Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36951
標題: 水稻抗褐飛蝨分子標誌之篩選與選殖
Screening and Cloning of Brown Planthopper Resisitance Markers in Rice (Oriza sativa)
作者: 尤淑娟
You, Su-Juan
關鍵字: rice;水稻;brown planthopper;褐飛蝨
出版社: 農藝學研究所
摘要: 
褐飛蝨是危害水稻最嚴重的害蟲之一,育成抗蟲品種為解
決蟲害最根本的防治方法。不抗褐飛蝨之水稻品種台農 67 號
(TNG67) 經疊氮化鈉 (sodium azide, NaN3) 誘變所得之品系中
TM6、D5、D6及D7 等品系經檢定具高度抗蟲性。為探討
誘變品系抗蟲基因之遺傳特性,以抗蟲品系 TM6、D5 及 D7
分別與不具抗蟲性之台農 67 號及台梗 2 號進行雜交,並逐
代進行褐飛蝨抗性檢定。遺傳分析結果,除 D5 x TNG67 組合
外,其他四個參試雜交組合,其 F2 後代均符合 3:1 抗感分
離比,顯示誘變品系之抗蟲現象係由單一顯性基因所控制。
利用聚合酵素鏈反應 (polymerase chain reaction, PCR) 為
基礎所建立之逢機擴大多型性 DNA (random amplified
polymorphism DNA, RAPD) 技術,針對抗 (D7)、感 (TNG 67)
褐飛蝨品系進行 DNA 多型性差異之篩選。經篩檢 680 個逢
機引子,獲得 48 個多型性片段。利用 dT 載體進行多型性
DNA 片段選殖,殖入片段以限制酵素切割,經回收、純化後
做為偵測 F2 族群之探針,探討多型性片段與褐飛蝨抗性之相
關性。其中以 OPB4 引子自 D7 品系增殖所選殖之多型性條
帶殖系 B4-450 進行限制酵素片段長度多型性 (restriction
fragment length polymorphism, RFLP) 分析結果,所有抗性 F2
個體均較感性個體多出一條帶,顯示可能與抗褐飛蝨特性相
關。推測B4-450 片段應為褐飛蝨抗性基因之部份片段,或與
基因緊密連鎖之 DNA 片段。而具抗蟲性之誘變品系 TM6 與
D5、D6 及 D7 呈現不同的 RFLP 圖譜顯示,疊氮化鈉誘變
可能產生兩種或兩種以上不同之抗褐飛蝨基因。
以 B4-450 殖系為探針,對具不同褐飛蝨抗性基因之水稻
種原 Mudgo (Bph 1)、ASD7 (bph 2)、Rathu Heenati (Bph 3)、
Ptb33 (bph 2 + Bph 3) 及 Babawee (bph 4),進行 RFLP 分析發
現,B4-450 應與褐飛蝨基因具有序列相似性 (homology),並
且可明顯區分不同抗性基因。因此,B4-450 片段將可直接應
用於育種,做為偵測及篩選抗褐飛蝨品種及基因型之分子標
誌。

Brown planthopper (BPH) is one of the major insects that damage rice in
most production areas. Breeding varieties with BPH resistance (BPH-R) is
considered to be the best strategy for pest control in rice production.
For this purpose, mutagenesis was conducted to obtain mutants with BPH
resistance. Four mutants, TM6, D5, D6, and D7, derived from TNG67
(BPH susceptible,BPH-S) using sodium azide, were resistant to BPH.
Genetics of this induced BPH resistance was studied by crossing BPH-R
mutants, TM6 and D7, to BPH-S lines, TNG67 and TK2. A segregation with
3 BPH-R to 1 BPH-S in response to BPH challenge were observed in four F2
populations. The results indicated that BPH resistance was controlled by
a dominant resistant gene.
The above mutants were used to isolate the genetic markers linked to
BPH resistance gene using random amplified polymorphic DNA (RAPD) - a
PCR-based technique. A total of 12,000 distinct fragments were amplified
by using 680 Operon random primers (10-mers) and 48 RAPDs (about 0.4%)
observed in D7 (BPH-R) were absent in TNG67 (BPH-S). The polymorphic
DNA fragments were cloned into a dT-vector to probe F2 DNAs. A 450bp
fragment amplified by B4 primer from D7 showed RFLP polymorphisms
between the parents of F2 populations. The segregation pattern of two
BPH-R mutants, TM6 and D7 was different, suggesting involovement of two
different BPH resistant genes. The tight linkage between the B4-450
fragment and the BPH resistance suggested that this fragment is located
at part of the resistant gene or closely linked to it.
Probing rice varieties, Mudgo (Bph 1), ASD7 (bph 2), Rathu Heenati
(Bph 3), Ptb33 (bph 2 + Bph 3), and Babawee (bph 4), from rice germplasm
containing various BPH resistance genes with B4-450 clone suggested that
the B4-450 fragment may have sequence homology to Bph genes. The distinct
RFLP patterns from rice containing various BPH resistance genes indicated
that the B4-450 fragment can be used as a molecular marker for breeding
the BPH resistance programs. The result also demonstrate a good example
for a marker assisted-selection (MAS) using molecular genetic techniques.
URI: http://hdl.handle.net/11455/36951
Appears in Collections:農藝學系

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