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

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.

Control of sexuality by the sk1 -encoded UDP-glycosyltransferase of maize

“…A candidate sk1 gene was identified within this interval by the characterization of a second sk1 allele ( sk1-rMu1 ) derived from active Mutator ( Mu ) maize lines ( 15 ). We used Mu -Taq, a genome sequencing approach that enriches for Mu -chromosomal junction fragments ( 16 ). Of the 179 total independent Mu junction fragments identified, 2 mapped within the coding sequence of GRMZM2G021786, a predicted gene located within the sk1 genetic interval, making it a candidate for the sk1 gene (Fig.…”

“…Taq α I libraries were created with genomic DNA extracted from four independent plants homozygous for the sk1-rMu1 allele. The custom adapters used for sk1-rMu1 cloning were of an earlier iteration than those described by Howard et al ( 16 ). One adapter incorporated a 4-bp barcode index, whereas the other was a common adapter (table S2).…”

“…Reactions were incubated at 20°C for 20 min. Excess adapters were removed using Microcon YM-50 columns (Millipore), as described by Howard et al ( 16 ). Duplicate 50-μl PCRs were performed to enrich each sample for sequencing.…”

LAZY Genes Mediate the Effects of Gravity on Auxin Gradients and Plant Architecture

“…The gene affected in a classic maize lazy mutant was recently identified and found to be a homolog of OsLAZY1 (Dong et al, 2013;Howard et al, 2014). Arabidopsis LAZY1 (AtLAZY1) is also required for gravitropism in the inflorescence stem and seedling hypocotyl (Yoshihara et al, 2013).…”

“…LAZY1 was shown to be required for auxin redistribution in response to gravitropic stimulation in rice (Oryza sativa; Godbole et al, 1999;Li et al, 2007;Yoshihara and Iino, 2007) and in maize (Zea mays; Dong et al, 2013). OsLAZY1, ZmLAZY1, and AtLAZY1 localize to the nucleus and at the cell periphery, probably including the plasma membrane (Dong et al, 2013;Yoshihara et al, 2013;Howard et al, 2014;Sasaki and Yamamoto, 2015). The rice Deeper Rooting1 (OsDRO1) is also a LAZY family gene (Yoshihara et al, 2013).…”