Background Forward genetic screens are a powerful approach for identifying the genes contributing to a trait of interest. However, mutants arising in genes already known can obscure the identification of new genes contributing to the trait. Here, we describe a strategy called Candidate gene-Sequencing (Can-Seq) for rapidly identifying and filtering out mutants carrying new alleles of known and candidate genes. Results We carried out a forward genetic screen and identified 40 independent Arabidopsis mutants with defects in systemic spreading of RNA interference (RNAi), or more specifically in root-to-shoot transmission of post-transcriptional gene silencing (rtp). To classify the mutants as either representing a new allele of a known or candidate gene versus carrying a mutation in an undiscovered gene, bulk genomic DNA from up to 23 independent mutants was used as template to amplify a collection of 47 known or candidate genes. These amplified sequences were combined into Can-Seq libraries and deep sequenced. Subsequently, mutations in the known and candidate genes were identified using a custom Snakemake script (https://github.com/Carroll-Lab/can_seq), and PCR zygosity tests were then designed and used to identify the individual mutants carrying each mutation. Using this approach, we showed that 28 of the 40 rtp mutants carried homozygous nonsense, missense or splice site mutations in one or more of the 47 known or candidate genes. We conducted complementation tests to demonstrate that several of the candidate mutations were responsible for the rtp defect. Importantly, by exclusion, the Can-Seq pipeline also identified rtp mutants that did not carry a causative mutation in any of the 47 known and candidate genes, and these mutants represent an undiscovered gene(s) required for systemic RNAi. Conclusions Can-Seq offers an accurate, cost-effective method for classifying new mutants into known versus unknown genes. It has several advantages over existing genetic and DNA sequencing approaches that are currently being used in forward genetic screens for gene discovery. Using Can-Seq in conjunction with map-based gene cloning is a cost-effective approach towards identifying the full complement of genes contributing to a trait of interest.

中文翻译：

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Can-Seq：一种用于识别已知和候选基因的新等位基因的 PCR 和 DNA 测序策略。

背景 正向遗传筛选是一种强有力的方法，用于鉴定对感兴趣的性状有贡献的基因。然而，已知基因中出现的突变体可能会掩盖对导致该性状的新基因的识别。在这里，我们描述了一种称为候选基因测序 (Can-Seq) 的策略，用于快速识别和过滤携带已知和候选基因的新等位基因的突变体。结果我们进行了前向遗传筛选，并鉴定了 40 个独立的拟南芥突变体，这些突变体在 RNA 干扰 (RNAi) 的系统传播方面存在缺陷，或者更具体地说，在转录后基因沉默 (rtp) 的根到茎传播方面存在缺陷。为了将突变体分类为代表已知或候选基因的新等位基因与携带未发现基因中的突变，来自多达 23 个独立突变体的大量基因组 DNA 被用作模板来扩增 47 个已知或候选基因的集合。这些扩增的序列被合并到 Can-Seq 文库中并进行深度测序。随后，使用定制的 Snakemake 脚本 (https://github.com/Carroll-Lab/can_seq) 识别已知和候选基因中的突变，然后设计 PCR 接合性测试并用于识别携带每个突变的个体突变体。使用这种方法，我们发现 40 个 rtp 突变体中有 28 个在 47 个已知或候选基因中的一个或多个中携带纯合无义、错义或剪接位点突变。我们进行了互补测试以证明几个候选突变是导致 rtp 缺陷的原因。重要的是，通过排除，Can-Seq 管道还确定了 rtp 突变体，这些突变体在 47 个已知和候选基因中的任何一个中都不携带致病突变，这些突变体代表了系统性 RNAi 所需的未发现基因。结论 Can-Seq 提供了一种准确、经济有效的方法，可将新突变体分类为已知基因和未知基因。与目前用于基因发现的正向遗传筛选的现有遗传和 DNA 测序方法相比，它具有几个优点。将 Can-Seq 与基于图谱的基因克隆结合使用是一种具有成本效益的方法，可用于识别有助于感兴趣性状的全部基因。将新突变体分类为已知基因和未知基因的经济有效的方法。与目前用于基因发现的正向遗传筛选的现有遗传和 DNA 测序方法相比，它具有几个优点。将 Can-Seq 与基于图谱的基因克隆结合使用是一种具有成本效益的方法，可用于识别有助于感兴趣性状的全部基因。将新突变体分类为已知基因和未知基因的经济有效的方法。与目前用于基因发现的正向遗传筛选的现有遗传和 DNA 测序方法相比，它具有几个优点。将 Can-Seq 与基于图谱的基因克隆结合使用是一种具有成本效益的方法，可用于识别有助于感兴趣性状的全部基因。