title: Validating Genome-Wide Association Candidates Controlling Quantitative Variation in Nodulation.
abstract: Genome-wide association (GWA) studies offer the opportunity to identify genes that contribute to naturally occurring variation in quantitative traits. However, GWA relies exclusively on statistical association, so functional validation is necessary to make strong claims about gene function. We used a combination of gene-disruption platforms (Tnt1 retrotransposons, hairpin RNA-interference constructs, and CRISPR/Cas9 nucleases) together with randomized, well-replicated experiments to evaluate the function of genes that an earlier GWA study in Medicago truncatula had identified as candidates contributing to variation in the symbiosis between legumes and rhizobia. We evaluated ten candidate genes found in six clusters of strongly associated single nucleotide polymorphisms, selected on the basis of their strength of statistical association, proximity to annotated gene models, and root or nodule expression. We found statistically significant effects on nodule production for three candidate genes, each validated in two independent mutants. Annotated functions of these three genes suggest their contributions to quantitative variation in nodule production occur through processes not previously connected to nodulation, including phosphorous supply and salicylic acid-related defense response. These results demonstrate the utility of GWA combined with reverse mutagenesis technologies to discover and validate genes contributing to naturally occurring variation in quantitative traits. The results highlight the potential for GWA to complement forward genetics in identifying the genetic basis of ecologically and economically important traits.
authors.affiliation: Department of Plant Pathology (S.J.C., R.D., N.D.Y.) and Department of Plant Biology (P.T., J.G., D.T., L.B., N.D.Y.), University of Minnesota, St. Paul, Minnesota 55108.|Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (P.A., N.J.B., D.F.V.); and.|Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, Minnesota 55108 (R.M.S.).
authors.forename: Shaun J
authors.identifier: 0000-0002-9528-3335
authors.initials: SJ
authors.lastname: Curtin
authors.affiliation: Department of Plant Pathology (S.J.C., R.D., N.D.Y.) and Department of Plant Biology (P.T., J.G., D.T., L.B., N.D.Y.), University of Minnesota, St. Paul, Minnesota 55108.|Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (P.A., N.J.B., D.F.V.); and.|Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, Minnesota 55108 (R.M.S.).
authors.forename: Peter
authors.initials: P
authors.lastname: Tiffin
authors.affiliation: Department of Plant Pathology (S.J.C., R.D., N.D.Y.) and Department of Plant Biology (P.T., J.G., D.T., L.B., N.D.Y.), University of Minnesota, St. Paul, Minnesota 55108.|Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (P.A., N.J.B., D.F.V.); and.|Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, Minnesota 55108 (R.M.S.).
authors.forename: Joseph
authors.initials: J
authors.lastname: Guhlin
authors.affiliation: Department of Plant Pathology (S.J.C., R.D., N.D.Y.) and Department of Plant Biology (P.T., J.G., D.T., L.B., N.D.Y.), University of Minnesota, St. Paul, Minnesota 55108.|Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (P.A., N.J.B., D.F.V.); and.|Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, Minnesota 55108 (R.M.S.).
authors.forename: Diana I
authors.identifier: 0000-0003-2981-1360
authors.initials: DI
authors.lastname: Trujillo
authors.affiliation: Department of Plant Pathology (S.J.C., R.D., N.D.Y.) and Department of Plant Biology (P.T., J.G., D.T., L.B., N.D.Y.), University of Minnesota, St. Paul, Minnesota 55108.|Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (P.A., N.J.B., D.F.V.); and.|Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, Minnesota 55108 (R.M.S.).
authors.forename: Liana T
authors.identifier: 0000-0002-0239-1071
authors.initials: LT
authors.lastname: Burghart
authors.affiliation: Department of Plant Pathology (S.J.C., R.D., N.D.Y.) and Department of Plant Biology (P.T., J.G., D.T., L.B., N.D.Y.), University of Minnesota, St. Paul, Minnesota 55108.|Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (P.A., N.J.B., D.F.V.); and.|Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, Minnesota 55108 (R.M.S.).
authors.forename: Paul
authors.initials: P
authors.lastname: Atkins
authors.affiliation: Department of Plant Pathology (S.J.C., R.D., N.D.Y.) and Department of Plant Biology (P.T., J.G., D.T., L.B., N.D.Y.), University of Minnesota, St. Paul, Minnesota 55108.|Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (P.A., N.J.B., D.F.V.); and.|Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, Minnesota 55108 (R.M.S.).
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Baltes
authors.affiliation: Department of Plant Pathology (S.J.C., R.D., N.D.Y.) and Department of Plant Biology (P.T., J.G., D.T., L.B., N.D.Y.), University of Minnesota, St. Paul, Minnesota 55108.|Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (P.A., N.J.B., D.F.V.); and.|Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, Minnesota 55108 (R.M.S.).
authors.forename: Roxanne
authors.initials: R
authors.lastname: Denny
authors.affiliation: Department of Plant Pathology (S.J.C., R.D., N.D.Y.) and Department of Plant Biology (P.T., J.G., D.T., L.B., N.D.Y.), University of Minnesota, St. Paul, Minnesota 55108.|Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (P.A., N.J.B., D.F.V.); and.|Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, Minnesota 55108 (R.M.S.).
authors.forename: Daniel F
authors.identifier: 0000-0002-4944-1224
authors.initials: DF
authors.lastname: Voytas
authors.affiliation: Department of Plant Pathology (S.J.C., R.D., N.D.Y.) and Department of Plant Biology (P.T., J.G., D.T., L.B., N.D.Y.), University of Minnesota, St. Paul, Minnesota 55108.|Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (P.A., N.J.B., D.F.V.); and.|Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, Minnesota 55108 (R.M.S.).
authors.forename: Robert M
authors.identifier: 0000-0002-8836-2924
authors.initials: RM
authors.lastname: Stupar
authors.affiliation: Department of Plant Pathology (S.J.C., R.D., N.D.Y.) and Department of Plant Biology (P.T., J.G., D.T., L.B., N.D.Y.), University of Minnesota, St. Paul, Minnesota 55108; neviny@umn.edu.|Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (P.A., N.J.B., D.F.V.); and neviny@umn.edu.|Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, Minnesota 55108 (R.M.S.) neviny@umn.edu.
authors.forename: Nevin D
authors.identifier: 0000-0001-6463-4772
authors.initials: ND
authors.lastname: Young
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D002874:Chromosome Mapping; D018506:Gene Expression Regulation, Plant; D055785:Gene Knockdown Techniques; D018745:Genome, Plant; D055106:Genome-Wide Association Study; D046913:Medicago truncatula / Q000235:genetics*; D016296:Mutagenesis / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009584:Nitrogen / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics; D055170:Plant Root Nodulation / Q000235:genetics*; D040641:Quantitative Trait Loci / Q000235:genetics*; D015203:Reproducibility of Results
chemical_list: D010940:Plant Proteins; D009584:Nitrogen