利用主要組織相容性複合體基因與微衛星標識探討臺灣穿山甲之遺傳多態性與結構

臺灣穿山甲（Manis pentadactyla pentadactyla）為臺灣的瀕危保育類動物，瀕危物種可能面臨遺傳多樣性的喪失的問題，進而提高滅絕的風險。因此檢測臺灣穿山甲族群之遺傳多態性與結構成為保育研究中重要的一環。著眼於 MHC 基因在免疫上扮演的重要功能，此基因於族群中的多態性可作為間接評估族群免疫適存度的指標。另外微衛星標識常被用於分析族群之遺傳結構，並已廣泛被用於野生動遺傳保育研究上。結合這兩個分子標識，將能以更廣泛的角度評估臺灣穿山甲族群之遺傳多態性與結構。本試驗開發出可用於 7 個不同臺灣穿山甲 MHC 基因座的檢測平台，分別能用於檢驗 5 個臺灣穿山甲第一型 MHC 基因座，包含類 HLA class I B-14 alpha chain 基因座、類 HLA class I A-11 alpha chain 基因座第二外顯子、類 HLA class I A-11 alpha chain 基因座第三外顯子、類 HLA class I G alpha chain 基因座，和類 HLA class I B-15 alpha chain 基因座；以及 2 個基因座第二型 MHC 基因座，包含類 HLA class II DQ alpha 2 chain 基因座，和類 DLA class II DR-1 beta chain 基因座之遺傳多態性檢測，同時也利用 15 組微衛星標識進行遺傳變異性分析。並以 92 隻由臺北市立動物園和特有生物研究保育中心，提供的救傷穿山甲樣本評估族群遺傳多態性。但在類 HLA class I B-14 alpha chain 基因座、類 HLA class I A-11 alpha chain 基因座第三外顯子、類 HLA class I G alpha chain 基因座，和類 HLA class I B-15 alpha chain 基因座中，因部分樣本萃取的 DNA 較少，僅使用其中 88 個樣本進行分析。在 MHC 基因座中，於類 HLA class I A-11 alpha chain 基因座第二外顯子，和類 HLA class I B-15 alpha chain 基因座呈現單型性。而在類 HLA class I B-14 alpha chain 基因座、類 HLA class I A-11 alpha chain 基因座第三外顯子、類 HLA class I G alpha chain 基因座，以及類 HLA class II DQ alpha 2 chain 基因座則呈現有限的多態性，僅有 2 個單套型。且相對頻率在類 HLA class II DQ alpha 2 chain 基因座中為99.5 和 0.5%；在第一型 MHC 基因的其餘 3 個基因座皆為99.6 和 0.4%。在這 4 個基因座中單套型多樣性指數（h）皆為 0.011 。另一方面，在類 DLA class II DR-1 beta chain 基因座則有檢測出多態性，共有 4 個單套型，相對頻率依序為 62.0、37.0、0.5 和 0.5%，在核苷酸序列上有 9 個變異點，並於預測的胺基酸序列間有 6 個變異點。單套型間的非同義鹼基置換率（dN）以及同義鹼基置換率（dS）依序分別為 0.026 和 0.017，顯示此基因座可能處於正向選汰壓力下（dN /dS > 1）。在類 DLA class II DR-1 beta chain 基因座中，觀測異質度（HO）為 0.413，而期望異質度（HE）則為 0.482。FIS 值為 0.144 顯示收集的樣本中在此基因座上，可能近親程度較高（FIS > 0）。FST 值為 -0.056 表示此基因座在各區域間呈現低度遺傳分化（FST < 0.05）。在類 DLA class II DR-1 beta chain 基因座之單套型多樣性指數（h）為 0.482，核苷酸多樣性指數（π）則為 0.010。以上結果指出，收集的樣本中在 MHC 基因上的多態性可能十分有限。在 15 組微衛星標識的檢測結果中，平均交替基因數（Na）和平均有效交替基因數，分別為 6.5±3.3 和 3.0±1.8；觀測異質度（HO）與期望異質度（HE）之平均值依序為 0.529±0.119 和 0.611±0.130；平均多態性訊息含量（PIC）為 0.553±0.149。另外收集的樣本中 FIS 值之平均值為 0.115±0.207，而在結合 15 組微衛星基因座之綜合個體鑑別率（P(ID)）與綜合近親個體鑑別率（P(ID)sib）依序分別為 1.074×10-11 和 2.522×10-5。以此 15 組微衛星標識分析族群遺傳結構，結果顯示臺灣穿山甲可能主要分為北部和中南部兩個保育管理單位。綜合上述結果，本試驗所開發之 MHC 基因型檢測平台，能有效使用於臺灣穿山甲 MHC 基因遺傳多態性分析。進一步結合 15 組微衛星標識的檢測結果，將能有效運用於臺灣穿山甲之遺傳監控。

關鍵字

臺灣穿山甲；主要組織相容性複合體；微衛星標識；族群遺傳分析

並列摘要

Formosan pangolin (Manis pentadactyla pentadactyla) is an endanger species of Taiwan. Declines in population size might lead to decrease in genetic diversity and raise extinction risk. Thus, measuring genetic diversity and genetic structure of Formosan pangolins in Taiwan becomes important in conservation programs. MHC plays a crucial role in immune response. Therefore, polymorphisms at these gene region would provide indicator of the immunological fitness of a population. Microsatellite markers have been utilized to evaluate genetic structure and widely applied to wildlife studies. Through combining information of genetic diversity obtained from different genetic markers, the diverse aspects of genetic structure for Formosan pangolins would be revealed. In this study, seven different markers, Formosan pangolin HLA class I B-14 alpha chain-like locus, HLA class I A-11 alpha chain-like locus exon 2, HLA class I A-11 alpha chain-like locus exon 3, HLA class I G alpha chain-like locus, HLA class I B-15 alpha chain-like locus, HLA class II DQ alpha 2 chain-like locus, and DLA class II DR-1 beta chain-like locus, for MHC genotyping of Formosan pangolin were developed. In addition, 15 sets of novel microsatellite markers were used to analyze the genetic variability of the population. Totally, 92 Formosan pangolins were collected from Taipei Zoo and Taiwan Endemic Species Research Institute and used to evaluate their genetic diversity. However, HLA class I B-14 alpha chain-like locus, HLA class I A-11 alpha chain-like locus exon 3, HLA class I G alpha chain-like locus, and HLA class I B-15 alpha chain-like locus loci only used 88 samples for analysis due to insufficient DNA extracted from some samples. In MHC markers, HLA class I A-11 alpha chain-like locus exon 2 and HLA class I B-15 alpha chain-like locus showed monomorphism. HLA class I B-14 alpha chain-like locus, HLA class I A-11 alpha chain-like locus exon 3, HLA class I G alpha chain-like locus, and HLA class II DQ alpha 2 chain-like locus showed limited polymorphism, since only two haplotypes were detected in each locus. The relative frequency of the two haplotypes were 99.5 and 0.5% in HLA class II DQ alpha 2 chain-like locus. Furthermore, the relative frequency of the other two haplotypes in HLA class I B-14 alpha chain-like locus, HLA class I A-11 alpha chain-like locus exon 3, and HLA class I G alpha chain-like locus were all 99.4 and 0.6%. In the four loci, the haplotype diversity (h) were all 0.011. On the contrary, DLA class II DR-1 beta chain-like locus showed slightly diversity. Four haplotypes of DLA class II DR-1 beta chain-like locus were detected and their relative frequency were 62.0, 37.0, 0.5 and 0.5% with nine polymorphic sites in the nucleotide sequence and six variable points in the amino acid sequence. Moreover, the non-synonymous substitution rate (dN) and synonymous substitution rate (dS) were 0.026 and 0.017 showed that this locus might be under positive selection (dN /dS >1). In DLA class II DR-1 beta chain-like locus, the observed heterozygosity (HO) and expected heterozygosity (HE) were 0.413 and 0.482. FIS was 0.144 which indicated that the population might have an inbreeding tendency (FIS > 0). The average differentiation among populations (FST) was -0.056. The haplotype diversity (h) was 0.482 and nucleotide diversity (π) was 0.010. Overall, the results indicated that the genetic diversity of MHC genes of Formosan pangolin among these samples may be limited. In the 15 sets of novel microsatellite markers, the average number of alleles (Na) and effective alleles (Ne) were 6.5±3.3 and 3.0±1.8, respectively. The average observed heterozygosity (HO) and expected heterozygosity (HE) were 0.529±0.119 and 0.611±0.130, respectively. The average polymorphic information content (PIC) was 0.553±0.149. The average FIS was 0.115±0.207. The total probability of identity (P(ID)) and the total probability of identity among sibs (P(ID)sib) were 1.074×10-11 and 2.522×10-5, respectively. The neighbor-joining (NJ) tree was constructed and indicated that the northern group of Formosan pangolin might differentiate from the central and southern groups. Our results indicated that the new MHC genotyping platform for Formosan pangolin is suitable for further MHC diversity analysis. Moreover, combining MHC genotyping platform and the 15 sets of novel microsatellite markers are appropriate tools to monitor the genetic structure of Formosan pangolins.