Can-Seq: a PCR and DNA sequencing strategy for identifying new alleles of known and candidate genes

Abstract

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 ://githu b.com/Carro ll-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.

RNA-Based Control of Fungal Pathogens in Plants

“…This is evident in the extensive gene duplication events that have occurred during the evolution of genes that encode core gene silencing machinery proteins and the sometimes overlapping but often distinct functionality of members of these gene families, as detailed below. For example, the model plant Arabidopsis encodes four DCLs, ten AGOs, and six RDRs, as well as an extensive suite of other related proteins that together aid in the specification of each RNA silencing pathway [33]. Moreover, sometimes the overlapping functions of members of gene families allow cross-talk to occur between interdependent sRNA pathways that are otherwise involved primarily in mediated silencing mechanisms at either the transcriptional or posttranscriptional level.…”