| Summary: | 碩士 === 國立臺灣大學 === 分子與細胞生物學研究所 === 99 === In recent years, mitochondrial DNA and microsatellite sequences are often used as the molecular markers for studying species evolution and population structure. However, to date, appropriate molecular markers for distinguishing closely related species have not been well-established. Our previous study demonstrated that the gene sequences of intermediate filaments (IFs) proteins, one of the major types of cytoskeleton, could separate vertebrates and invertebrates into two different groups in phylogenetic
reconstruction. In addition, each of the vertebrate IF genes was separated into different subgroups corresponding to their IF types. Furthermore, within each subgroup, the
evolutionary relationship of different species is parallel to that of species. These results suggested that the evolution of IF genes is closely related to the evolution of vertebrate species. In this study, the gene of type III intermediate filament protein GFAP (glial fibrillary acidic protein) was selected as the molecular marker. Due to its specific expression in the central nervous system, it is postulated that its selective pressure from the external environment is minimum, thus it is more likely to maintain neutral. To prove that the GFAP gene is an appropriate molecular marker, we collected the GFAP sequences of each species from the Ensembl database. Subsequently, we generated the phylogenetic tree of the GFAP gene sequences of 19 animal species by Bayesian inference (BI). The results was in agree with the currently accepted classification. Therefore, the validity of GFAP gene as a molecular marker is confirmed. Recently, it has been shown that introns have relatively large nucleotide variability, and can be easily amplified with primers placed in the adjacent exons. Thus, there are increasing number of studies using intronic sequence to investigate the phylogenetic relationship of species. In 2010, Igea et al., by applying several reasonable filters, selected 224 intronic sequences that belongs to several mammalian species, including human, chimpanzee, macaque, dog and cattle, and successfully distinguish the phylogenetic relationship of these species. Therefore, in this study, it is anticipated to characterize the appropriate introns of GFAP gene for distinguishing the closely related species. By aligning the 8 intronic sequence of GFAP gene of five primate species, including human, chimpanzee, gorilla, gibbon and macaque, it was revealed that the length of the intron 2 fragment are the same among five primate species. In addition, the phylogenetic tree of these five primate species reconstructed with GFAP intron 2 by UPGMA method was also in consensus with currently established phylogenetic relationship. Thus, the intron 2 sequence was selected as the molecular marker for the current study. The traditional classification for mammals is based on fossil
evidence. On the other hand, classification of fishes is mainly based on morphology of the extant species. Thus, the aim of this study is to examine the phylogenetic relationship of species under a unified standard by examining GFAP intron 2 sequences of primates and fishes. In our study, fishes of several different families and genera in the suborder Scombroidei were selected, and their GFAP genes sequences were amplified by PCR. After sequencing, their lengths and variation were compared. The results revealed the length of GFAP intron 2 is different in each of the five fish families. Surprisingly, although from the same family of Scombridae, the length of GFAP intron 2 of the yellowfin tuna, bigeye tuna, albacore tuna,, Pacific bluefin tuna (Thunnus) and the seer fish (Scomberomorus) is distinct from that of spotted mackerel (Scomber) and frigate mackerel (Auxis). Then, we reconstructed the phylogenetic tree by intron 2 sequence from each of the fishes, the results revealed one single group among the four fishes of Scombridae and fishes of Trichiuridae. Moreover, among the Scombridae, spotted
mackerel and frigate mackerel are closer to each other, but are distinct from the Thunns and seer fish. Taken together, the results suggest that the evolutionary relationship of
Scombridae and Trichiuridae are closer than the currently accepted phylogenetic classification. Furthermore, it is also proposed that spotted mackerel, frigate mackerel
should be separated from Thunns, seer fish, and regrouped into a new family. Besides, the protein coding region of GFAP exon 1 was also included in our analysis. The results
showed that the evolutionary rate of exon 1 is higher than intron 2, thus it can be used to clarify the phylogenetic relationship of closely related fish species. In summary, the current study confirmed the validity of GFAP intron 2 and exon 1 as the molecular marker for closely related species. We have also re-examined the phylogenetic relationship of mammals and fishes, and the results shows that GFAP may have the potential to serve as
one of the species barcode genes.
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