Myoglobin intron variation in the Gouldian Finch Erythrura gouldiae assessed by temperature gradient gel electrophoresis

Abstract

In this study we show how the use of exon‐primed, intron‐crossing (EPIC) polymerase chain reaction (PCR) of a diploid intronic region, in conjunction with temperature gradient gel electrophoresis (TGGE), can be used to detect and rapidly assess allelic variation at the nucleotide level. We developed passerine‐specific primers to amplify and sequence a 762 bp region including the second intron of the myoglobin gene in the Gouldian Finch, Erythrura gouldiae. A POLAND plot based on this sequence indicated that TGGE in combination with heteroduplex analysis (TGGE/HA) should reveal nucleotide variation in the 160 bp low‐melting domain. Sequencing of the entire fragment from 19 Er. gouldiae revealed five nucleotide substitution differences within the low‐melt domain, all of which could be detected and differentiated by TGGE/HA, and an additional substitution in a section of the high‐melt domain which characterised another allele. A total of 181 individuals from four populations were screened for these six alleles.