Validating Genome-Wide Association Candidates Controlling Quantitative Variation in Nodulation

Curtin, Shaun J.; Tiffin, Peter; Guhlin, Joseph; Trujillo, Diana; Burghart, Liana T; Atkins, Paul; Baltes, Nicholas J.; Denny, Roxanne; Voytas, Daniel F.; Stupar, Robert M.; Young, Nevin D.

doi:10.1104/pp.16.01923

Cited by 79 publications

(65 citation statements)

References 54 publications

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“…Furthermore, none of the T 1 plants exhibited the striking pleiotropic phenotypes associated with mutation of the hen1 locus in Arabidopsis and rice (Abe et al ., ; Chen et al ., ). Using an identical reagent platform to introduce mutations to other targeted M. truncatula genes, we have previously observed high mutagenesis rates of 50%–75% in the T 0 generation plants (Curtin et al ., ). It was therefore surprising that only in‐frame mutations were recovered at reduced mutagenesis efficiencies.…”

Section: Discussionmentioning

confidence: 97%

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CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula

Curtin

Xiong

Michno

et al. 2017

Plant Biotechnology Journal

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147

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SummaryProcessing of double‐stranded RNA precursors into small RNAs is an essential regulator of gene expression in plant development and stress response. Small RNA processing requires the combined activity of a functionally diverse group of molecular components. However, in most of the plant species, there are insufficient mutant resources to functionally characterize each encoding gene. Here, mutations in loci encoding protein machinery involved in small RNA processing in soya bean and Medicago truncatula were generated using the CRISPR/Cas9 and TAL‐effector nuclease (TALEN) mutagenesis platforms. An efficient CRISPR/Cas9 reagent was used to create a bi‐allelic double mutant for the two soya bean paralogous Double‐stranded RNA‐binding2 (GmDrb2a and GmDrb2b) genes. These mutations, along with a CRISPR/Cas9‐generated mutation of the M. truncatula Hua enhancer1 (MtHen1) gene, were determined to be germ‐line transmissible. Furthermore, TALENs were used to generate a mutation within the soya bean Dicer‐like2 gene. CRISPR/Cas9 mutagenesis of the soya bean Dicer‐like3 gene and the GmHen1a gene was observed in the T0 generation, but these mutations failed to transmit to the T1 generation. The irregular transmission of induced mutations and the corresponding transgenes was investigated by whole‐genome sequencing to reveal a spectrum of non‐germ‐line‐targeted mutations and multiple transgene insertion events. Finally, a suite of combinatorial mutant plants were generated by combining the previously reported Gmdcl1a, Gmdcl1b and Gmdcl4b mutants with the Gmdrb2ab double mutant. Altogether, this study demonstrates the synergistic use of different genome engineering platforms to generate a collection of useful mutant plant lines for future study of small RNA processing in legume crops.

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Section: Discussionmentioning

confidence: 97%

“…Guide RNAs for each target were engineered using a PNK oligo annealing assay and cloned into three binary vectors using Gateway™ cloning reactions. A detailed assembly method has been previously published (Curtin et al ., ).…”

Section: Methodsmentioning

confidence: 97%

CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula

Curtin

Xiong

Michno

et al. 2017

Plant Biotechnology Journal

Self Cite

147

View full text Add to dashboard Cite

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“…(). GWAS have also been used to probe the genetic basis of plant mutualisms with microbes, such as the relationships between legumes and their nodule‐forming bacteria (Stanton‐Geddes et al ., ; Curtin et al ., ).…”

Section: Previous Workmentioning

confidence: 97%

Genome‐wide association studies on the phyllosphere microbiome: Embracing complexity in host–microbe interactions

et al. 2019

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Summary Environmental sequencing shows that plants harbor complex communities of microbes that vary across environments. However, many approaches for mapping plant genetic variation to microbe‐related traits were developed in the relatively simple context of binary host–microbe interactions under controlled conditions. Recent advances in sequencing and statistics make genome‐wide association studies (GWAS) an increasingly promising approach for identifying the plant genetic variation associated with microbes in a community context. This review discusses early efforts on GWAS of the plant phyllosphere microbiome and the outlook for future studies based on human microbiome GWAS. A workflow for GWAS of the phyllosphere microbiome is then presented, with particular attention to how perspectives on the mechanisms, evolution and environmental dependence of plant–microbe interactions will influence the choice of traits to be mapped.

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“…genes identified by a genome-wide association study to be associated with symbiotic nitrogen fixation (Curtin et al, 2017). The gRNAs were assembled (in the order from 1 to 6) into the direct cloning T-DNA vectors that use either Csy4 or tRNA processing enzymes (see Supplemental Methods, Protocols 3A and 3B), and after transformation, DNA from randomly selected calli was tested by PCR for potential chromosomal deletions.…”

Section: Targeted Deletion Of 58 Kb In M Truncatula Using the Trna Amentioning

confidence: 99%

A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants

et al. 2017

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We report a comprehensive toolkit that enables targeted, specific modification of monocot and dicot genomes using a variety of genome engineering approaches. Our reagents, based on transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, are systematized for fast, modular cloning and accommodate diverse regulatory sequences to drive reagent expression. Vectors are optimized to create either single or multiple gene knockouts and large chromosomal deletions. Moreover, integration of geminivirus-based vectors enables precise gene editing through homologous recombination. Regulation of transcription is also possible. A Web-based tool streamlines vector selection and construction. One advantage of our platform is the use of the Csy-type (CRISPR system yersinia) ribonuclease 4 (Csy4) and tRNA processing enzymes to simultaneously express multiple guide RNAs (gRNAs). For example, we demonstrate targeted deletions in up to six genes by expressing 12 gRNAs from a single transcript. Csy4 and tRNA expression systems are almost twice as effective in inducing mutations as gRNAs expressed from individual RNA polymerase III promoters. Mutagenesis can be further enhanced 2.5-fold by incorporating the Trex2 exonuclease. Finally, we demonstrate that Cas9 nickases induce gene targeting at frequencies comparable to native Cas9 when they are delivered on geminivirus replicons. The reagents have been successfully validated in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), Medicago truncatula, wheat (Triticum aestivum), and barley (Hordeum vulgare).

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Validating Genome-Wide Association Candidates Controlling Quantitative Variation in Nodulation

Cited by 79 publications

References 54 publications

CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula

CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula

Genome‐wide association studies on the phyllosphere microbiome: Embracing complexity in host–microbe interactions

A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants

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