Dual-Probe FISH on Butterfly Chromosomes: Step-By-Step Technique

Objective: Karyotype diversity in some groups of Lepidoptera is associated with chromosomal rearrangements that are difficult to detect due to the structural features of lepidopteran chromosomes—the absence of a localized centromere. Methods: This article describes a technique for conducting fluorescence in situ hybridization (FISH) with two probes on chromosomes of Lepidoptera representatives. Blue butterflies of the genus Polyommatus (subgenus Agrodiaetus, family Lycaenidae) were chosen as the model organism. 18S rDNA and telomeric repeats (TTAGG)n were used as markers. The distinctive feature of this technique is the use of probes of different lengths—short (28 nucleotides) and long (1100 nucleotides)—labeled with different reporter molecules: a probe conjugated with fluorochrome (Cy3) was used for detecting telomeric repeats, while a biotinylated probe requiring immunochemical detection was used for detecting 18S rDNA. Results: Data on the localization of 18S rDNA and telomeric repeat markers for Polyommatus (A.) admetus malievi were obtained. Conclusions: The described technique allows for the independent construction of specific probes for the studied material and successful conduct of dual-probe FISH with probes significantly differing in length on butterfly chromosomes (Lepidoptera, Rhopalocera).