台灣山羌核型及衛星DNA之研究

羌屬或稱barking deer，在分類上可分為6種，分別是Muntiacus. muntjak, M. reevesi, M. rooseveltorum, M. crinifrons, M. feae, M. atherodes。台灣山羌(M. reevesi micrurus)為本土保育類動物，屬於M. Reevesi中兩個亞種之一，過去僅少數研究報告，探討其生態、行為的表現、棲息地、人工豢養和野生習性之不同，其分子細胞遺傳學的研究則尚無報告，本論文應用G-banding帶狀技術的分析，確定台灣山羌染色體的數目為2n = 46，其核型帶狀與另一個亞種中國山羌(M. reevesi reevesi)相當接近。另外，從台灣山羌的基因組中選殖了二段鹿科衛星DNA，一段約為1.5 kb，稱為FM sat I，另一段為1 kb，稱為FM 1 kb。其中FM sat I的序列與中國山羌sat I DNA (C5)之相似性高達82%；FM 1kb的DNA序列則和已發表之鹿科衛星DNA都不相同，僅與最近選殖自中國山羌的衛星DNA (CM 1 kb)有很高的相似性(98 %)。以台灣山羌衛星DNA (FM sat I )為探針進行南方氏雜交反應，可見到DNA片段是以0.75-0.8 kb為單元體的規則性梯狀排列，至於FM 1 kb DNA的南方點漬圖則呈現出不規則的形式。利用FM sat I及FM 1 kb為探針分別與台灣山羌染色體進行螢光原位雜交反應，除了第三對染色體及Y染色體的著絲點偵測不到訊號外，其餘染色體的著絲點都可偵測到FM sat I的訊號。另外，一些染色體臂上也有FM sat I螢光雜交的訊號，如第1-3對染色體各有兩處interstitial (FM sat I)的訊號，而尚有三對較小的體染色體各有一處interstitial訊號反應。觀察(FM 1 kb)在台灣山羌染色體上的分佈，也是除了第三對染色體及Y染色體著絲點沒有訊號反應之外，其餘的染色體皆有FM 1 kb的訊號反應，而染色體臂上interstitial雜交訊號則與FM sat I的反應相同。當同時以FM sat I和FM 1 kb為探針做原位雜交反應時發現，雖然兩種探針的訊號都同時出現在著絲點的位置上，但FM 1 kb的訊號比起FM sat I則較接近染色體的末端。由上述結果發現，台灣山羌和中國山羌無論是在核型、著絲點衛星DNA序列以及衛星DNA在染色體上的分佈情形，都有相當高的相似性，證明此二種山羌的血緣關係非常相近，被分為M. reevesi之二種亞種是相當合理。期望藉由以上資料的建立，能夠提供一些資訊對於探討台灣山羌演化的相關研究有所幫助。

Asian muntjac or barking deer consists six known species; Muntiacus muntjak, M. reevesi, M. rooseveltorum, M. crinifrons, M. feae, and M. atherodes. The native endangered Formosan muntjac (M. reevesi micrurus) in Taiwan is classified as one of the two subspecies of M. reevesi. In the past, there has been some documentations regarding the morphology, ecology, behavior, habitat and captivity of this subspecies, and yet no molecular cytogenetic study has been reported. The present thesis research therefore is aimed to gather molecular cytogenetic data of the Formosan muntjac. Based on the G-banding analysis, the chromosome number of this subspescies is identified as 2n = 46 and in general its G-banbed karyotype is resemble to the DAPI-banded karyotype of the other subspecies, the Chinese muntjac (M. reevesi reevesi). Two satellite DNA clones designated as FM sat I and FM 1kb were generated from PCR amplification of Formosan muntjac genomic DNA using primer sequences obtained from Chinese muntjac satellite I (C5) (Lin et al. 1991) and white tailed deer satellite II clone (OvDII) DNA, respectively. Sequence comparison revealed that FM sat I and C5 shared 82% homology. No significant sequence homology was found between FM 1kb and any reported cervid satellite DNA. However, very high sequence similarity was observed between FM 1kb and CM 1kb (98%) (Li et al. unpublished). Southern hybridization using FM sat I as a probe on Formosan muntjac genomic DNA digested with several restriction endonucleases revealed prominent bands in a 0.75- 0.8 register confirming the nature of satellite DNA. On the other hand, irregular banding patterns were observed using the FM 1kb as a probe. FISH study using either FM sat I and/or FM 1kb as probes on Formosan muntjac chromosome preparations revealed strong FM sat I hybridization signals on the centromeric regions of all chromosomes with the exception of a pair of chromosome 3 and the Y chromosome. Interstitial signals by FM sat I were also observed in six pairs of autosomes. Among those, two interstitial signals were found in each chromosome of three pairs of the largest chromosomes in the complement. One interstitial signal was found in each chromosome in the remaining three pairs of chromosomes with smaller size. FISH with FM 1kb probe showed hybridization signals on centromeric regions of all chromosomes except on chromosomes 3 pairs and the Y chromosome. Interstitial signals were similar to those seen with FM sat I. Co-hybridization with both satellite DNA probes revealed that although both satellite DNA sequences located at the centromeric/pericentromeric regions, the satellite DNA FM 1kb appeared located near the distal end of the chromosome, whereas, the FM sat I was located more proximally. Based on the results of karyotypic analysis, satellite DNA sequencing and FISH signals distribution, it is reasonable to suggest that Formosan muntjac and Chinese muntjac are closely related.