Identification of the maize gravitropism gene lazy plant1 by a transposon-tagging genome resequencing strategy.
Since their initial discovery, transposons have been widely used as mutagens for forward and reverse genetic screens in a range of organisms. The problems of high copy number and sequence divergence among related transposons have often limited the efficiency at which tagged genes can be identified. A method was developed to identity the locations of Mutator (Mu) transposons in the Zea mays genome using a simple enrichment method combined with genome resequencing to identify transposon junction fragments. The sequencing library was prepared from genomic DNA by digesting with a restriction enzyme that cuts within a perfectly conserved motif of the Mu terminal inverted repeats (TIR). Paired-end reads containing Mu TIR sequences were computationally identified and chromosomal sequences flanking the transposon were mapped to the maize reference genome. This method has been used to identify Mu insertions in a number of alleles and to isolate the previously unidentified lazy plant1 (la1) gene. The la1 gene is required for the negatively gravitropic response of shoots and mutant plants lack the ability to sense gravity. Using bioinformatic and fluorescence microscopy approaches, we show that the la1 gene encodes a cell membrane and nuclear localized protein. Our Mu-Taq method is readily adaptable to identify the genomic locations of any insertion of a known sequence in any organism using any sequencing platform.
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, United States of America.
Thomas P
TP
Howard
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, United States of America.
Andrew P
AP
Hayward
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, United States of America.
Anthony
A
Tordillos
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, United States of America.
Christopher
C
Fragoso
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, United States of America.
Maria A
MA
Moreno
International Center for Tropical Agriculture, Cali, Colombia.
Joe
J
Tohme
Department of Cell and Molecular Biology, University of Rhode Island, Kingston, Rhode Island, United States of America.
Albert P
AP
Kausch
Department of Cell and Molecular Biology, University of Rhode Island, Kingston, Rhode Island, United States of America.
John P
JP
Mottinger
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, United States of America.
Stephen L
SL
Dellaporta
D000483:Alleles; D001483:Base Sequence; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004251:DNA Transposable Elements / Q000235:genetics*; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017343:Genes, Plant / Q000235:genetics*; D018745:Genome, Plant / Q000235:genetics*; D015698:Genomic Library; D006112:Gravitation; D018522:Gravitropism / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D018520:Plant Shoots / Q000235:genetics / Q000502:physiology; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D012689:Sequence Homology, Nucleic Acid; D003313:Zea mays / Q000235:genetics* / Q000502:physiology
D004251:DNA Transposable Elements; D018744:DNA, Plant; D008565:Membrane Proteins; D010940:Plant Proteins; D004262:DNA Restriction Enzymes