Can-Seq: a PCR and deep sequencing strategy for identifying new alleles of candidate genes required for systemic RNA silencing in Arabidopsis

JL Cao, NR Gursanscky, SJ Fletcher, C Taochy, M Coleman, L Makeough, U Dressel, N Mitter and BJ Carroll

  1. School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD, 4072, Australia
  2. Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, QLD, 4072, Australia

Forward genetic screens are used to identify the genes required for biological traits. However, new alleles arising in known genes can obscure the full repertoire of genes that are required for a trait. We carried out a forward genetic screen and identified 41 independent Root-to-shoot Transmission of Post-transcriptional gene silencing (rtp) mutants of Arabidopsis. To filter out rtp mutants that carried mutations in known or related genes, we developed a PCR-based sequencing approach called Can-Seq (Candidate gene-Sequencing). Leaves of up to 25 independent rtp mutants were bulked, and extracted DNA was used as template to amplify 47 candidate genes. PCR amplicons were bulked, sequenced and candidate mutations identified. PCR zygosity tests were used to identify the rtp mutant carrying each candidate mutation. 30 of the 41 rtp mutants carried homozygous mutations in one or more candidate genes. Complementation tests confirmed that the candidate mutations were the causative mutation in several rtp mutants. Importantly, 11 rtp mutants did not carry mutations in candidate genes, and therefore represent novel genes required for systemic RNA silencing.