Identification of the Maize Gravitropism Gene lazy plant1 by a Transposon-Tagging Genome Resequencing Strategy

Thomas P Howard,Anthony Tordillos,Stephen L Dellaporta,Christopher Fragoso,Maria A Moreno,John P Mottinger,Andrew P Hayward,Albert P Kausch,Joe Tohme

doi:10.1371/journal.pone.0087053

Abstract

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.

Highlights

The maize Mutator (Mu) is one of the most aggressively mobile transposon families yet characterized in any organism
Given the high read coverage of current instrumentation –200 million or more pairedend reads – these terminal inverted repeats (TIR)-containing fragments could be readily identified by computational methods and mapped to a reference genome
The Mu-Taq method identified three out of four alleles known to be caused by Mu insertions and four out of nine alleles that were potentially caused by insertion of a Mu element

Summary

Introduction

The maize Mutator (Mu) is one of the most aggressively mobile transposon families yet characterized in any organism. Mutator lines were first described as a genetic system that increased the mutation rate by 30-fold [1]. The first mutant allele to be cloned and characterized from a Mutator line contained a 1.4 kb insertion (later named Mu1) in the alcohol dehydrogenase (adh1) gene of maize [2]. When Robertson’s Mu lines were examined by Southern hybridization, plants were shown to possess between 10–70 copies of Mu-related sequences [4]. These early studies suggested that active transposable elements were the genetic basis for the high mutation rates found in Robertson’s Mutator lines

Methods

Results

Conclusion