臺灣犬隻新微衛星標幟之開發與貓隻微衛星標幟之檢測

Abstract

近年來臺灣的動物保護意識逐漸高漲，因此犬、貓因近親繁殖而使得遺傳缺陷疾病之發生率有提高的現象，備受大眾所關注。本試驗之目的主要藉由開發適宜國內犬、貓族群分析之新微衛星標幟（microsatellite markers），並建立套組，供進行犬、貓遺傳結構之檢測。 本試驗採用選擇性雜合法（selective hybridization），將含有重複序列片段（repeat units）之探針（probes）與一公一母之犬隻基因體 DNA 樣本進行雜合（hybridization），以建立微衛星標幟豐富化之基因庫（microsatellite-enriched genomic library）。其中，在 230 個陽性選殖菌落中，共有 65 個菌落可經由 Primer 3 Plus 軟體設計出針對重複片段之引子序列（primer sequence）。將所得之引子組進行包含三個品種：74隻臺灣米格魯（Beagle）、8 隻日本米格魯、17 隻比熊（Bichon）及14 隻雪納瑞（Schnauzer），共計 113 隻犬隻 DNA 樣本之微衛星基因座（loci）多態性檢測。試驗結果顯示，篩選出 14 組新微衛星標幟在三個犬隻品種皆具有多態性，且其重複片段之種類大多為雙核苷酸（dinucleotide）。14 組新微衛星基因座之交替基因數（number of alleles, Na） 與有效交替基因數（number of effective alleles, Ne）之平均及標準偏差各為 6.3±3.4 和 3.6±1.6；期望異質度（expected heterozygosity, HE）與觀測異質度（observed heterozygosity, HO）之平均及標準偏差分別為 0.662±0.162 與 0.567±0.136；多態性訊息含量（polymorphic information content, PIC）之平均及標準偏差為 0.612±0.179。另外，FIS 之平均及標準偏差為 0.002±0.128，FST 之平均及標準偏差為 0.212±0.103，且 FIT 之平均及標準偏差為 0.209±0.173。前述變量（parameter）顯示這些新篩選的微衛星標幟具有高度多態性，大幅提升個體鑑別之效力：全部新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 1.7×10-12 與 1.6×10-5。另外，此 14 組新微衛星標幟之親子排除率（power of exclusion, PE）為 99.98%。利用鄰位連接（neighbor-joining）法根據遺傳距離所繪製之親緣關係樹中可觀察到，此 14 組新微衛星標幟可將試驗族群區分為三個明顯的主要群集（clusters），即可依照品種區分為雪納瑞、比熊及米格魯；其中，米格魯族群又可分為臺灣族群與日本族群。再者，利用主座標分析法（principal coordinate analysis, PCoA）分析試驗族群間的相異程度，其所繪製之 3D 圖顯示，試驗犬隻可正確地依據品種不同而被區分出來，並且可明確地區分出不同來源的米格魯犬隻，而第一座標軸、第二座標軸及第三座標軸分別可解釋 44.15、26.35 及 19.97％ 的遺傳變異。 本研究另外從文獻中擇取 22 組重複片段為四核苷酸（tetranucleotide）之高多態性微衛星標幟，進行 25 隻孟加拉豹貓（Bengal）之基因型分析。試驗結果顯示，只有 14 組微衛星標幟在此貓隻品種可成功增幅且具有多態性。所有基因座之交替基因數與有效交替基因數之平均及標準偏差各為 5.8±1.8 和 3.7±1.3；而期望異質度與觀測異質度之平均及標準偏差分別為 0.726±0.095 與 0.660±0.157；多態性訊息含量之平均及標準偏差為 0.669±0.104；而其 FIS 之平均及標準偏差為 0.097±0.167。另外，綜合個體鑑別率與綜合近親個體鑑別率分別為 9.7×10-14 與 1.1×10-4。而此 14 組微衛星標幟之親子排除率為 99.99%。藉由前述各變量所顯示之高度多態性與兩項個體鑑別率，證實此 14 組微衛星標幟足夠作為臺灣之孟加拉豹貓族群的遺傳監控與個體鑑別工具。 綜合所述，本試驗所應用之高多態性犬、貓微衛星標幟，可供進行國內犬、貓之族群遺傳結構檢測與個體鑑別。

Along with the rise of animal protection consciousness in Taiwan, the public pays more attention on dog and cat genetic deficiencies due to inbreeding in the pet market. The goal of this study was to isolate novel microsatellite markers and develop a set of microsatellite markers for monitoring their genetic structure of the domestic dog and cat populations in Taiwan. The microsatellite-enriched genomic library was constructed from one male and one female dog DNA samples by selective hybridization with mixed probes containing different repeat units. There were 230 positive clones selected, and only 65 clones could be desiged primers for single repeats by the Primer 3 Plus software. A total of 113 DNA samples of three dog breeds, including 74 Beagles from Taiwan, 8 Beagles from Japan, 17 Bichon, and 14 Schnauzer, were used in the following polymorphic tests. The results showed that only 14 sets of novel microsatellite markers were polymorphic in the three breeds; among those markers, the majority of repeat unit was dinucleotide. The average number of alleles (Na) and the average number of effective alleles (Ne) were 6.3±3.4 and 3.6±1.6, respectively. The average expected heterozygosity (HE) and average observed heterozygosity (HO) were 0.662±0.162 and 0.567±0.136, respectively. The estimated of average polymorphic information content (PIC) was 0.612±0.179. The FIS was 0.002±0.128; the FST was 0.212±0.103 and the FIT was 0.209±0.173. The high level of genetic diversity observed in these novel microsatellite markers provided a high discriminating power. The estimated probability of identity (P(ID)) and the probability of identity among sibs (P(ID)sib) of the 14 sets of novel microsatellite markers amounted to 1.7×10-12 and 1.6×10-5, respectively. Furthermore, power of exclusion (PE) of the 14 sets of novel microsatellite markers in this study is 99.98%。The neighbor-joining trees were constructed among the three breeds on the basis of the genetic distance estimated from the 14 sets of novel microsatellite markers. The configuration indicated that the 14 sets of novel microsatellite markers were sufficient to correctly cluster the three dog breeds — Beagle, Bichon, and Schnauzer. Still, the subgroups of Japanese Beagle within the Beagle cluster were clearly separated from Taiwanese Beagle. Principal coordinate analysis (PCoA) based on the allele frequency of 14 loci was used to visualize the dissimilarities among these experiment populations. The 3D plot showed that the dogs could be accurately separated by these 14 loci baled on different breeds; moreover, the Beagles from different sources were also distinguished. The first, the second, and the third principal coordinates could be used to explain 44.15, 26.35 and 19.97% of the genetic variation. In the second part of this study, genomic DNA samples were extracted from blood samples of 25 Bengal cats. Twenty-two sets of highly polymorphic microsatellite markers containing tetranucleotide repeat unit were chosen from a previous study. The results showed that there were only 17 sets of microsatellite markers amplified and 14 sets of them showed polymorphism in the Bengal cat population tested. The average Na and the average Ne were 5.8±1.8 and 3.7±1.3, respectively. Also, the average HE and HO per locus were 0.726±0.095 and 0.660±0.157, respectively. Average PIC was 0.669±0.104, and FIS was 0.097±0.167. P(ID) and P(ID)sib of all 14 sets of markers were 9.7×10-14 and 1.1×10-4, respectively. PE of all 14 sets of microsatellite markers is 99.99%。These parameters showed that the 14 loci could sufficiently monitor the genetic structure and individual identity of Bengal population in Taiwan. The results of this study could be used as powerful examination methods for genetic background structure of the domestic dog and cat populations in Taiwan.