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...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2020-02-01
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| Series: | Plant Methods |
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| Online Access: | https://doi.org/10.1186/s13007-020-0555-0 |
| _version_ | 1818946126172127232 |
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| author | Jiangling Cao Nial R. Gursanscky Stephen J. Fletcher Anne Sawyer Mehershad Wadia Lachlan McKeough Marek Coleman Uwe Dressel Christelle Taochy Neena Mitter Hervé Vaucheret Bernard J. Carroll |
| author_facet | Jiangling Cao Nial R. Gursanscky Stephen J. Fletcher Anne Sawyer Mehershad Wadia Lachlan McKeough Marek Coleman Uwe Dressel Christelle Taochy Neena Mitter Hervé Vaucheret Bernard J. Carroll |
| author_sort | Jiangling Cao |
| collection | DOAJ |
| description | 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://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. |
| first_indexed | 2024-12-20T08:10:03Z |
| format | Article |
| id | doaj.art-89d6dbecf9734a4198a7e8fefd8e0267 |
| institution | Directory Open Access Journal |
| issn | 1746-4811 |
| language | English |
| last_indexed | 2024-12-20T08:10:03Z |
| publishDate | 2020-02-01 |
| publisher | BMC |
| record_format | Article |
| series | Plant Methods |
| spelling | doaj.art-89d6dbecf9734a4198a7e8fefd8e02672022-12-21T19:47:18ZengBMCPlant Methods1746-48112020-02-0116111210.1186/s13007-020-0555-0Can-Seq: a PCR and DNA sequencing strategy for identifying new alleles of known and candidate genesJiangling Cao0Nial R. Gursanscky1Stephen J. Fletcher2Anne Sawyer3Mehershad Wadia4Lachlan McKeough5Marek Coleman6Uwe Dressel7Christelle Taochy8Neena Mitter9Hervé Vaucheret10Bernard J. Carroll11School of Chemistry and Molecular Biosciences, The University of QueenslandSchool of Chemistry and Molecular Biosciences, The University of QueenslandSchool of Chemistry and Molecular Biosciences, The University of QueenslandSchool of Chemistry and Molecular Biosciences, The University of QueenslandSchool of Chemistry and Molecular Biosciences, The University of QueenslandSchool of Chemistry and Molecular Biosciences, The University of QueenslandSchool of Chemistry and Molecular Biosciences, The University of QueenslandSchool of Chemistry and Molecular Biosciences, The University of QueenslandSchool of Chemistry and Molecular Biosciences, The University of QueenslandQueensland Alliance for Agriculture and Food Innovation, The University of QueenslandInstitut Jean-Pierre Bourgin, UMR 1318, INRA AgroParisTech CNRS, Université Paris-SaclaySchool of Chemistry and Molecular Biosciences, The University of QueenslandAbstract 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.https://doi.org/10.1186/s13007-020-0555-0Forward geneticsCandidate gene-Sequencing (Can-Seq)Post-transcriptional gene silencing (PTGS)Root-to-shoot transmission of PTGS (RTP)Map-based gene cloningRNA interference (RNAi) |
| spellingShingle | Jiangling Cao Nial R. Gursanscky Stephen J. Fletcher Anne Sawyer Mehershad Wadia Lachlan McKeough Marek Coleman Uwe Dressel Christelle Taochy Neena Mitter Hervé Vaucheret Bernard J. Carroll Can-Seq: a PCR and DNA sequencing strategy for identifying new alleles of known and candidate genes Plant Methods Forward genetics Candidate gene-Sequencing (Can-Seq) Post-transcriptional gene silencing (PTGS) Root-to-shoot transmission of PTGS (RTP) Map-based gene cloning RNA interference (RNAi) |
| title | Can-Seq: a PCR and DNA sequencing strategy for identifying new alleles of known and candidate genes |
| title_full | Can-Seq: a PCR and DNA sequencing strategy for identifying new alleles of known and candidate genes |
| title_fullStr | Can-Seq: a PCR and DNA sequencing strategy for identifying new alleles of known and candidate genes |
| title_full_unstemmed | Can-Seq: a PCR and DNA sequencing strategy for identifying new alleles of known and candidate genes |
| title_short | Can-Seq: a PCR and DNA sequencing strategy for identifying new alleles of known and candidate genes |
| title_sort | can seq a pcr and dna sequencing strategy for identifying new alleles of known and candidate genes |
| topic | Forward genetics Candidate gene-Sequencing (Can-Seq) Post-transcriptional gene silencing (PTGS) Root-to-shoot transmission of PTGS (RTP) Map-based gene cloning RNA interference (RNAi) |
| url | https://doi.org/10.1186/s13007-020-0555-0 |
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