Variations of Random Amplified Polymorphic DNA (RAPD) Patterns among Field Populations of Plasmodiophora brassicae
Variations of Random Amplified Polymorphic DNA (RAPD) Patterns among Field Populations of Plasmodiophora brassicae
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甘蓝根霉田间种群随机扩增多态性 DNA (RAPD) 模式的变异
DOI：
10.3186/jjphytopath.63.179
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
M. Kameya
中科院分区：
文献类型：
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作者：
S. Yano;Shuhei Tanaka;S. Ito;M. Kameya
芸苔根肿病（Plasmodiophora brassicae Woronin）是十字花科蔬菜根肿病的病原菌，包括致病性不同的田间种群。虽然这些种群的小种通常是用Williams 16）和布克扎奇等人2）提出的鉴别寄主来鉴定的，但这些方法需要很长时间。艾德最近报道了日本15个田间种群的同工酶和细胞可溶性蛋白质的差异。根据这些变异，这些种群中的一部分与其他种群明显不同。基于DNA的技术的发展提供了大量额外的遗传标记。随机扩增多态性DNA（RAPD）可用于估计包括植物病原真菌在内的多种生物的遗传变异。RAPD技术可能比同工酶和可溶性蛋白质分析更适用于检测甘蓝型油菜群体的遗传变异。此外，RAPD PCR（聚合酶链式反应）使用极少量的DNA样品。我们认为，一些RAPD引物可用于检测芸苔群体间的遗传变异。Moller和Harling 9）也应用RAPD技术来区分芸苔疫霉分离株，但他们只检测了三个分离株。本文利用RAPD-PCR技术对日本16个芸苔群体的遗传变异进行了研究。从日本7个县的16个田间采集的大白菜、甘蓝、花椰菜和西兰花的根肿病瘿中获得16个田间群体（表1）。在这些群体中，13个被鉴定为威廉姆斯4号小种，2个为9号小种，1个为1号小种。已确认其中5种对大白菜抗根肿病（CR）品种具有致病性，11种对大白菜抗根肿病（CR）品种无致病性11，12，未公开数据）根肿病虫瘿在-40 ° C下储存，直至制备休眠孢子。将从根肿病五倍子制备的每个种群的休眠孢子（1·~ 101（）个孢子6）悬浮在2 ml提取缓冲液（150 mM EDTA和50 mM Tris-HCl，pH 8.0）中，并与Ig玻璃珠一起涡旋6分钟以破坏休眠孢子。通过前述方法从匀浆中纯化基因组DNA 6）。从试剂盒A（Operon Technologies，阿拉米达，CA，USA）获得20种引物（OPA 01至20），从DNA寡聚体（12）组A-1（和子Pure Chemical，Osaka，Japan）获得12种引物（A01至12）。PCR扩增及其产物的检测基本上根据威廉姆斯等人15）进行。扩增反应在25 μ 1溶液中进行，所述溶液含有25 ng基因组DNA、2.5 μ 1 10 μ 1反应缓冲液（连接到聚合酶试剂盒）、2.5 U Takara Taq聚合酶（Takara，日本）、各200 μ M dCTP、dGTP、dATP和dTTP以及0.4 μ M引物。扩增在Astec Thermal Cycler PC-700和PC-800中进行，程序为40个循环，包括94 ℃ 1 min，10 ℃ 2 min和73 ℃ 1 min。在此，T是每种引物的最佳退火温度，A01为34 ℃; A02，04为38 ℃; A03，05-08为40 ℃; A09，11为36 ℃; A10为42 ℃; A12为32 ℃; OPA 01 -20为29 ℃。通过在1.2%（w/ v）琼脂糖凝胶中电泳来解析扩增产物的10 μ l样品，并通过用溴化乙锭染色来检测。测试样品的所有扩增反应和电泳运行包括来自群体HA 1的样品作为参考。每个样品至少重复检查两次。32个引物中有31个引物扩增出特异性条带，其中29个引物扩增出的条带在16个供试群体中表现出多态性。引物A02无扩增产物，OPA 01和06也有微弱的扩增产物。最常见的是具有四到五个条带的简单图案。底漆A12
Plasmodiophora brassicae Woronin, causal fungus of clubroot disease of crucifers, includes various field popu lations with different pathogenicity. Although the races of these populations are usually identified using differential hosts as proposed by Williams16) and Buc zacki et al.2), these methods take a long time. We report ed recently that isozymes and cellular soluble proteins varied significantly among fifteen field populations from Japan. A part of these populations were clearly distin guishable from other populations on the basis of these variations 13). The development of DNA-based technologies is pro viding a plentiful array of additional genetic markers. Random amplified polymorphic DNAs (RAPDs) have been useful for estimating genetic variation in a wide range of organisms, including plant pathogenic fungi. RAPDs may prove to be more useful for detecting genetic variation in P. brassicae populations than iso zyme and soluble protein analyses. In addition, RAPD PCR (polymerase chain reaction) uses an extremely small amount of DNA sample. We suggested previously that some RAPD primers might be available for the detection of genetic variability among P. brassicae populations6). Moller and Harling9) have also applied RAPDs in distinguishing P. brassicae isolates, but they examined only three isolates of the fungus. In the pres ent paper, we evaluate genetic variation in 16 popula tions of P. brassicae from Japan using RAPD-PCR. Sixteen field populations of P. brassicae were obtained from clubroot galls of Chinese cabbage, cabbage, cauliflower and broccoli which were collected from sixteen fields in seven prefectures of Japan (Table 1). Of these populations, 13 were identified as Williams'16) race 4, two as race 9 and one as race 1. Five have been confirmed to be pathogenic and eleven to be nonpatho genic to clubroot-resistant (CR) cultivars of Chinese cabbage11,12,unpubllshed data) Clubroot galls were stored at -40•Ž until preparation of resting spores. Resting spores (1•~1010 spores) of each population prepared from clubroot galls6) were suspended in 2ml of extraction buffer (150mM EDTA and 50mM Tris-HCI, pH8.0) and vortexed together with 1g of glass beads for 6min in order to disrupt the resting spores. Genomic DNA was purified from the homogenate by the method described previously6). Twenty primers (OPA01 to 20) were obtained from kit A (Operon Technologies, Alameda, CA, USA) and 12 primers (A01 to 12) from DNA Oligomer (12) Set A-1 (Wako Pure Chemical, Osaka, Japan). The PCR amplification and the detection of its products were fundamentally performed according to Williams et al.15). Amplification reactions were performed in 25 ul of a solution containing 25ng of genomic DNA, 2.5ƒÊl of 10•~ Reaction buffer (attached to polymerase kit), 2.5 U of Takara Taq polymerase (Takara, Japan), 200•~ƒÊM each of dCTP, dGTP, dATP and dTTP and 0.4ƒÊM of primer. Amplification was carried out in an Astec Thermal Cycler PC-700 and PC-800, programmed for 40 cycles that consisted of 1 min at 94•Ž, 2min at T•Ž, and 1min at 73•Ž. Here, T is the optimum annealing temperature for each primer, 34•Ž for A01; 38•Ž for A02, 04; 40•Ž for A03, 05-08; 36•Ž for A09, 11; 42•Ž for A10; 32•Ž for A12; 29•Ž for OPA01-20. A 10ƒÊl sample of the amplification products was resolved by electrophoresis in a 1.2% (w/ v) agarose gel and detected by staining with ethidium bromide. All amplification reactions and electrophoretic runs of test samples included the sample from the population HAl as reference. The examination was repeated at least two times for each sample. Thirty-one of 32 primers generated PCR amplification products; 29 of them gave reproducible electrophoretic banding patterns which showed polymorphisms among the 16 tested field populations of P. brassicae. On the other hand, the primer A02 gave no amplification prod uct, and the other two primers, OPA01 and 06, also gave very faint products. Simple patterns with four to five bands were most frequently observed. The primer A12