In order to analyze potential genetic defects in P. apollo individuals
with deformed or reduced wings, total DNA was isolated from samples
of legs of either normal (9 butterflies) or malformed (23 butterflies) insects.
In addition, for external control experiments, DNA was isolated
analogously from wild-type P. rapae (7 butterflies) and P. napi (5 butter-
flies) individuals.
The quality of DNA templates were proved by PCR reactions with
primers for amplification of fragments of dpp, hh and ptc genes
(Table 1). The product of amplification of the fragment of the wingless
(wg) gene, obtained with the wg primers and using DNA from wildtype
P. apollo was cloned into a plasmid vector, and nucleotide sequence
of the clone has been determined. The obtained sequence (455 bp long)
was found (on the basis of Blast search) to be 99% identical to a fragment
of the wg gene of both Hyposcada illinissa (Lepidoptera: Papilionidae)
and Oleria cyrene (Lepidoptera: Papilionidae). The P. apollo wg gene
fragment sequence has been deposited in GenBank (Accession no.
HM213842).
We have analyzed the presence of the PCR product after reactions
with the wg primers when DNA templates were derived from wildtype
P. rapae or P. napi (external controls), wild-type P. apollo, and malformed
P. apollo with either deformed wings, reduced wings or extremely
reduced wings. In all tested samples obtained from P. rapae
and P. napi, the specific reaction product has been detected (Table 2).
This was the case also in 8 out of 9 samples obtained from wild-type
P. apollo. However, completely different results were obtained for malformed
P. apollo. No specific wg amplification product could be observed
in all samples derived from butterflies with reduced or extremely
reduced wings, and such a product was detected only in 1 out of 10
samples from individuals with deformed wings (Table 2).