開發新穎微衛星標識及檢測平臺進行臺灣畜禽種原之管理與應用

種原保存與管理就是遺傳資源的保存與管理，詳細的品種內與品種間的多態性的分子資訊已被廣泛接受是有效管理經濟動物遺傳資源所必需的。分析畜禽族群的遺傳特性和結構是建構維持穩定動物品質與保持族群遺傳多態性的長期計畫的基本步驟，本研究的目的為開發高多型性微衛星標識套組以監控及分析臺灣實驗兔、家鵝與菜鴨族群之遺傳結構。總共 18 個微衛星標識用來進行 5 個兔族群的遺傳分析，18 個微衛星標識的交替基因數、有效交替基因數、觀察異質度、期望異質度和FIS平均值分別是 5.50、2.437、0.442、0.568 和 0.232，代表此組微衛星標識具有高多態性，而這些臺灣主要實驗兔族群有近親趨勢。同時分子變異分析（analysis of molecular variance, AMOVA）分析顯示這些實驗兔族群有高的族群內歧異（83%），中等的族群間歧異（FST = 0.18），貝氏群集分析最可能的群集數是4 （K = 4），主成分分析（principal component analysis, PCA）也大致呈現4個群組，LRI 與 BC 兩族群在PC3時相對接近，STRUCTURE 分析中，此兩族群在 K = 4 時無法分開。個體親緣關係樹中5族群可分開，唯 LRI、ADIB 與 BC 三族群有少部分個體重疊。因此，為了降低FIS，本研究建議此三個紐西蘭白兔族群間可互相交配或由外界引進新的基因。18 個微衛星標識相當適合分析臺灣實驗兔族群的種原關係與遺傳結構，利用這些資訊，各兔場可進行適當的配種計畫以維持小族群內最高的多態性，生產高品質的實驗用兔。臺灣的白羅曼鵝和華鵝族群是使用本研究最新篩選的 14 個新微衛星標識和 7 個已知的微衛星標識偵測遺傳特性，21 個微衛星標識偵測彰化種畜繁殖場（CAPS）鵝群的交替基因數、有效交替基因數、觀察異質度、期望異質度和多態性訊息含量平均值分別是 11.09、5.145、0.499、0.745 和 0.705。加入民間鵝場的鵝隻，以 AMOVA 和 PCA 分析顯示白羅曼群集較緊密，而華鵝的群集較鬆散。在親緣關係樹中白羅曼鵝族群間的 bootstrap 值幾乎都小於 70%，而CAPS 白羅曼鵝族群在貝氏群集分析中，K = 6 時會分裂成兩個次族群，各民間鵝場的白羅曼族群之遺傳結構則是互相類似。華鵝族群方面，CAPS 族群和民間鵝場族群在親緣關係樹中分屬兩個不同的群集，甚至經多態性分析，民間鵝場族群的個體之間的遺傳特性較不一致。基於本研究的結果，CAPS 白羅曼鵝族群應依據各鵝隻基因型座進行適當的配種，以防次族群的出現。為了種原管理與更穩定的品質，CAPS 應輸出華鵝並導入民間鵝場正確的育種計畫。從菜鴨分離 17 個全新的微衛星標識，加上 7 個已知的微衛星標識，24 個引子對被設計用來分析畜產試驗所菜鴨族群的遺傳特性。交替基因數、有效交替基因數、觀察異質度、期望異質度和多態性訊息含量，平均值分別是 11.29、5.370、0.591、0.746 和 0.708。AMOVA 和 PCA 分析顯示褐色菜鴨群集較緊密，而白色菜鴨群集較鬆散。在褐色菜鴨貝氏群集分析中， 6 個褐色菜鴨族群在 K = 6 時可被分為 6 個群集，同樣地，白色菜鴨在貝氏群集分析中 3 個白色鴨族群在 K = 3 時可被分為3個群集。個體親緣關係樹出現 8 個群集，且每個個體可全部被分派至該族群中。根據分析結果，24 個微衛星標識有能力在族群彼此相似度高的情況下，分析族群內與族群間的多態性，我們認為除了畜產試驗所外，應該將此微衛星標識套組推廣至種鴨場，定期監控與分析鴨群的遺傳特性變化，以保育品系並確保品質。關聯性分析結果顯示，17個新微衛星中有11個基因座與生長或繁殖性能相關，其中有10個交替基因與第二子代（F2）母鴨的生長性狀相關，數個交替基因與F2母鴨的繁殖性狀相關，顯示未來可以藉由檢測此些微衛星標識的基因型來做為鴨隻之遺傳育種選拔。

關鍵字

種原管理；家鵝；遺傳多態性；實驗用兔；微衛星標識；菜鴨

並列摘要

Preservation and management of breeds is the preservation and management of genetic resources. It is widely accepted that detailed molecular data on within- and between-breed diversity are essential for effective management of genetic resources. An analysis of the genetic characteristics and structure of livestock populations would thus be a fundamental step in building a long-term management program for maintaining stable animal quality and preserving the genetic variation among the populations. The aim of this study was to create a set of microsatellite markers with high polymorphism for the genetic monitoring and genetic structure analysis of local rabbit, goose and Tsaiya duck populations. A set of 18 microsatellite markers was designed for genetic analysis of 5 rabbit populations. The average values for the allele number (Na), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (HE), and Wright’s fixation index (FIS) were 5.50, 2.437, 0.442, 0.568, and 0.232, respectively. These results revealed that this set of microsatellite markers has high diversity and that the major local populations have a tendency toward inbreeding. At the same time, analysis of molecular variance results showed that the laboratory rabbits used in Taiwan have maintained a high level of within-population genetic differentiation (83%). The genetic differentiation among clusters was moderate (FST=0.18), and Bayesian cluster analysis showed that the most likely number of groups was 4 (K=4). Principal component analysis (PCA) also showed four divergent clusters. The LRI and BC NZW populations were not separated when K=4 was used in a STRUCTURE software analysis and also hard to split until PC3 in PCA. The individual unrooted phylogenetic tree showed that the 5 populations were separated except that some individuals from the LRI NZW population overlapped with the ADIB NZW and BC NZW populations. As such, in order to counteract the reduced FIS (0.232) and maximize heterozygosity, the three NZW populations could be interbred or have new genes introduced into them. The set of microsatellite markers used herein was useful for studying the relationships and genetic diversities among these rabbit populations of Taiwan. Based on the resulting data, rabbit farms in Taiwan could select parental stocks for planned mating in the future as part of strategies to preserve and restore the rational breeding of laboratory rabbits. 14 novel microsatellite loci were isolated. In addition to seven known loci, two multiplex sets were constructed for the detection of genetic variations in White Roman and Chinese goose populations. The Na, the Ne, the Ho, the HE, and the PIC were 11.09, 5.145, 0.499, 0.745, and 0.705, respectively. The results of AMOVA and PCA indicated a contracting White Roman cluster and a spreading Chinese cluster. In White Roman populations, the bootstrap values of the private farm populations were almost below 70% in the phylogenetic tree and the Changhua Animal Propagation Station (CAPS) populations were depleted to roughly two sub-populations when K was set equal to 6 in the Bayesian cluster analysis. The founders of private farm populations had a similar genetic structure. Among the Chinese geese populations, the CAPS populations and private populations represented different clads of the phylogenetic tree and individuals from the private populations had uneven genetic characteristics according to various analyses. Based on this study’s analyses, we suggest that the CAPS should institute a proper breeding strategy for White Roman geese in order to avoid further clustering. In addition, for preservation and stable quality, the Chinese geese in the CAPS should be popularized and the aforementioned proper breeding scheme should be introduced to geese breeders. 17 novel microsatellite loci were isolated. In addition to seven previously known loci, 24 primer pairs were constructed for the detection of genetic variations in Tsaiya duck populations in Livestock Research Institute. The average values for the Na, the Ne, the Ho, the HE, and the PIC were 11.29, 5.370, 0.591, 0.746, and 0.708, respectively. The results of AMOVA and PCA indicated a contracting brown Tsaiya duck cluster and a spreading white Tsaiya duck cluster. The brown Tsaiya ducks and the white Tsaiya ducks with Pekin ducks were just split to six clusters and three clusters when K was set equal to 6 and 3 in the Bayesian cluster analysis. The individual phylogenetic tree revealed eight clads, and each individual was assigned to its own population. By the results of this study, the 24 microsatellite markers exhibited a high capacity to analyze relationships of inter- and intra-population in those populations with a relatively limited degree of genetic diversity. We suggest that duck farms in Taiwan could use the new microsatellite set to monitor the genetic characteristics and structures of their Tsaiya duck populations at various intervals in order to ensure quality breeding and preservation. The results of association study showed that 11 of 17 sets of novel microsatellite markers were associated with growth and reproductive performance. 10 alleles had significant effect on growth performance of F2 male ducks; several alleles had significant effect on reproductive performance of F2 female ducks. It indicated that these potential novel microsatellite markers could be used on marker-assisted selection of ducks.