involve hundreds to thousands of individuals.