水鹿表現序列微衛星開發與跨物種基因型鑑定之遺傳研究應用

Abstract

臺灣水鹿（Rusa unicolor swinhoii）與臺灣梅花鹿（Cervus nippon taiouanus）皆為臺灣原生之鹿科動物。臺灣鹿茸產業中常使用上述兩種鹿生產鹿茸，其中以水鹿鹿茸為主。而早期業者因紅鹿鹿角碩大，常將紅鹿與梅花鹿雜交配種以提高梅花鹿茸產量。是否在畜養的過程中有紅鹿基因滲入到本土鹿族群中，過去還未有可進行相關遺傳檢測與監控DNA分子標記。 動物因環境變遷、人為等因素導致異種雜交的情形並不少見，檢測時常使用的分子標記為微衛星標記（microsatellite markers）。微衛星又稱為簡單重複序列（simple sequence repeats, SSRs），以1至6個核苷酸作為重複單元並連續出現數次，此類序列廣泛分佈於基因體中，包含表現序列（expressed sequence tag）與非表現序列之中。微衛星在基因體中的高豐富度、高多形性且為共顯性，使其成為最常被使用的分子標記之一。依微衛星標記的序列來源，可分為基因體微衛星（genomic SSR）與表現序列微衛星標記（EST-derived SSR, EST-SSR），其中基因體微衛星的序列在跨物種的基因型鑑定常因引子的低保守性導致增幅困難，相形之下表現序列微衛星因其源於表現序列，在演化上有較高的保守性，使在跨物種鑑定時有更高的基因型鑑定成功率。 本試驗主要的目的在獲得臺灣水鹿鹿茸轉錄體（transcriptome）mRNA序列、篩選具多形性之表現序列微衛星標記，並將這些微衛星標記用於臺灣水鹿、臺灣梅花鹿、商業鹿場家養紅鹿以及雜交個體的基因型檢定，以檢測EST-SSR跨物種轉移能力與上述三種鹿種間雜交的情形。以次世代定序所得之水鹿鹿茸cDNA庫設計出55組可增幅微衛星基因座的引子對，並驗證其在水鹿DNA可增幅出專一性且具有核苷酸重複單元的PCR產物。後以45隻臺灣水鹿、30隻臺灣梅花鹿與15隻臺灣家養紅鹿進行基因型鑑定，分別有21個、11個與17個基因座在這些鹿種具多形性，其中使用分別在三鹿種具多形性之25個微衛星標記進行後續統計分析。 結果顯示三鹿種間有顯著的分化指數（FST ≧ 0.3798），顯示此套微衛星標記組具顯著區分三種鹿種的效力。為測試此套微衛星組在檢測雜交的效力，另使用4隻從外表型判斷為紅鹿、依粒線體DNA控制區域判斷為紅鹿、Y染色體鋅指序列判斷為臺灣梅花鹿之個體進行基因型鑑定與分派檢定。使用主座標分析（principal coordinates analysis）發現在解釋能力為58.16%之下，三種鹿的個體皆被正確分配到同一物種的群中，而其中兩個雜交個體座落位置介於紅鹿群與梅花鹿群中。以Structure軟體進行分派檢定，進一步分析此兩個雜交個體的雜交程度，其祖先分別有0.43與0.30的比例被分配到梅花鹿，證明本次開發的微衛星標記組可檢測出雜交個體之雜交程度。分別以正確率（percentage of correctly assigned）與分化係數對單個標記進行排序分別挑選出10個標記進行分派檢定，亦具有檢驗雜交程度的能力，但依何種標準排序較佳仍須未來評估。 在基因的編碼序列中具多形性的EST-SSR，經轉譯後會產生不同長度、不同胺基酸序列的胜肽。本研究篩選出具多形性、且EST-SSR座落於編碼序列中之基因座，各個對偶基因經預測轉譯後之胺基酸序列，未發生閱讀框架移動，但胺基酸序列確實因微衛星序列的重複次數不同而產生蛋白質分子量多形性。是否這些EST-SSR多型性影響蛋白質功能，需要進一步的探討。 本研究開發的25個多形性微衛星標記，可用於研究水鹿、梅花鹿與紅鹿之間的物種鑑別、基因交流與雜交檢定，亦可作為家養鹿科動物的遺傳結構分析與育種管理。未來將可被用來探討EST-SSR多型性是否造成特定蛋白質功能性的差異，作為預測鹿茸產量的分子標記。另外，所開發的標記未來可被利用在臺灣本土鹿種的保育生物學研究。

Formosan sambar (Rusa unicolor swinhoii) and Formosan sika deer (Cervus nippon taiouanus), both deer are native to Taiwan. Formosan sambar is classified into the second category of protected species. While, Formosan sika deer was extinct in wild before 1969, and the captive sika deer in Yen-shan zoo were reintroduced into the wild in 1994 and grew a stable population in Kenting National Park. At the same time, since the antler velvet is a precious traditional Chinese medicine, the two deer species are also domesticated for antler velvet production and keep in deer farm. The red deer (Cervus elaphus) were introduced and cross breed with sika deer for better antler velvet performance by Taiwan deer farmers. However, it leads to a concern that whether the gene flow of red deer had introgressed into native sika deer. Still now, absent of effective nuclear genetic markers with cross-species transferability had hampered the cross-breeding diagnosis between different deer species. Simple sequence repeats (SSRs) are short sequence of nucleotides tandemly repeat in genome DNA, including coding sequence and non-coding sequence. SSRs are powerful molecular markers due to its high genomic abundance, high level of polymorphism and co-dominance inheritance. Genomic SSRs has been limited for the low reproducibility when performing cross-species genotyping by PCR amplification. Hence, the highly conserved transcriptome-derived SSR markers have been developed from expressed sequence tags (EST-SSRs) for its high transferability to related species and thereby serving as reliable markers and also for their functional study. The aim of this study is to obtain polymorphic EST-SSRs from sambar antler velvet and develop a set of microsatellite marker system for identifying sambar, sika deer, red deer, and their crossbreeds. The cDNA sequences were obtained from antler velvet mRNA by reverse transcription and Next Generation Sequencing. A total of 65,074 unigenes was assembled. Primer pairs for amplifying EST-SSRs are designed from the sequence data. After validation of the PCR specific amplification, 55 loci were selected. The capillary electrophoresis was applied for individual genotyping by amplifying the loci with fluorescent labeled primer pairs. After the genotype screening of three deer species, we found that 21 loci showed polymorphism among 45 sambars, 13 loci among 30 sika deer, and 17 loci among 15 red deer, respectively. The structure analysis, F-statistic (FST) analysis, and Principal Coordinates Analysis (PCoA) were applied to examine the ability of the selected loci to distinguish these 3 deer species. In addition, two of red deer and sika hybrid individuals were chosen as a cross-breeding control. The individuals among 3 species were clearly assigned to their own populations both in structure and PCoA. While the two cross-breed deer showed the porportion of 0.43 and 0.30 being assigned to sika population in structure analysis. Besides, these 2 individuals located at positions between sika deer group and red deer group in PCoA. These results indicated that the set of newly developed polymorphic EST-SSR marker system could diagnose the deer species and the hybridization. With the polymorphism of DNA sequence, whether the functions of gene will be influence still need further studies. The unigenes, which the newly-developed microsatellite markers located, were annotated against NCBI nt database. The deduced amino acid sequences of different alleles at the CDS-derived locus were aligned with the annotated gene. Though the polymorphism of SSRs, there was no frameshift in the sequence but the different repeat of specific amino acid. A set of EST-SSR marker system for genotyping sambar, sika deer, and red deer has been developed. In the present study, we proved the set of marker system can be applied for diagnosis the sambar, sika deer, red deer, and their hybrids.