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表 1. 螢光原位雜交反應使用的探針。
探針 來源 插入片段大小 標定抗原 標定方法 染色
1. 5S rDNA
2. 45S rDNA
3. Arabidopsis-type telomeric sequences
表 2. 姬蝴蝶蘭粗絲期染色體核型分析統計數據。
編號 細胞數a 總長度(µm) 相對長度(%) 真染色質(µm) 異染色質(µm) 長臂(µm) 短臂(µm) Arm Ratiob 中心節位置C 1 5 32.07 ± 5.49 7.82 ± 0.42 21.38 ± 4.58 10.69 ± 0.98 21.60 ± 5.33 10.47 ± 2.80 2.21 ± 0.90 sm 2 4 29.08 ± 3.03 7.46 ± 0.34 24.69 ± 2.64 4.39 ± 0.93 18.06 ± 1.62 11.02 ± 1.79 1.66 ± 0.22 m 3 5 29.43 ± 4.22 7.20 ± 0.21 23 ± 4.47 6.44 ± 0.75 17.73 ± 1.92 11.71 ± 3.20 1.59 ± 0.40 m 4 5 28.29 ± 2.83 6.96 ± 0.44 20.38 ± 2.11 7.91 ± 1.25 18.66 ± 2.99 9.62 ± 0.79 1.96 ± 0.43 sm 5 5 26.29 ± 4.77 6.43 ± 0.59 19.96 ± 4.05 6.33 ± 1.59 15.09 ± 2.17 11.19 ± 2.92 1.39 ± 0.26 m 6 5 24.56 ± 4.25 6.00 ± 0.51 19.11 ± 3.19 5.45 ± 1.44 16.48 ± 3.89 8.08 ± 1.21 2.07 ± 0.52 sm 7 5 24.75 ± 3.64 6.07 ± 0.59 16.79 ± 3.81 7.95 ± 1.94 16.67 ± 2.25 8.07 ± 1.77 2.11 ± 0.28 sm 8 5 25.50 ± 7.73 6.19 ± 1.22 17 ± 6.07 8.5 ± 1.91 17.39 ± 6.92 8.10 ± 2.45 2.33 ± 1.11 sm 9 5 21.70 ± 2.42 5.32 ± 0.07 15.94 ± 2.33 5.76 ± 0.69 15.17 ± 1.35 6.51 ± 1.90 2.51 ± 0.84 sm 10 5 21.62 ± 2.57 5.30 ± 0.16 15.22 ± 2.24 6.4 ± 0.49 14.47 ± 1.98 7.14 ± 1.31 2.07 ± 0.41 sm 11 5 19.01 ± 2.70 4.66 ± 0.25 10.49 ± 1.69 8.52 ± 1.25 11.20 ± 1.96 7.07 ± 2.57 1.86 ± 0.65 sm 12 5 19.83 ± 2.70 4.86 ± 0.22 13.39 ± 2.33 6.44 ± 0.61 10.72 ± 1.76 8.64 ± 0.88 1.29 ± 0.15 m 13 4 17.18 ± 4.18 4.04 ± 0.60 12.88 ± 4.07 4.3 ± 0.29 14.22 ± 3.33 3.68 ± 1.50 4.03 ± 0.66 st 14 5 18.20 ± 2.84 4.46 ± 0.36 13.29 ± 1.69 4.9 ± 1.32 13.00 ± 1.94 5.20 ± 1.26 2.50 ± 0.50 sm 15 5 16.51 ± 2.14 4.04 ± 0.10 9.28 ± 1.21 7.23 ± 1.12 9.33 ± 1.07 7.19 ± 1.16 1.31 ± 0.14 m 16 5 17.17 ± 3.03 4.20 ± 0.43 9.78 ± 2.79 7.39 ± 0.41 9.50 ± 1.66 7.67 ± 1.45 1.25 ± 0.12 m 17 5 16.74 ± 2.14 4.15 ± 0.74 10.40 ± 2.50 6.34 ± 0.59 11.95 ± 1.49 4.31 ± 0.80 2.81 ± 0.32 sm 18 5 15.56 ± 3.56 3.78 ± 0.39 8.63 ± 2.34 6.93 ± 1.35 9.50 ± 2.61 6.06 ± 1.10 1.56 ± 0.28 m 19 5 13.65 ± 2.07 3.34 ± 0.28 7.24 ± 1.41 6.41 ± 0.88 8.24 ± 1.88 5.40 ± 0.27 1.52 ± 0.29 m a. 供作分析的 5 個細胞中，其中 2 個細胞各只有 18 對染色體，分別缺少第 2 對及第 13 對，故細胞的樣本數為 4。
b. 臂比 arm ratio 數值係長臂(q) /短臂(p)所獲得。
c. 英文字縮寫: m: metacentric ; sm: submetacentric ; st: subtelocentric，係參考 Rho et al., (2012)文獻。
表 3. 姬蝴蝶蘭中期染色體長度統計分析。
表 4. 姬蝴蝶蘭相異細胞中內含 45S rDNA 訊號的染色體細部分析。
細胞 長度 真染色質 異染色質 長臂 短臂 Arm Ratio 中心節
1 16.54 12.86 3.68 13.36 3.18 4.2 st 2 18.45 12.54 5.91 12.19 6.26 1.95 sm 3 17.61 13.29 4.32 13.79 3.82 3.61 st 4 25.64 16.5 9.14 18.58 7.06 2.63 sm 5 21.09 13.29 7.8 16.03 5.06 3.17 st 6 19.06 13.23 5.83 14.57 4.49 3.24 st 7 19.61 14.49 5.12 14.93 4.68 3.19 st 8 10.06 7.8 2.26 6.79 3.27 2.08 sm 9 17.16 13.68 3.48 12.68 4.48 2.83 sm
平均 18.36 13.08 5.28 13.66 4.7 3.0 st
標準偏差 4.1 2.3 2.17 3.21 1.29 0.71
*表中長度、真染色質、異染色質與長短臂單位皆為 µm。
圖 1. 姬蝴蝶蘭粗絲期染色體型態觀察。(A) 以 1% Acetocarmine 染色觀察花粉 細胞染色體型態，細胞壁尚未破壞，可觀察到較完整的細胞結構；(B) 以光學 顯微鏡觀察染色體型態；(C) 將姬蝴蝶蘭粗絲期染色體以 DAPI 染色後，以螢 光顯微鏡 CCD camera 拍照收集圖像；(D) 以影像軟體編輯圖檔。Scale bar = 10 µm。
圖 2. 姬蝴蝶蘭粗絲期染色體模式核型圖(第一組核型)。以影像軟體處理後，呈 現完整的 19 對染色體，有些染色體收縮良好且染色均勻，能夠觀察到清晰的異 染色質結。染色體以 DAPI 作對比染色，箭頭所指之處為第 19 對染色體，是所 有染色體中長度最小者。Scale bar = 10 µm。
圖 3. 供作核型分析之細胞染色體圖。從製備完成的染色體玻片中挑選染色體完 整，異染色質清晰且染色體分散程度較均勻的細胞，以 PI 作對比染色，於螢光 顯微鏡下拍攝收集影像並進行圖檔的編輯，讓異染色質以及染色體型態更為清 楚。(A) 第二組核型；(B) 第三組核型；(C) 第四組核型；(D) 第五組核型。Scale bar = 10 µm。
圖 4. 觀察姬蝴蝶蘭單離染色體末端次級收縮區。箭頭所指的地方有一段尚未收 縮完全的染色體。染色體以 DAPI 作對比染色，Scale bar = 10 µm。
圖 5. 觀察姬蝴蝶蘭中期染色體型態。以 DAPI 作對比染色，於螢光顯微鏡下拍 攝並進行影像處理。圖中可發現中期染色體大小型態相似，有些染色體染色較 深。Scale bar = 1 µm。
圖 6. 以螢光原位雜交分別將 45S rDNA 與端粒序列定位在姬蝴蝶蘭染色體。
(A)、(B) 以 biotin-16-dUTP 標定 45S rDNA，再以 Rodamin 偵測，於相異細胞 中染色體的末端觀察到極為明顯的紅色訊號；(C) 以 dig-11-dUTP 標定 45S rDNA，再以 FITC 偵測，染色體的末端亦觀察到綠色訊號。藉由觀察 45S rDNA 可判斷衛星體的染色體。(D) 探針為阿拉伯芥型的端粒序列(TTTAGGG)n，以 Rodamin 紅色螢光偵測後發現單離染色體的末端皆出現微弱的訊號(箭頭處)。
染色體以 DAPI 作對比染色，Scale bar = 10 µm。
圖 7. 以雙標的螢光原位雜交將兩種重複性序列定位在姬蝴蝶蘭粗絲期染色體。
綠色訊號代表 45S rDNA，紅色訊號為(TTTAGGG)n，染色體以 DAPI 作對比染 色，Scale bar = 10 µm。
圖 8. 以螢光原位雜交將 5S rDNA 定位在姬蝴蝶蘭粗絲期染色體。以 FITC 偵 測螢光訊號，可發現到 5S rDNA 皆位於染色體的末端。染色體以 PI 作對比染 色。Scale bar = 10 µm。
圖 9. 獨特性序列 SOC1 基因利用螢光原位雜交定位在姬蝴蝶蘭粗絲期染色體。
(A) 以 FITC 偵測螢光，於粗絲期染色體上可觀察到明顯的綠色訊號，包含兩 個明顯的點以及一個較微弱的點，而圖檔上方染色間期也發現到一對明顯的訊 號，包含兩個清晰的點。(B) 相異細胞中觀察 SOC1 基因訊號。染色體以 DAPI 作對比染色。Scale bar = 10 µm。
圖 10. 獨特性序列 SOC1 基因定位在姬蝴蝶蘭單離的粗絲期染色體上。(A) 單 離染色體上觀察到 SOC1 的訊號，包含一個相當明顯的點以及兩個較微弱的點；
(B) 以影像軟體編輯圖檔，可發現到 SOC1 基因位於染色體短臂的真染色質上。
染色體以 DAPI 作對比染色。Scale bar = 10 µm。
圖 11. 姬蝴蝶蘭核型結構模式圖。
*. SOC1 基因距離第一對染色體短臂末端約 5.16 µm。
*. CMT(chromomethylase)基因位置係參考 林(2012) 論文，未發表。(CMT 基因距離第六對染色體長臂末端約 1.23 µm)。
*. EFS(Early Flowering in Short days)基因位置係參考 周(2012) 論文，未發表。(EFS 基因距離第十對染色體長臂末端約 1.13 µm)。