Implementation of two high through-put techniques in a novel application: detecting point mutations in large EMS mutated plant populations.
Implementation of two high through-put techniques in a novel application: detecting point mutations in large EMS mutated plant populations.
复制标题
DOI：
10.1186/1746-4811-5-13
复制
发表时间：
2009-10-07
期刊：
影响因子：
5.1
通讯作者：
Bachem CW
中科院分区：
文献类型：
--
作者：
Gady AL;Hermans FW;Van de Wal MH;van Loo EN;Visser RG;Bachem CW
突变群体的建立以及定向突变检测策略已成功应用于大量生物，包括植物界的许多物种。作为浆果类植物的模式植物，番茄的研究投入了大量精力。随着番茄测序项目的进展，反向遗传学成为一个明显且可实现的目标。
在此我们描述了用1% EMS处理番茄（Solanum lycopersicum）种子以及一个新的番茄突变群体的培育。为了提高定向突变检测通量，一种自动化的种子DNA提取方法与用于植物TILLING（定向诱导基因组局部突变技术）的新型突变检测平台相结合。我们采用了两种用于人类遗传诊断的技术：构象敏感毛细管电泳（CSCE）和高分辨率DNA熔解分析（HRM）来对DNA混合样本进行突变筛选。传统的TILLING涉及关键且耗时的步骤，如内切核酸酶消化反应和凝胶电泳。使用CSCE或HRM时，唯一需要的步骤是在毛细管电泳或DNA熔解曲线分析之前进行一次简单的PCR。在此我们描述了一个番茄突变群体的培育、两个用于植物的多态性检测平台的建立以及首次筛选的结果，包括群体中的突变密度以及使用HRM筛选DNA混合样本时假阳性率的估计。
这些结果表明，CSCE和HRM是用于DNA混合样本中突变检测的快速、经济且灵敏的技术，因此能够快速鉴定突变群体中的新等位基因变体。首次筛选的结果表明，诱变处理是有效的，每个二倍体基因组的平均突变检测率为每1000株系1.36个突变/千碱基。
The establishment of mutant populations together with the strategies for targeted mutation detection has been applied successfully to a large number of organisms including many species in the plant kingdom. Considerable efforts have been invested into research on tomato as a model for berry-fruit plants. With the progress of the tomato sequencing project, reverse genetics becomes an obvious and achievable goal. Here we describe the treatment of Solanum lycopersicum seeds with 1% EMS and the development of a new mutated tomato population. To increase targeted mutant detection throughput an automated seed DNA extraction has been combined with novel mutation detection platforms for TILLING in plants. We have adapted two techniques used in human genetic diagnostics: Conformation Sensitive Capillary Electrophoresis (CSCE) and High Resolution DNA Melting Analysis (HRM) to mutation screening in DNA pools. Classical TILLING involves critical and time consuming steps such as endonuclease digestion reactions and gel electrophoresis runs. Using CSCE or HRM, the only step required is a simple PCR before either capillary electrophoresis or DNA melting curve analysis. Here we describe the development of a mutant tomato population, the setting up of two polymorphism detection platforms for plants and the results of the first screens as mutation density in the populations and estimation of the false-positives rate when using HRM to screen DNA pools. These results demonstrate that CSCE and HRM are fast, affordable and sensitive techniques for mutation detection in DNA pools and therefore allow the rapid identification of new allelic variants in a mutant population. Results from the first screens indicate that the mutagen treatment has been effective with an average mutation detection rate per diploid genome of 1.36 mutation/kb/1000 lines.
登录
查看更多内容
影响因子：
3.7
作者：
Rigola D;van Oeveren J;Janssen A;Bonné A;Schneiders H;van der Poel HJ;van Orsouw NJ;Hogers RC;de Both MT;van Eijk MJ
通讯作者：
van Eijk MJ
影响因子：
4.4
作者：
Davies, H;Dicks, E;Wooster, R
通讯作者：
Wooster, R
影响因子：
7.2
作者：
Meissner, R;Jacobson, Y;Levy, A
通讯作者：
Levy, A
影响因子：
5.4
作者：
Lehmensiek, Anke;Sutherland, Mark W.;McNamara, Raymond B.
通讯作者：
McNamara, Raymond B.
影响因子：
11.6
作者：
IRISH, VF;SUSSEX, IM
通讯作者：
SUSSEX, IM