Transposon Mutagenesis and Analysis of Mutants in UniformMu Maize (Zea mays)

Peng Liu1, Donald R. McCarty1, Karen E. Koch1

1 Horticultural Sciences Department and Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, Florida
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20029
Online Posting Date:  September, 2016
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Abstract

A wealth of maize mutants is now available with known sites of transposon insertions in over 45% of maize genes. Materials can be obtained free of charge from the UniformMu public resource through MaizeGDB.org or directly from the Maize Genetics COOP Stock Center. Specific mutants can be sought online based on gene‐sequences of interest. A key feature of the UniformMu resource is the uniformity of wild‐type controls, which facilitates characterization of mutant phenotypes. Methods developed for construction (transposon mutagenesis), analysis and utilization of the resource are described here. These include the high‐throughput Mu‐seq genotyping protocol that enables both forward and reverse approaches for linking genotypes‐to‐phenotypes. © 2016 by John Wiley & Sons, Inc.

Keywords: maize; Mu‐seq; transposon; UniformMu; W22

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Access and Growth of UniformMu Lines
  • Basic Protocol 2: PCR Analysis of UniformMu Lines for Transpose‐Genotyping of Individual Families
  • Basic Protocol 3: Analysis of UniformMu Lines: Using Mu‐Seq for High‐Throughput Transposon Genotyping
  • Basic Protocol 4: Construction of a Custom Population for Transposon Mutagenesis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Access and Growth of UniformMu Lines

  Materials
  • Online access to MaizeGDB.org
  • Locus names or sequence information for target genes
  • Seeds from selected UniformMu lines
  • Field and/or greenhouse space for growing maize: The greenhouse should ideally be able to provide 14 hr of light at or above 700 µmol m−2 s−1 with an average temperature of 28°C during the day and 21°C during the night

Basic Protocol 2: PCR Analysis of UniformMu Lines for Transpose‐Genotyping of Individual Families

  Materials
  • Primers: Mu‐Terminal Inverted Repeat (TIR) and two sets of gene‐specific primers
  • PowerPlant Pro DNA Isolation kit (QIAGEN, cat. no. 13400−50)
  • Wild‐type W22 DNA
  • UFMu plants
  • Dimethyl sulfoxide (DMSO), optional
  • 1% Agarose gel
  • TAE buffer
  • Thermo Scientific MassRuler DNA Ladder Mix (Thermo Scientific, cat. no. SM0403)
  • GoTaq DNA Polymerase (Promega, cat. no. M3008)
  • Wide Mini‐Sub Cell GT Cell (Bio‐Rad)
  • Thermal cycler
  • Gibco BRL Life Technologies model 250 Power Supply
  • UV light

Basic Protocol 3: Analysis of UniformMu Lines: Using Mu‐Seq for High‐Throughput Transposon Genotyping

  Materials
  • UFMu stock line
  • Liquid nitrogen
  • 70% (v/v) ethanol
  • Extraction buffer (see recipe)
  • Phenol (Fisher, cat. no. 116885)
  • Chloroform solution (24 part chloroform; Fisher cat. no. 066906), 1 part 3‐methylbutanol (Sigma, cat. no. 19392)
  • 3 M NaOAc (pH 5.2)
  • Isopropanol
  • TE (10 mM Tris·Cl, 1 mM EDTA, pH 8.0)
  • Ribonuclease A solution (1 mg/ml; Thermo Scientific, cat. no. AB‐0549)
  • Ice
  • 100% (v/v) Ethanol
  • 5 M NH 4Ac.
  • Agarose gels (e.g., 1.2% agarose; Invitrogen Ultrapure)
  • TAE buffer
  • DNA ladder
  • 3 M Na acetate
  • Elution buffer (EB; Qiagen)
  • Agencourt Ampure XP Magnetic Beads (Beckman Coulter)
  • Roche 454 FLX Titanium General Library Preparation kit (Roche)
  • NEB Quick Blunting kit
  • Zymo Research Zymoclean‐5 DNA Clean and Concentrator kit (Zymo Research)
  • 50 mM NaCl
  • Roche Rapid DNA Ligation kit (Roche Applied Science)
  • Zymo Research Zymoclean Gel DNA Recovery kit (Zymo Research)
  • phiX DNA (Illumina)
  • CoorsTek Ceramic mortar (cat. no. 60322) pestle (cat. no. 603203), 400‐ml capacity
  • 50‐ml Phase‐Lock gel tubes (Eppendorf, cat. no. 06‐443‐18)
  • Thermo Forma swinging‐bucket centrifuge (model 5530 1LGP)
  • 37°C incubator
  • TPX tube (Diagenode)
  • Biorupter UCD200 instrument (Diagenode)
  • BioRad SmartSpec 3000 or NanoDrop 1000 instrument
  • Zymo Research Zymoclean‐25 DNA Clean and Concentrator column (Zymo Research)
  • TapeStation D1000 system
  • NexSeq 500 instrument
  • Standard alignment programs (e.g., BLASTN, BOWTIE or BWA)

Basic Protocol 4: Construction of a Custom Population for Transposon Mutagenesis

  Materials
  • Field
  • Green house
  • UF W22 inbred (Maize Genetics Cooperation Stock Center, cat. no. X17EA)
  • UF bz1‐mum9 (MuDR+) (Maize Genetics Cooperation Stock Center, cat. no. 919JA)
  • 70% ethanol
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Figures

Videos

Literature Cited

Literature Cited
  Bray, R.A. and Brink, R.A. 1966. Mutation and Paramutation at the R Locus in Maize. Genetics 54:137‐149.
  Dooner, H.K. and Nelson, O.E. 1979. Interaction among C , R and Vp in the control of the Bz glucosyltransferase during endosperm development in maize. Genetics 91:309‐315.
  Hunter, C.T., Suzuki, M., Saunders, J., Wu, S., Tasi, A., McCarty, D.R., and Koch, K.E. 2014 Phenotype to genotype using forward‐genetic Mu‐seq for identification and functional classification of maize mutants. Front. Plant. Sci. 4:545. doi: 10.3389/fpls.2013.00545.
  Li, Y., Segal, G., Wang, Q., and Dooner, H.K. 2013. Gene tagging with engineered Ds elements in maize. Methods Mol. Biol. 1057:83‐99. doi: 10.1007/978‐1‐62703‐568‐2_6.
  May, B.P., Liu, H., Vollbrecht, E., Senior, L., Rabinowicz, P.D., Roh, D., Pan, X.K., Stein, L., Freeling, M., Alexander, D., and Martienssen, R. 2003. Maize‐targeted mutagenesis: A knockout resource for maize. Proc. Natl. Acad. Sci. U.S.A. 100:11541‐11546. doi: 10.1073/pnas.1831119100.
  McCarty, D.R., Suzuki, M., Hunter, C., and Koch, K.E. 2013a. Genetic and molecular analysis of UniformMu transposon insertion lines. Methods Mol. Biol. 1057:157‐166. doi: 10.1007/978‐1‐62703‐568‐2_11.
  McCarty, D.R., Latshaw, S., Wu, S., Suzuki, M., Hunter, C.T., Avigne, W.T., and Koch, K.E. 2013b. Mu‐seq: Sequence‐based mapping and identification of transposon induced mutations. PLoS One 8:e77172. doi: 10.1371/journal.pone.0077172.
  McCarty, D.R., Settles, A.M., Suzuki, M., Tan, B.C., Latshaw, S., Porch, T., Robin, K., Baier, J., Avigne, W., Lai, J, Messing, J. Koch, K.E., and Hannah, L.C. 2005. Steady‐state transposon mutagenesis in inbred maize. Plant J. 44:52‐61. doi: 10.1111/j.1365‐313X.2005.02509.x.
  McClintock, B. 1950. The origin and behavior of mutable loci in maize. Proc. Natl. Acad. Sci. U.S.A. 36:344‐355. doi: 10.1073/pnas.36.6.344.
  Neuffer, M.G. 1993. Growing maize for genetics studies. In The Maize Handbook (M. Freeling and V. Talbot, eds.) pp. 197‐208. Springer, New York.
  Schnable, P.S. et al. 2009. The B73 maize genome: Complexity, diversity, and dynamics. Science 326:1112‐1115. doi: 10.1126/science.1178534.
  Vollbrecht, E., Duvick, J., Schares, J.P., Ahern, K.R., Deewatthanawong, P., Xu, L., Conrad, L.J., Kikuchi, K., Kubinec, T.A., Hall, B.D., Weeks, R., Unger‐Wallace, E., Muszynksi, M., Brendel, V.P., and Brutnell, T.P. 2010. Genome‐wide distribution of transposed dissociation elements in maize. Plant Cell 22:1667‐1685. doi: 10.1105/tpc.109.073452.
  Walbot, V. and Qüesta, J. 2012. Using MuDR/Mu transposons in directed tagging strategies. Methods Mol. Biol. 1057:143‐155. doi: 10.1007/978‐1‐62703‐568‐2_10.
  Williams‐Carrier, R., Stiffler, N., Belcher, S., Kroeger, T., Stern, D.B., Monde, R.A., Coalter, R., and Barkan, A. 2010. Use of Illumina sequencing to identify transposon insertions underlying mutant phenotypes in high‐copy Mutator lines of maize. Plant J. 63:167‐177.doi: 10.1111/j.1365‐313X.2010.04231.x.
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