2 Generating Mutants; Forward Genetics 1. Generating your own mutantsEMSGamma/X-raysTransposon Tagging2. Public Collections of MutantsDatabases of EMS-generated mutantsDatabases of (likely) transposon-induced mutantsThe collection at Maize Genetics Cooperation Stock CenterTaken from University Communications Publications
3 EMS Mutagenesis For creating a broad spectrum of new mutants For creating new alleles of an existing mutantFor identifying suppressors and enhancers of a particular mutant phenotypeTaken from University Communications Publications
4 EMS Mutagenesis of Pollen Paraffin oil pollen treatment with EMS-- M.G. Neuffer--”Mutagenesis” in The Maize Handbook (M. Freeling and V. Walbot, eds) 1993Treated pollen is applied to silksF1 (M1) seed is heterozygous for EMS-generated mutationsPlant M1 seed, self each plant, collect and store M2 seedPlant seeds from each M2, observe mutants segregatingA population of 3,000-5,000 M2 families should yield multiple mutants for every locus (successful treatments yield frequencies exceeding 1 recessive mutant per locus per 1000 M2 families analyzed).Taken from University Communications Publications
5 ra1 is a mutation that strongly affects tassel and ear architecture EMS Mutagenesis of ramosa1ra1 is a mutation that strongly affects tassel and ear architectureTwo main purposes for EMS mutagenesis of ra1:To find intragenic mutations in the RA1 locus (new alleles)To find new loci involved in the RA1 pathway
6 Erik Vollbrecht (unpublished) ENHANCER/SUPPRESSOR SCREENING ON A WEAK ra1 ALLELE (ra1-RS)ra1 weak phenotypera1-RS allele is due to a 2bp insertion at the very beginning of the coding sequence and encodes for a slightly shorter proteinErik Vollbrecht (unpublished)2bp insertionra1-RS166 aa9 aa
7 EMS MUTAGENESIS AND SCREENING EMS treated pollenM0M1M2 familiesra1 weak~ 1100 M2 families screened so far for enhanced/suppressed phenotypes(not a complete genome coverage)Identified 16 families segregating new mutations affecting branching:10 show an enhanced branched ear phenotype but no obvious change in tassel phenotype2 show an enhanced branched ear and tassel phenotype3 show a suppressed branched tassel phenotype1 shows an enhanced tassel phenotype, but no effect on ear
9 M2 FAMILIESSEGREGATING FOR ENHANCED EAR AND TASSEL BRANCHING Enhanced branched tassel and ear phenotypes00F20-39200F20-139Are the mutations intragenic or extragenic?Can sequence the ra1 locus and look for new mutations
10 SEQUENCING OF THE RA1 LOCUS FROM NEW MUTANTS -1 kbRA 21RA 50RA 13RA 442bp insertionra1-RS176 aa-2,9 kbRA 100F20-120Sequenced ra1 gene from DNA of segregating mutant plants in M2 familiesHave sequenced ra1 for 12 out of the 16 familiesResultsFound one new allele of ra1!Indeterminate spikelet? Barren inflorescence?00F family has a point mutation in ra1 locus in the coding sequence causing a missense mutation (a G-C transvertion) resulting in an amino acid change from Leu/Phe at position 160)The mutation resides in a known repression domainThe mutation seems to affect ear branching only; no significant enhancement in tassel branching phenotype!00F20-120
11 What was obtained from the EMS mutagenesis of ra1-RS? Potentially, several new loci involved in the branching pathways have been identified; more are likely to be identified with continued screening.One new interesting allele of ra1.Now the hard work beginsAre they allelic to each other?Complementation tests2. Do the enhancers increase branching in standard maize inbreds?Back cross to different inbred lines.3. Are these suppressors/enhancers known loci affecting branching?Mapping and QTL4. What is the developmental defect in the enhanced families?Careful phenotypic analysis by SEM of immature ear and tassel
12 Generating Mutants; Forward Genetics 1. Generating your own mutantsEMSGamma/X-raysTransposon TaggingTaken from University Communications Publications
13 Gamma/X-rays for Mosaic and Clonal Analysis Means of determining whether a gene acts in a cell autonomous or non-autonomous manner.To study the relationship of cell lineages during development of the plant.An example from a study of the control of leaf epidermal cell morphology.Frank, MJ et al Development 130:Taken from University Communications PublicationsHake and Sinha1994 in The Maize Handbook
17 Generating Mutants; Forward Genetics 1. Generating your own mutantsEMSX-raysDirected Transposon TaggingTaken from University Communications Publications
18 Directed Transposon Tagging A classic and (still current) means of gene cloning in maize!SPM, AC and Mutator all proven sources for transposon tagging.Mutator has higher mutation frequency than AC or SPM, so ofteneasier to find Mutator-induced mutations, but high copy numberoften complicates identifying and cloning tagged gene.AC and SPM-tagged alleles are often easier to clone due to lowercopy number in genome.Lets look at an example of transposon tagging with Mutator.Taken from University Communications Publications
19 Directed Transposon Tagging An example---cloning the male sterile silky1 (si1) gene using Mutator.si1 y1/si1 y1 X Si1 Y1/Si1 Y1, Mu-active(white seed/silky tassel) X (Yellow seed/normal tassel)all Yellow seed (y1/Y1)si1 y1/Si1 Y1 (99.99%) Normal Phenotypesi1 y1/si1-mum Y1 (rare mutant 1/10000)X Si1 y1/Si1 y1Taken from University Communications PublicationsYellow seed have tagged si1 allele (Si1 y1/si1-mum Y1)Grow plants from yellow seed and backcross to si1 y1/si1 y1. Plant out seeds, score for silky vs normal tassel phenotype.Prepare DNA from leaves of each type.Perform co-segregation analysis to identify transposon linked to your mutant phenotype.
20 Southern Blot Hybridized with Mu1 Probe (normal tassel)(silky tassel (si1/si1-mum))
21 Generating Mutants; Forward Genetics 1. Generating your own mutantsEMSGamma/X-raysTransposon Tagging2. Public Collections of MutantsDatabases of EMS-generated mutantsDatabases of (likely) transposon-induced mutantsThe collection at MGSCTaken from University Communications Publications
22 Public Collections of Mutants Maize GDB (http://www.maizegdb.org/)(http://www.maizegdb.org/phenotype.php)Has a vast collection of mutants and associated alleles, linked to references containing background information about locus.Maize Inflorescence Architecture Project(http://www.maizegdb.org/ems-phenotype.php)A new project primarily cataloging EMS-generated inflorescence (ear and tassel) mutants but also documenting other adult and seedling mutant phenotypes.Taken from University Communications PublicationsGenerated in defined inbred backgrounds (B73 and A619) and B73/Mo17 hybrid. Shooting for M2 families.
23 Public Collections of Mutants Databases of (likely) transposon-induced mutantsMaize Gene Discovery Project (RescueMu Phenotype Data In MaizeGDB) (http://www.maizegdb.org/rescuemu-phenotype.php) A database of over 31,000 mutants from screens of RescueMu-containing/Mutator lines. Includes seed, seedling, and various stages of adult phenotypes. F2 seeds for any phenotype can be ordered from MGCSC.Functional Genomics of Maize Endosperm(http://www.endosperm.org/) A database of seed mutants from Mutator-containing lines in a W22 inbred background. All mutants are in a Mu-off state. Order seed through web address.Photosynthetic Mutant Library (PML)(http://pml.uoregon.edu)Phenotype data of Mu-induced, non-photosynthetic maize mutants.Taken from University Communications Publications
24 Public Collections of Mutants The collection at the Maize Genetics Cooperation Stock Center(http://w3.aces.uiuc.edu/maize-coop/)A fantastic catalog of well maintained stocks of maize mutants of all types, some dating back to the early 1900s. The source for reference alleles.Linkage groups often available (eg. o2 gl1 v5 ra1 on 7S)Repository for mutant stocks from many genome projects, including EMS mutants from the Maize Inflorescence Architecture Project and Maize Gene Discovery Project.Seed requests can be made on line, and orders are filled within a couple of days!!Taken from University Communications Publications
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