Project part funded by NSF award. Click for full details.

Regional Mutagenesis Utilizing Activator (Ac) in Maize

In many model genetic systems such as yeast, Drosophila and mice, homologous recombination can be exploited to target genes for disruption or to precisely engineer them. Unfortunately, this technology is not yet available to study or engineer plant genomes. Instead, plant geneticists rely heavily on insertional mutagenesis to define the roles of plant genes. In maize (corn), transposable elements have had a long history of helping geneticists understand the maize genome. In the 1940s transposable elements were discovered by Barbara McClintock as agents that serve to modify and restructure genomes. Today transposable elements are serving as tools for gene discovery. Through recent industry- and government-sponsored initiatives rapid progress is being made at genome-wide insertional mutagenesis. In our lab we are using the well-characterized transposon Activator (Ac) to clone and characterize genes in the maize genome.

GO DIRECT TO CURRENT AC MAP

Why did we choose the Ac transposon?

What is our strategy for genome-wide insertional mutagenesis?

How many Ac elements have we cloned and mapped?

How have we isolated the flanking sequences of new Ac elements (protocols)?

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Acknowledgements

This project is possible through the generous support of the National Science Foundation Plant Genome Research Program and Monsanto. Individuals who contributed to the development of these lines include:

Brutnell lab: Tom Brutnell (Project Leader), Judy Kolkman (Ac genetics, cloning, sequence analysis, web design and data management), Liza Conrad (Ac genetics, cloning and data management), Paul Lewis (Ac cloning), Kevin Ahern (Ac genetics, cloning and sequence analysis), Phyllis Farmer (cloning, sequence analysis and RFLP mapping), Ruairidh Sawers (Web design and management).

Monsanto: Paul Chomet, Mystic Research, Monsanto Co. (Project Leader), Kristine Hardeman (Ac genetics, sequence analysis and data management), Sara Lebejko (Sequence analysis, SNP mapping), Alessandra Frizzi (Ac cloning, sequence analysis), Brian Hauge, Cereon Genomics, Cambridge, MA (SNP-based mapping).

Why Ac?

The maize germplasm pool displays an incredible degree of phenotypic plasticity. Mutant alleles and, in particular, transposon-induced alleles will often show a great deal of phenotypic variability depending on the genetic background or effects of modifiers that happen to cosegregate with them. In the most severe cases, a mutant phenotype in one genetic background is completely suppressed in another, either due to genetic redundancy or suppression of the transposon insertion.

One way to simplify genetic analysis is to utilize plants from the same genetic background for mutagenesis. Near- isogenic comparisons of mutant and wild-type individuals often provides more insight into gene function than if comparisons are made between distantly related individuals. This is especially true when determining the contribution of a single gene to a multigenic trait, such a flowering time or yield.

The Mutator family of transposable elements is the most active maize transposable element characterized to date and is currently being used in several gene tagging programs in maize. In order to maintain a high level of Mutator activity, these lines are often maintained in heterogeneous genetic backgrounds, complicating detailed phenotypic analyses. Furthermore, the high forward mutation rate associated with Mutator can result in families segregating multiple mutant phenotypes. Thus, although Mutator insertions can be quickly identified, it may take several years before near isogenic comparisons can be made between various Mu-induced alleles.

In this project we will exploit the tendency of Ac to move to closely linked sites in the genome, to provide a tool for regional mutagenesis. The low forward mutation rate associated with Ac will enable near-isogenic comparisons between any mutants recovered in this program. By distributing Ac elements at 10 cM intervals throughout the maize genome, any mapped gene, EST or QTL will serve as a target for Ac mutagenesis. Each Ac will be maintained in an inbred W22 germplasm as a single active element at a well-defined genetic and physical map position. These non-transgenic lines will be freely available to the public through the Maize Genetics Cooperation Stock Center, University of Illinois.

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Strategy

A two-step strategy will be used to distribute Ac throughout the maize genome. In the first step, unlinked transpositions will first be selected from several donor Ac elements. These elements will then be genetically positioned using publicly available recombinant inbred populations. A group of approximately 50 lines each containing an Ac at a well-defined map position will then be used in the second round of selections. In the second step, linked transpositions will be selected and again mapped using recombinant inbred populations. When complete, we hope to have a set of approximately 200 Ac containing lines. Each line will contain a single active Ac element at a well-defined map position. The complete set will provide approximately 10 cM coverage throughout the maize genome.

Step1-Distribute Ac elements throughout genome

Step 2-Generate linked transpositions from mapped donor Ac's

Selecting transpositions

As the copy number of Ac in the genome increases, excisions of autonomous Ac or the non-autonomous Ds element are delayed. This feature of Ac, known as the negative dosage effect, can be exploited to monitor copy number changes of Ac in the genome. In a cross between a plant homozygous for an Ac insertion (Ac/Ac) and a Ds tester line (Ds/Ds), most kernels will inherit a single active Ac from the megaspore gametophyte and a single Ds from the microspore gametophyte resulting in a characteristic coarse spotting pattern. Transpositions that are inherited with the donor Ac element can be readily identified as finely spotted or near colorless kernels (arrow).

Mapping transpositions

In the first round of selections, transpositions are selected from donor Ac elements located on chromosome 1S. Finely spotted kernels (arrow) are selected and crossed to the Ds tester line. We then utilize the negative dosage effect of Ac to determine linkage of the transposed Ac (tr-Ac) to the donor. If the two Ac elements are closely linked in cis, then most kernels will inherit one of the two parental chromosomes resulting in either finely spotted kernels (two AcÕs) or completely colorless kernels (no Ac).If the two Ac elements are unlinked, then approximately half of the kernels will contain a single copy of Ac resulting in a characteristic coarse spotting pattern.

Approximate genetic distance between two linked Ac elements can easily be determined, by simply counting the recombinants (coarse spotted) and dividing by the total number of kernels. Coarsely spotted kernels are selected from unlinked Ac elements (Step 1) or from linked elements (Step 2) and plants genotyped by DNA blot analysis to determine which of the two Ac elements are inherited.

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