Introduction

The maize transposable elements Ac/Ds were the first transposable elements discovered by Barbara McClintock over 50 years ago. Over the years, these elements have been extensively characterized through classical genetic and molecular genetic studies that have 1) elucidated the mechanism of transposition 2) examined the modes of Ac/Ds regulation and 3) explored the utility of Ac/Ds as gene cloning and characterization tools. We have exploited this tremendous foundation to develop a two-component Ac/Ds gene tagging platform for maize.

Through NSF funding, members of the Brutnell and Vollbrecht groups are now developing a series of Ds-containing lines for use in both forward and reverse genetic screens. The goal of the project is to create a collection of approximately 10,000 Ds insertions distributed uniformly throughout the maize genome. DNA flanking each Ds insertion will be cloned and sequenced providing a precise physical location for each insertion. These insertions will be integrated into genomic assemblies and displayed graphically through web links at PlantGDB by members of the Brendel group.

How It Works

Click here for slides outlining the project goals and methods.

Progress Reports

March, 2007

Cloning fDs

To facilitate the cloning of Ds flanking sequences we developed a high-throughput (96-well plate format) method to isolate high-quality DNA from seedling tissues. Using this method we have been able to isolate up to 30 ug of high quality DNA from each seedling prep. This is sufficient for all subsequent DNA blot and IPCR amplification protocols. Thus, we are now screening each family individually in DNA blot analysis. To identify trDs elements and enrich for Ds insertions in the hypomethylated, low copy regions of the maize genome, DNA is digested with a methylation sensitive restriction enzyme prior to DNA blot analysis. We estimate that we can detect nearly 90% of the trDs elements using the two restriction enzymes, PvuII and SacII. As of Jan. 31, 2007, we have isolated DNA and performed DNA blot analysis using PvuII and SacII on 3509 families and identified approximately 900 Ds-containing fragments that are of a suitable size for amplifying by IPCR. We have amplified 664 fragments representing germinal Ds insertions. In December 2006 and January 2007 325 fragments were amplified using IPCR by the Brutnell group and were sent to the Vollbrecht lab for sequencing. We anticipate that from this point forward 200-250 fDs sequences/month will be amplified by the Brutnell lab.

Sequence analysis of fDs

Over the past year, we have developed scripts, software and protocols in anticipation of a large number of fDs sequences. Sequence tracefiles from each shipment of fragments are first batch processed by scripts and software that renames tracefiles according to their parent maize lines, removes low quality, vector and Ds sequences, and assembles paired reads into contigs. These cleaned assemblies are then inspected manually for template content and where appropriate (i.e., in most cases) for agreement with the size expectations from DNA blot analysis and IPCR steps carried out at BTI, an important quality control measure. The bioinformatics pipeline then generates and exports fasta files for each shipment, which are compared by BLAST against our cumulative fDs database before we append the new, unique sequences to the database. When the cumulative database consisted of 387 fDs in mid-February, the "unique-only" database consisted of 372 fDs sequences (96%) that were isolated only once, while nine were isolated twice (2-copy), three were 3-copy, two were 4-copy, and one fDs was 8-copy. This analysis indicates that insertion hotspots or cryptic or other preexisting Ds elements are rare. The current dataset of 469 fDs assemblies will be deposited to the GSS section of GenBank in March 2007.

Database development

As a means to visualize fDs:BAC matches in PlantGDB, we have developed a new annotation track for the ZmGDB genome browser that shows the match between fDs and BAC sequence in a genome context view. The new track will be implemented when we release our next iteration of BAC annotation, scheduled for March 2007.

February 1, 2006

Our work to date has focused on developing the genetic materials necessary to create large populations of transposed Ds (tDs) insertions and on improving molecular techniques to identify and clone tDs flanking sequences. We have also generated a small population of approximately 1000 families, each carrying a unique tDs insertion generated from excision of Ds at the r-sc:m3 locus. We are now screening this population to identify tDs insertions that are unlinked to the r locus. Thus far, we have screened approximately 400 families and have identified 79 tDs insertions that segregate independently from the r locus. Over the next 6 months, we will attempt to clone DNA flanking these insertions using an inverse PCR technique and begin integrating flanking sequences into genomic assemblies.

Relevant Publications

Conrad, L.J. and T. P. Brutnell (2005) Ac-immobilized, a stable source of Activator transposase that mediates sporophytic and gametophytic excision of dissociation elements in maize. Genetics 171: 1999-2012. [Full Text]

Activator mutagenesis of the maize genome

Through NSF support, several Ac elements have been distributed throughout the genome. These elements have been positioned relative to molecular markers on the IBM2 framework maps and are available through the Maize Genetics Cooperative Stock Center. DNA flanking some of these Ac lines have also been integrated into GSS assemblies and can be searched below.

Genome-wide mutagenesis of maize with Dissociation

Sequence matches to maize GSS contigs have been identified for several Ds flanking sequences - please follow the links below to identify sequences and their most significantly matching maize GSS contig.