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  • 學位論文

利用微衛星標識與粒線體DNA探討臺灣地區穿山甲之遺傳結構與多態性

Genetic Diversity and Genetic Structure of Formosan Pangolins in Taiwan Based on Microsatellite Markers and Mitochondrial DNA

指導教授 : 王佩華
共同指導教授 : 林恩仲(En-Chung Lin)

摘要


臺灣穿山甲(Formosan pangolin, Manis pentadactyla pentadactyla )屬於臺灣特有種,在國內屬於珍貴稀有保育動物,利用分子遺傳標識了解族群的遺傳變異性(genetic variability)與遺傳結構(genetic structure),有助於進行族群遺傳管理與遺傳保育。而目前分子標識中以微衛星標識較為廣泛應用,因此本試驗之目的為開發適用於臺灣穿山甲族群之微衛星標識,以作為遺傳監測的有力工具。另外,粒線體 DNA 控制區域也同樣被選為分子遺傳標識對臺灣穿山甲進行分析,期望結合核內與核外的遺傳資訊,得到更完整的臺灣穿山甲族群遺傳結構。 本試驗挑選 18 組自馬來西亞穿山甲(Manis javanica )所開發的微衛星基因座,作為跨物種微衛星標識進行研究,結果顯示,僅有 5 組微衛星基因座可在臺灣穿山甲 DNA 成功增幅並具有高多態性,與應用於原物種的結果比較,交替基因數(number of alleles, Na)、平均觀測異質度(observed heterozygosity, HO)與平均期望異質度(expected heterozygosity, HE)皆有所下降。因此,本試驗遂以選擇性雜合法(selective hybridization),將含有重複序列片段(repeat units)之探針(probe)與一公一母之臺灣穿山甲基因體 DNA進行雜合,建構微衛星序列豐富基因庫,從中挑選出 10 組新的微衛星基因座。結合此 10 組新微衛星標識與 5 組跨物種微衛星標識,檢測 36 隻臺灣穿山甲之遺傳變異,結果顯示此 15 組微衛星標識的交替基因數(Na)與有效交替基因數(number of effective alleles, Ne)之平均值各為 4.9±2.2 與 2.8±1.5;觀測異質度(HO)與期望異質度(HE)之平均值分別為 0.585±0.170 及 0.600±0.142;多態性訊息含量(polymorphic information content, PIC)之平均值為 0.538±0.154。FIS的平均值為 0.011±0.244,代表此 36 隻臺灣穿山甲的近親程度不嚴重。結合 15 組微衛星基因座之綜合個體鑑別率(probability of identity, P(ID))與綜合近親個體鑑別率(probability of identity among sibs, P(ID)sib)分別為 2.916×10-11 與 3.261×10-5;另外,此 15 組新微衛星標識之親子排除率(power of exclusion, PE)為 98.02%。以 15 組微衛星標識分析臺灣穿山甲族群遺傳結構結果顯示,南投地區的穿山甲可以被區分成不同的群集。 在臺灣穿山甲粒線體 DNA的研究方面,本試驗成功增幅的控制區域序列長度為 915 bp,序列分析結果顯示,在 36 隻臺灣穿山甲樣本中,共可發現 5 種單套型(haplotype),單套型多樣性指數(haplotype diversity, h)及核苷酸多樣性指數(nucleotide diversity, π)在中部地區穿山甲最高(0.810與0.00189)。以單套型所繪製的最小關聯網狀圖(minimum spanning network)結果顯示,單套型E僅出現於南投地區的穿山甲。 綜合所述,本研究開發出 15 組微衛星基因座,不僅可作為遺傳監控的重要分子遺傳標識,亦可用於身分辨識與推測系譜。此外,結合微衛星標識與粒線體控制區域的多態性分析結果顯示,南投地區的臺灣穿山甲與其他地區有較明顯的分化情形,可視為獨立的保育管理單位,而整體穿山甲族群之近親程度不嚴重,也沒有遺傳歧異度喪失的情形,但仍應持續進行遺傳監測以進行遺傳保育。

並列摘要


Formosan pangolin(Manis pentadactyla pentadactyla )is a rare species which is endemic to Taiwan. To manage and conserve this species, information of the genetic variability and genetic structure is important. In molecular genetic markers, microsatellite marker seems to be the most sultable markers for this purpose. Therefore, the objective of this study was to develop suitable microsatellite markers for Formosan pangolins to process genetic monitoring. On the other hand, the mitochondrial DNA control region was also used as molecular genetic marker. Through combining informations of genetic diversity obtained from nuclear DNA and mitochondrial DNA, the more complete genetic structure of Formosan pangolins would be revealed. Corss-species microsatellite markers isolated from Manis javanica were used in this study. The results showed that there were only 5 sets of microsatellite loci could amplified successfully and displayed high polymorphisms in a total of 18 loci in Formosan pangolin DNA. The number of alleles(Na), observed heterozygosity(HO), and expected heterozygosity(HE)were lower when compared with the markers used in Manis javanica. Therefore, we isolated 10 sets of novel microsatellite markers from one male and one female Formosan pangolins DNA using a selective hybridization method with mixed probes containing different repeat units from a microsatellite- enriched library. The 10 sets of novel microsatellite markers and 5 sets of cross-species microsatellite markers were combined to analyze the genetic variability of 36 Formosan pangolins. The results showed that the average number of alleles(Na) and the average number of effective alleles(Ne)were 4.9±2.2 and 2.8±1.5, respectively. The average observed heterozygosity(HO)and average expected heterozygosity(HE)were 0.585±0.170 and 0.600±0.142, respectively. The average polymorphic information content(PIC)was 0.538±0.154. The average FIS was 0.011±0.244, indicating the low inbreeding level. The total propability of identity(P(ID))and total probability of identity among sibs(P(ID)sib)were 2.916×10-11 and 3.261×10-5, respectively. Furthermore, the power of exclusion(PE)of the 15 sets of microsatellite markers was 98.02%. The results of using these 15 sets of microsatellite markers to analyze genetic structure of Formosan pangolins indicated that the individuals originated from Nantou could be separated from others. On the aspect of mitochondrial DNA studies, we amplified 915 bp control region sequence. We observed 5 haplotypes in 36 Formosan pangolin samples. The haplotype diversity(h)and nucleotide diversity(π)of Formosan pangolins were highest in central region(0.810 and 0.00189, respectively). The results of minimum spanning network showed that haplotype E was only existed in pangolins originated from Nantou region. In conclusion, we developed 15 sets of microsatellite markers for Formosan pangolins. these tools were not only useful for genetic monitoring but also suitable for identifying individuals or inferring the parentage coordinating. Moreover, the microsatellite markers and mitochondrial DNA data showed that Formosan pangolins in Nantou region could be considered as a independent management unit of conservation. The inbreeding level was low in total Formosan pangolin population and it displayed sufficient genetic diversity. But the genetic monitoring should be carried on.

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