Myoglobin intron variation in the Gouldian Finch Erythrura gouldiae assessed by temperature gradient gel electrophoresis.
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.
Heslewood
M M
MM
Centre for Conservation Technology, School of Resource Science and Management, Southern Cross University, Lismore, NSW, Australia. mheslewo@scu.edu.au
Elphinstone
M S
MS
Tidemann
S C
SC
Baverstock
P R
PR
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D001717:Birds / Q000235:genetics*; D004247:DNA; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005787:Gene Frequency; D014644:Genetic Variation; D007438:Introns; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000235:genetics*; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D013696:Temperature
D017931:DNA Primers; D009211:Myoglobin; D004247:DNA