以簡單重複序列分子標誌探討花椰菜品種及白菜類的遺傳變異

Genetic Variation of Brassica oleracea L. var. botrytis and B. rapa L. in Taiwan as Depicted by Simple Sequence Repeats Markers

陳怡婷(Yi-Ting Chen)

指導教授 ： 曹幸之

共同指導教授 ： 楊堯文(Yau-Wen Yang)

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.00158

摘要

本研究利用真核生物基因體中普遍存在的簡單重複序列(Simple Sequence Repeats DNA, SSRs DNA)做為分子標誌(包含Intra-SSR標誌及Inter-SSR標誌)，探討23個台灣花椰菜(Brassica oleracea L. var. botrytis)品種及六個白菜亞種類群(包括Brassica rapa L. ssp. chinensis Chinensis group，Japonica group，Narinosa group，Parachinensis group，B. rapa L. ssp. pekinensis，及B. rapa L. ssp. rapifera)的遺傳變異。依據春作及秋作植株的結球時間及育種者提供的資訊，花椰菜23品種可分成7個早生類型品種、6個中生類型品種及10個晚生類型品種。以27組SSR專一性引子對(primer pairs)進行品種間遺傳變異分析，共得到91個Intra-SSR標誌。其中9組引子對所得共11個標誌在所有供試材料間呈單型性(monomorphic)。另18組引子對所得80個標誌中，有6組引子對所得共19個標誌在23個花椰菜品種間是單型性，只能區分花椰菜與外群；另外12組引子對共獲得61個標誌在花椰菜品種間具有多型性(polymorphic)，佔所有標誌的67.0%；其中有9個標誌只在特定類型的品種中出現。以6個SSR型式的引子(primers)在供試花椰菜共得到169個Inter-SSR標誌，其中1個標誌為單型性，63個標誌僅能區別花椰菜及外群，另外105個是花椰菜品種間的多型性標誌(佔62.1%)，7個標誌只在特定類型品種中具有。
以80個於花椰菜及外群間具多型性的Intra-SSR標誌及169個Inter-SSR標誌分別計算花椰菜品種間的遺傳變異(genetic variation)，及進行群叢分析(clustering)。由Intra-SSR標誌所得花椰菜品種間的遺傳變異為0.055~0.491，由Inter-SSR標誌所得遺傳變異為0~0.446。兩種標誌均顯示紫花椰菜與白花椰菜間的遺傳變異較大，成熟性相同的品種間遺傳變異較小，‘慶農台寶60天’(中生，F1)與其中生型及晚生型的父母本間的遺傳變異，小於其父母本之間的遺傳變異。花椰菜與外群中的青花菜遺傳變異最小，與白菜類間的遺傳變異最大。分析所得之NJ、UPGMA與Bayesian樹狀圖，皆將紫花椰菜品種及白花椰菜品種分成兩大群，白花椰菜群內早生類型品種聚集一起，此結果與合併兩種標誌再以Bayesian方法分析所得的品種分群結果一致。白花椰菜晚生類型品種在Intra-SSR標誌所得之群叢圖中聚為一群，但在Inter-SSR標誌分析及兩種標誌合併分析所得Bayesian圖中則無法群聚在一起，但可以與早生型品種分開。
以相同之27組Intra-SSR引子對，用於探討六種白菜類(共28個品種)的親緣關係，並以兩個千寶菜(B. napus L.)做為外群，共得到91個Intra-SSR標誌。其中9組引子對所得9個標誌在30個供試品種間呈單型性。另18組引子對產生82個標誌中有3個標誌為單型性，79個標誌呈多型性，這其中有1個標誌在白菜類品種中為單型性，6個標誌只出現在2個千寶菜品種中，其餘72個標誌則在六個白菜亞種類及類群中具多型性(佔所有標誌的79.1%)。6個標誌為亞種或類群的特有標誌。
以18組引子對所得82個Intra-SSR標誌分析白菜品種及千寶菜的親緣關係。在六種白菜類的遺傳變異為0.211~0.862，以小白菜類群(Brassica rapa L. ssp. chinensis Chinensis group)與蕪菁亞種(ssp. rapifera)間差異最大，各類群內的遺傳變異最小。千寶菜與各白菜種類間的遺傳變異為0.827~0.920。以NJ、UPGMA及Bayesian方法進行群叢分析，將白菜類及千寶菜清楚分群；三種群叢方法皆可將不結球白菜(ssp. chinensis Chinensis group)品種聚為一大群，且小白菜品種又大致依夏季及冬季生產分群區別。小油菜(ssp. chinensis Japonica group)、菜苔(ssp. chinensis Parachinensis group)、烏塌菜(ssp. chinensis Narinosa group)、結球白菜(ssp. pekinensis)及蕪菁(ssp. rapifera)在NJ及UPGMA之群叢分析圖可分群區別，但以Bayesian方法則僅能區分出小油菜、菜苔及烏塌菜而已。顯示Intra-SSR標誌鑑別不結球白菜品種間遺傳變異效果不及利用RAPD標誌所得結果(Chuang et al., 2004)。
本研究顯示Intra-SSR及Inter-SSR標誌可用於分析花椰菜及白菜類的遺傳變異，所得資訊可提供品種改良時雜交育種親本選擇以及種原收集保存的參考。花椰菜品種在Intra-SSR標誌分析得到較Inter-SSR標誌分析更多的類型特有標誌，可協助花椰菜類型或品種鑑別之用。

關鍵字

白菜類 ； 簡單重複序列分子標誌 ； 花椰菜 ； 遺傳變異

並列摘要

Genetic diversity and phylogenetic relationship among 23 Cauliflower (Brassica oleracea var. botrytis L.) cultivars and six groups of B. rapa L. have been separately studied using simple sequence repeats (including Intra-SSR marker and Inter-SSR marker), a DNA sequence type ubiquitous in most eukaryotic genomes.
Based on the timing of curd development, cultivars can be classified into early maturity type, medium maturity type and late maturity types. Among eighty intra-SSR markers were generated from eighteen primer pairs. Among cauliflower cultivars tested, nineteen markers are monomorphic, sixty one markers are polymorphic, and nine markers are specific in certain maturity types. In addition, one hundred and sixty nine inter-SSR markers were generated from six SSR-type primers and one hundred and five (62%) ones are polymorphic among the cauliflower cultivars tested. Seven inter-SSR markers are specific for certain cauliflower types.
Eighty Intra-SSR markers and 169 Inter-SSR markers were scored and compiled separately as a data matrix. The genetic variations among cauliflower cultivars based on Intra-SSR markers range from 0.055 to 0.491, the corresponding variation based on Inter-SSR markers are between 0 and 0.446. The variation was larger between purple cauliflowers and white cauliflowers than that within group of the same curd color. The cluster analyses based on NJ, UPGMA and Bayesian method separate all accessions into purple type and white cauliflower type, with early maturity type of white cauliflower being further grouped in a clade when Intra-SSR or Inter-SSR markers were analyzed separately. The Bayesian dendrogram obtained from two types of markers combined shows similar clustering. Several late maturity white cauliflowers are grouped together in the dendrogram of Intra-SSR markers, but not in that of the Inter-SSR markers.
Eighteen primers were selected from the same 27 Intra-SSR primer pairs of cauliflowers to detect the difference of 28 cultivars among six B. rapa L. groups with two San-Po Tsai (B. napus L.) as outgroups. Eighty-two Intra-SSR markers were generated and two markers are monomorphic for all tested taxa. Four markers are present in San-Po Tsai only. In addition, four markers are monomorphic and seventy-two markers were polymorphic for all tested B. rapa L. Six are specific for certain subspecies or groups of B. rapa L.
Pair-wise genetic variations for 28 B. rapa L. accessions based on 82 intra-SSR markers range from 0.211 to 0.862, with greater variation between Chinensis group of ssp. chinensis and ssp. rapifera. Summer accessions in the Chinensis group of ssp. chinensis can be separated from the rest of other accessions with high confidence levels in this study. B. rapa L. ssp. chinensis Japonica group, ssp. chinensis Parachinensis group, ssp. chinensis Narinosa group, Chinese Cabbage (ssp. pekinensis) and turnip(ssp. rapifera) can each form separate groups in NJ and UPGMA dendrograms, but not by Bayesian dendrogram.
The results of this study showed that both intra-SSR and inter-SSR markers can be used to establish phylogenetic relationship in different cauliflower cultivars. In addition, intra SSR markers were shown to be able to detect the genetic relationship among B. rapa L. groups although it was less effective as inter-SSR or RAPD markers could do as described by Chuang et al. (2004).

並列關鍵字

S ； Genetic Variation ； Califlower ； Brassica rapa L.

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