Sun Liang, Ge Yinbing, Sparks J Alan, Robinson Zachary T, Cheng Xiaofei, Wen Jiangqi, Blancaflor Elison B
Noble Research Institute LLC, Ardmore, OK, United States.
Front Genet. 2019 Jul 25;10:685. doi: 10.3389/fgene.2019.00685. eCollection 2019.
Transfer (T)-DNA insertions in mutants isolated from forward genetic screens are typically identified through thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR), inverse PCR, or plasmid rescue. Despite the popularity and success of these methods, they have limited capabilities, particularly in situations in which the T-DNA is truncated. Here, we present a next generation sequencing (NGS)-based platform to facilitate the identification of complete and truncated T-DNA insertions. Our method enables the detection of the corresponding T-DNA insertion orientation and zygosity as well as insertion annotation. This method, called TDNAscan, was developed to be an open source software. We expect that TDNAscan will be a valuable addition to forward genetics toolkits because it provides a solution to the problem of causal gene identification, particularly genes disrupted by truncated T-DNA insertions. We present a case study in which TDNAscan was used to determine that the recessive mutant isolated in a forward genetic screen of T-DNA mutagenized plants encodes a class II FORMIN.
从正向遗传筛选中分离出的突变体中的转移（T）-DNA插入通常通过热不对称交错聚合酶链反应（TAIL-PCR）、反向PCR或质粒拯救来鉴定。尽管这些方法很受欢迎且取得了成功，但它们的能力有限，特别是在T-DNA被截断的情况下。在这里，我们提出了一个基于下一代测序（NGS）的平台，以促进完整和截断的T-DNA插入的鉴定。我们的方法能够检测相应的T-DNA插入方向和纯合度以及插入注释。这种方法称为TDNAscan，被开发为开源软件。我们预计TDNAscan将成为正向遗传学工具包的一个有价值的补充，因为它为因果基因鉴定问题提供了一个解决方案，特别是被截断的T-DNA插入破坏的基因。我们展示了一个案例研究，其中TDNAscan被用于确定在T-DNA诱变植物的正向遗传筛选中分离出的隐性突变体编码一种II类formin。
