Vandenbussche Michiel, Janssen Antoine, Zethof Jan, van Orsouw Nathalie, Peters Janny, van Eijk Michiel J T, Rijpkema Anneke S, Schneiders Harrie, Santhanam Parthasarathy, de Been Mark, van Tunen Arjen, Gerats Tom
Radboud University, IWWR/Plant Genetics, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands.
Plant J. 2008 Jun;54(6):1105-14. doi: 10.1111/j.1365-313X.2008.03482.x. Epub 2008 Mar 13.
BLAST searchable databases containing insertion flanking sequences have revolutionized reverse genetics in plant research. The development of such databases has so far been limited to a small number of model species and normally requires extensive labour input. Here we describe a highly efficient and widely applicable method that we adapted to identify unique transposon-flanking genomic sequences in Petunia. The procedure is based on a multi-dimensional pooling strategy for the collection of DNA samples; up to thousands of different templates are amplified from each of the DNA pools separately, and knowledge of their source is safeguarded by the use of pool-specific (sample) identification tags in one of the amplification primers. All products are combined into a single sample that is subsequently used as a template for unidirectional pyrosequencing. Computational analysis of the clustered sequence output allows automatic assignment of sequences to individual DNA sources. We have amplified and analysed transposon-flanking sequences from a Petunia transposon insertion library of 1000 individuals. Using 30 DNA isolations, 70 PCR reactions and two GS20 sequencing runs, we were able to allocate around 10 000 transposon flanking sequences to specific plants in the library. These sequences have been organized in a database that can be BLAST-searched for insertions into genes of interest. As a proof of concept, we have performed an in silico screen for insertions into members of the NAM/NAC transcription factor family. All in silico-predicted transposon insertions into members of this family could be confirmed in planta.
包含插入侧翼序列的可通过BLAST搜索的数据库彻底改变了植物研究中的反向遗传学。到目前为止，此类数据库的开发仅限于少数模式物种，并且通常需要大量的人力投入。在这里，我们描述了一种高效且广泛适用的方法，该方法经过调整以鉴定矮牵牛中独特的转座子侧翼基因组序列。该程序基于用于收集DNA样本的多维混合策略；分别从每个DNA池中扩增多达数千个不同的模板，并通过在其中一个扩增引物中使用池特异性（样本）识别标签来确保对其来源的了解。所有产物合并到一个单一样本中，随后用作单向焦磷酸测序的模板。对聚类序列输出的计算分析允许将序列自动分配给各个DNA来源。我们从一个包含1000个个体的矮牵牛转座子插入文库中扩增并分析了转座子侧翼序列。通过30次DNA分离、70次PCR反应和两次GS20测序运行，我们能够将大约10000个转座子侧翼序列分配到文库中的特定植物。这些序列已被整理到一个数据库中，可通过BLAST搜索以查找插入到感兴趣基因中的情况。作为概念验证，我们对NAM/NAC转录因子家族成员的插入进行了电子筛选。所有电子预测的该家族成员的转座子插入在植物中都得到了证实。
