以不同性別專一引子鑑定鸚鵡性別以及利用cytochrome b與12S ribosomal RNA序列鑑定鸚鵡種別

Abstract

Approximately 60% of avian species are sexually monomorphic, and most of the parrots could not be sexed by feather colors or any other morphological characteristics in appearances. It has been reported that specific DNA sequences could be amplified by using polymerase chain reaction (PCR) with the specific primers of chromodomain helicase DNA binding protein 1 (CHD-1) gene. In birds, there are two genes related to CHD-1, CHD-W and CHD-Z, which are located in chromosome W and Z, respectively. Since these two genes are evolutionarily conserved, and their sequences vary sex specific manner, application of the differences between the nucleotide sequences could be used for sex identification. Multiple primers, including P1/P2/P3, P4/P5, P2/P8, 2550F/2718R, primer1/2 and 1272H/1237L have been designed according to the sequence of introns or exons in CHD-1 gene. By using different combinations of these specific primers for PCR, 19 species of examined parrots could be successfully determined in this study. Amplification and sequencing fragments of mitochondrial DNA (mtDNA) genes, such as cytochrome oxidese I (COI), cytochrome b (Cytb), 12S ribosomal RNA (12S) and 16S ribosomal RNA (16S) are widely used for species identification. In this study, Cytb and 12S were tried to identify different species of parrots. Comparisons of sequences were conducted by using the BLASTn portal within the National Centre for Biotechnology Information (NCBI) database. The threshold value for sequence similarities of Cytb or 12S rRNA genes between analyzed samples and database is 98%. Twenty-six species of parrots were analyzed in this study. Results showed that the sequences of Cytb gene in 19 species of examined parrots could be found in NCBI. Furthermore, 13 species of analyzed parrots have the high similarity in 12S gene published in database. However, 2 species were found neither the sequences of cyt b nor 12S rRNA published in NCBI. Hence, in addition by comparison of mtDNA to identify parrot species is an accurate method, uploading the unpublished sequences would help the integrity of database. In addition to use mitochondrial DNA for species identification, random amplified polymorphic DNA (RAPD) were used to identify 17 different parrot species. In this study, 40 random primers were employed. One of these primer, OPH-17, was successfully amplified a specific band in Orthopsittaca manilata. After analyzing 8 sequences amplified from the same individual, there are no significant correlations among these 8 sequences. Thus, it is not possible to find species specific sequence using the 40 random primers in this study. More stricted conditions way are further tried to inprove reproducibility.