Identification of microsatellite markers for Plasmopara viticola and establishment of high throughput method for SSR analysis
Identification of microsatellite markers for Plasmopara viticola and establishment of high throughput method for SSR analysis
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DOI：
10.1023/a:1022565405974
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发表时间：
2003-02-01
影响因子：
1.8
通讯作者：
Gessler, C
中科院分区：
文献类型：
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作者：
Gobbin, D;Pertot, I;Gessler, C
卵菌纲葡萄生单轴霉 (Plasmopara viticola) 是葡萄树霜霉病 (Vitis spp.) 的致病微生物。为了建立研究霜霉病动态和遗传结构的技术，开发了葡萄霜霉病的共显性、中性、高重复性和多态性微卫星标记。选择了五种标记，其中两种具有 (TC)(n) 重复（位点 BER 和 ISA），两种具有 (TC)(n)(AC)(n) 重复（位点 CES 和 REX），一种具有 (CT)(n)(CTAT)(n) 重复（位点 GOB）。简单序列重复 (SSR) 标记揭示了从受感染的意大利葡萄园收集的 190 个油斑（疾病症状）中存在不同程度的多态性。多态性最高的 SSR 标记 GOB 显示 43 个等位基因（Nei 预期的基因多样性 H-e = 0.89），而 CES、ISA、BER 和 REX 分别显示 14 个（H-e = 0.71）、4 个（H-e = 0.57）、3 个（H-e = 0.24）和 1 个等位基因（H-e = 0）。开发了一种高通量 DNA 提取方法，无需任何分离程序即可直接在宿主中对这种专性病原体进行分子分析。油点的质量和数量不影响SSR分析。在 Beckman-Coulter 2000XL 测序仪上通过电泳分离扩增的 SSR 位点并进行自动分析。本研究的目的是开发能够对霜霉病种群进行高通量分析的分子生物学工具和方法。
The Oomycete Plasmopara viticola is the causal organism of downy mildew on grapevine (Vitis spp.). In order to set up the techniques for investigating downy mildew disease dynamics and genetic structure, co-dominant, neutral, highly reproducible and polymorphic microsatellite markers for P. viticola were developed. Five markers, two with a (TC)(n) repeat (loci BER and ISA), two with a (TC)(n)(AC)(n) repeat (loci CES and REX) and one with a (CT)(n)(CTAT)(n) repeat (locus GOB), were selected. Simple sequence repeat (SSR) markers revealed different degrees of polymorphism within 190 oil spots (disease symptoms) collected from an infected Italian vineyard. The most polymorphic SSR marker GOB showed 43 alleles (Nei's expected gene diversity H-e = 0.89) while CES, ISA, BER and REX showed 14 (H-e = 0.71), 4 (H-e = 0.57), 3 (H-e = 0.24) and 1 allele (H-e = 0), respectively. A high throughput DNA extraction method, that allowed molecular analysis of this obligate pathogen directly in the host without any isolation procedure, was developed. The quality and quantity of oil spots did not influence the SSR analysis. Amplified SSR loci were separated by electrophoresis on a Beckman-Coulter 2000XL sequencer and automatically analysed. The objective of this study was to develop molecular biological tools and methods that allow high throughput analysis of the downy mildew populations.