表皮附肢物性狀在不同分支上的反覆改變，顯示了由環境和基因因素導致的平行演化。在家禽中，人為豢養的雞和鴿子都出現了毛腳性狀的品系，已知的研究發現這些家禽腳上鱗片轉成羽毛的現象是因為PIXT1上游增強子Pen發生缺失所造成。在刺魚的研究，同樣也因為PITX1上游增強子的遺失而造成後期凸刺性狀消失，且發現是因為其增強子Pel含有Z-DNA結構造成斷裂。Z-DNA在基因體裡容易形成特殊結構使促成突變和染色體重組、變動，這可能可以解釋為什麼在不同的家禽品種中反覆發生了在差不多位置的序列缺失導致相同的性狀改變。為了確認Z-DNA在家禽毛腳性狀中扮演的角色，我們會在實驗中檢視Z-DNA在Pen缺失附近和整個基因體之中的密度，並進行Z-DNA的結構和染色體脆弱度實驗。同時，使用RCAS感染系統降低胚胎後腳的PITX1表現量。我們的實驗結果顯示Z-DNA確實在Pen附近有較高的比例且容易使附近的序列斷裂，Z-DNA序列在雞和鴿子的長度、數量、及分布情形的差異可能是Pen缺失長度不同的原因；然而在基因功能的實驗中，降低PITX1表現的胚胎並沒有觀察到明顯的毛囊生成，我們推測可能是因為羽毛發育過於複雜無法透過單一基因的抑制產生部分的身分轉換。此研究結果檢視了Z-DNA在基因體穩定性的影響是如何調控家禽表皮附肢物的性狀改變。

Repeated transformation of traits in skin appendages reveals ecological and genetic factors shaping parallel evolution. A genetic study has shown that the conversion from scales to feathers on feet of pigeons and chicken results from a deletion in the PITX1 upstream enhancer, Pen. A study in stickleback fish indicates that pelvic reduction is contributed by distinct Z-DNA structures, which are enriched at mutation hotspots, so we assume that DNA fragility may explain the repeated loss at PITX1 enhancer in some domestic breeds of chicken as well. To validate the role of Z-DNA in feathered feet, the avian RCAS retrovirus transgenic system was applied to knockdown PITX1 in chicken embryos’ feet. Furthermore, the correlation between Pen deletion and non B-DNA structure would be illustrated in Z-DNA density predictions and DNA structural determination tests. In the functional study of PITX1, embryos with PITX1 suppression did not show obvious feather-germs. Thus, we speculate that suppression of one single gene could not form feathers in chicken feet. Meanwhile, our results in Z-DNA analysis indicate that Z-DNA exists on Pen both in pigeons and chickens with the differences in number, length, and density, which might lead to the discrepancy of loci and scale of deletion between the pigeon and the chicken. Therefore, this study inspected the importance of Z-DNA in genomic stability and how it regulates traits of skin appendages among several avian species.

摘要 ii
Abstract iii
致謝 iv
Contents v
1. Introduction 1
2. Materials and methods 5
2.1 Z-DNA motifs prediction and Z-DNA density analysis 5
2.2 S1 nuclease test 5
2.3 YAC assay 6
2.4 Chicken breeds resource and embryos collection 7
2.5 Paraffin embedding, tissue section, and HE staining 7
2.6 RCAS system 8
2.7 Fluorescence detection 9
3. Results 11
3.1 Z-DNA motifs prediction and Z-DNA density analysis 11
3.2 Z-DNA structure determination and fragility assay 12
3.3 Knock-down expression of PITX1 through RCAS system 12
4. Discussion 14
5. Table 17
6. Figure 21
7. Reference 29
8. Supplementary data 33

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