Pyrograms showing sequence analysis (SQA) of 24-base fragments of the COI gene. Group 1; (A) Parental: An. sinensis F0, (B) F1: An. sinensis x An. kleini -- > hybrid F1, (C) F5: hybrid F4 x An. sinensis -- > hybrid F5, (D) F10: hybrid F9 x An. sinensis -- > hybrid F10, and (F) F1: An. kleini x An. sinensis -- > hybrid F1. Group 2; (E) Parental: An. kleini F0 and (G) F5: hybrids F4 x An. kleini -- > hybrid F5 using pyrosequencing. Theoretical pyrogram patterns (top of each panel) and representative raw data (bottom of each panel) of control DNA extracted from each An. sinensis and An. kleini by pyrosequencing are shown. Pyrosequencing was performed by addition of enzyme (E), substrate (S), and four different nucleotides. The letters under the black bars show the dispensation (Disp:) order. The actual sequence detected by pyrosequencing is indicated below the panels after “Seq”. The Y-axis represents the level of fluorescence emitted by incorporating a nucleotide base, and the X-axis represents the total number of bases added at that point in time; A, T, C, G nucleotide bases. The light gray areas show the pyrogram for identifying each of the two groups of An. sinensis and An. kleini.