Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36912
標題: RAPD及AFLP分子標誌技術在紫錐菊遺傳變異之研究
Using RAPD and AFLP analyses to assess genetic diversity of Echinacea
作者: 莊淑貞
Chuang, Su-Jean
關鍵字: RAPD;遺傳歧異度;AFLP;genetic diversity;Echinacea;紫錐菊;分子標誌
出版社: 農藝學系所
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摘要: 
紫錐菊是台灣新興的藥用保健植物為常異交多年生作物,基因組體具有一定程度的異質性。本研究主要利用RAPD 與 AFLP技術對引進紫錐菊種/品種/變種進行鑑定,同時也嘗試建立 RAPD分子標誌與紫錐菊植體化學成份間之關係。此外,本研究也探討以RAPD 及AFLP來鑑定微體繁殖紫錐菊種苗之變異與實際應用的可行性。
試驗結果顯示,以RAPD及AFLP對具藥用效果的紫錐菊引進種/品種/變種進行遺傳歧異性及親緣關係分析,可使用 DNA 混合樣本。混合樣品數無論RAPD或AFLP分析均以10株、15株或20株涵蓋最大的分離條帶表現度(87~90 %),對種或族群鑑別最具代表性。親緣關係探討中結合混合DNA樣品於RAPD分析技術,以Jaccard’s相似性係數評估, E. purpurea種內之相似係數均在0.85以上,其中E. purpurea (s)與E. purpurea cv. Magnus之相似性更高達0.93,E. purpurea與其他種則在0.36~0.41之間。E. pallida var. pallida與E. atrorubens var. paradoxa及E. angustifolia之各種/變種/品種(系)的相似性為0.41~0.49被歸為一群。親緣關係探討中結合混合DNA樣品於AFLP分析,以Jaccard’s相似性係數評估,參試的紫錐菊種/變種/品種中E. purpurea種內之四個種/變種/品種其相似係數均在0.86以上,其中E. purpurea (s)與E. purpurea cv. Magnus之相似係數更高達0.90。參試紫錐菊種/變種/品種的親緣關係為E. atrorubens var. paradoxa與 E. pallida 的親緣關係最近,其次是E. angustifolia而與E. purpurea親緣關係最遠。Mantel測驗結果顯示RAPD與AFLP都是理想的紫錐菊親緣關係的分析方法,而且150個分子標誌數目的分析結果等同300個與400個標誌的分析。
RAPD評估紫錐菊植體化學成份的變異情形係以逢機取得集團選拔的植株59株為材料,以Folin & Ciocalteau’s酚類試劑及HPLC法分析植體化學成份含量,花、葉部各含不同程度的總酚類含量、烷醯胺8+9含量及咖啡酸衍生物含量,再與以44組RAPD逢機引子所獲得表現單株間多型性66個片段比對。結果發現多型性RAPDs標誌中,14個標誌與植體化學成份含量間的相關達顯著或極顯著。以SAS逐步迴歸統計建立複迴歸模式,其中葉部總酚類含量及葉部咖啡酸衍生物含量複迴歸迴歸方程式,逐步選取的分子標誌,多數為相同(87.5%及77.8%),花部各植體化學成份的複迴歸方程式決定係數(0.75-0.83)均高於葉部(0.43-0.71)。以另外11個單株之植體化學成份的實測值及RAPD標誌之迴歸模式估算值,來驗證迴歸模式。結果發現花部總酚類含量估算值與實測值間的相關最高為0.891且達1%極顯著水準,其次為葉部總酚類含量的估算值與實測值間的相關為0.534且達5%顯著水準。因此以RAPD分子標誌預估花部、葉部總酚類含量應可應用於紫錐菊育種程序的早期估算選拔。
在利用RAPD及AFLP偵測微體繁殖再生植株體細胞變異方面,以10條RAPD逢機引子進行檢定之結果顯示,6組微體繁殖組包括母株共56個單株個體中,3個微體繁殖再生植株發生體細胞變異且都在同一組微體繁殖組中,其餘5組再生植株均無體細胞變異的現象。本研究將RAPD分析未檢出體細胞變異的五組微體繁殖再生植株與其母株共45個單株為材料進一步以AFLP分析,結果顯示所測試40個再生植株中,有35個再生植株未表現或僅表現微小變異,與母株的Jaccard相似性係數介於0.9881~0.9986之間。顯示AFLP為檢測紫錐菊Echinacea purpurea微體繁殖苖體細胞變異的有效偵測工具。AFLP同樣也可以用於田間觀察圃中微體繁殖植株之體細胞變異分析。本研究以紫錐菊Echinacea purpurea繁殖系T5-9所衍生的組織培養植株的田間觀察圃共1080植株,對植株外表形態正常與異常的單株進行體細胞變異分析。結果發現外表形態正常的植株佔98.8% (1067株),外表形態異常的植株佔1.20%(13株)。正常株之Jaccard’s相似性係數為介於1.000~0.9898之間。外表形態異常的植株與正常株間的Jaccard’s相似性係數為介於0.9896~0.3115之間,這些異常株均可觀察到外表形態的差異。

Echinacea, which has recently been introduced into Taiwan, is a perennial medicinal plant that tends to be out-crossing and produce highly heterozygous progenies. The objectives of the present study were to utilize RAPD and AFLP markers to evaluate phylogenetics of eight Echinacea species/varieties/cultivars, to predict phytochemical levels in parts of Echinacea purpurea individual plant through multiple-regression analysis and to assess somaclonal variation among Echinacea purpurea plants regenerated by organogenesis from leaf explants.
The results indicated that bulked DNA samples combined RAPD and AFLP analyses could be used to assess the phylogenetics of eight Echinacea species/varieties/cultivars. Bulked-DNA samples of 10 plants, 15 plants and 20 plants gained 87~90% fragments obtained after PCR of RAPD and AFLP analyses in species of E. pallida and E. purepurea. Jaccard's similarity, based on RAPD analysis, showed beyond 0.85 within 4 E. purpurea cultivars/varieties and E. purpurea with E. purpurea cv. Magnus up to 0.93 in one cluster group. The similarity between E. purpurea and the rest species/varieties /cultivars was from 0.36 up to 0.41. The other one E. pallida var. pallida with E. atrorubens and E. angustifolia was 0.41 and 0.49 in the same cluster group. Jaccard's similarity based on AFLP analysis, showed beyond 0.86 within 4 E. purpurea cultivars/varieties and E. purpurea with E. purpurea cv. Magnus up to 0.90 in one cluster group. The most phylogenetic relationship of tested Echinacea species/ varieties/ cultivars was between E. atrorubens var. paradoxa and E. pallida, next was E. angustifolia and the lowest was E. purpurea. The numbers of molecular markers used for assessment were also affected at Jaccard's similarity coefficients. Mantel test revealed that both RAPD and AFLP were capable of assessing the genetic relationships among the tested Echinacea species/varieties /cultivars species. Phylogenetic relationship of tested Echinacea species/ varieties/ cultivars constructed based on 400 markers of RAPD analysis was same as 150 markers of AFLP analysis.
RAPD markers were also used to predict phytochemical levels in parts of Echinacea purpurea individual plant through multiple-regression analysis. Phytochemical levels in various parts of fifty-nine individual plants were analyzed by methods of HPLC and Folin-Ciocalteau reagent. Genomic DNA was extracted and PCR-analyzed using 44 RAPD primers. The results showed that 14 out of 66 RAPD polymorphic markers had significant correlation with phytochemical levels in parts of Echinacea purpurea individual plant at 1% or 5% statistical level. After multiple-regression analysis, there were 87.5% and 77.8% RAPD markers identical for total phenolics and caffeic acid derivatives levels in leaf part. In regression analysis, coefficient of determination in floret parts (0.75-0.83) higher than in leaf parts (0.43-0.71). Phytochemical levels in various parts of additional 11 individual plants were also analyzed by methods of HPLC and Folin-Ciocalteau reagent, and were compared to that of predicted values using multiple regression equations for validation. Correlation coefficient of total phenolics levels between observed and predicted values in floret part was 0.891, which was significant at 1% level. Correlation coefficient between observed and predicted values of total phenolics levels in leaf part was 0.534, which was also significant at 5% level. Thus, using RAPD marker seems to be capable of predicting the level of total phenolics in leaf and floret parts of Echinacea purpurea in a marker assisted selection program.
Moreover, RAPD and AFLP analysis were performed to assess somaclonal variation among Echinacea purpurea plants regenerated by organogenesis from leaf. In RAPD analysis by using 10 arbitrary primers, 3 out of 10 regenerated plants from donor plant D3-9 showed somalclonal variation based on the polymorphic profiles. The rest of regenerated plants from other donor plants showed no detectable somaclonal variation. The five donor groups with 45 regenerants were further subjected to AFLP analysis. The results revealed that only 35 regenerated plants showed no or minor somaclonal variations with Jaccard's similarity coefficients of 0.9881~0.9986 between donors and regenerants. Thus, AFLP was a very sensitive and reliable technique to detect somaclonal variation in Echinacea tissue culture system. Furthermore, it can be used to assess the somaclonal variation in field-grown Echinacea purpurea regenerants. In the present study, 1080 TC-regenerants of Echinacea purpurea clone T5-9 by organogenesis from leaf explants were planted in the field. AFLP analysis was performed to assess DNA variation among 1067 morphologically normal plants (98.80%) and 13 abnormal plants (1.20%). The Jaccard's similarity among morphological normal plants ranged between 0.9898 and 1.000. The other 11.22% of normal plants were 0.9898 up to 0.998. The Jaccard's similarity analysis for abnormal plants ranged between 0.3115 and 0.9896. Morphological differences were notable for abnormal plants.
URI: http://hdl.handle.net/11455/36912
其他識別: U0005-2407200814030300
Appears in Collections:農藝學系

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