|Yi, Gibum - IOWA STATE UNIVERSITY|
|Luth, Diane - IOWA STATE UNIVERSITY|
|Goodman, Timothy - IOWA STATE UNIVERSITY|
|Becraft, Philip - IOWA STATE UNIVERSITY|
Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 21, 2008
Publication Date: June 1, 2009
Citation: Yi, G., Luth, D., Goodman, T.D., Lawrence, C.J., Becraft, P.W. 2009. High-throughput Linkage Analysis of Mutator Insertion Sites in Maize. Plant Journal. 58(5):883-892. Interpretive Summary: Mutations are used to understand gene function, which ultimately defines plant traits and productivity; however, it is difficult to determine which gene was mutated when a mutant variety is identified. Here we describe a method to identify the causative mutation affecting gene function within mutant populations and describe how the method could be applied in a high-throughput manner. This should be useful to basic biologists, and will increase the rate at which we can discover agronomically important genes that can be used to improve maize.
Technical Abstract: Insertional mutagenesis is a cornerstone of functional genomics. High copy transposable element systems such as Mutator (Mu) in maize afford the advantage of high forward mutation rates but pose a challenge for identifying the particular element responsible for a given mutation. Several large mutant collections have been generated in Mu-active genetic stocks, but current methods limit the ability to rapidly identify the causal Mu insertions. Here we present a method to rapidly assay Mu insertions that are genetically linked to a mutation of interest. The method combines elements of MuTAIL (Mutator Thermal Asymmetrically InterLaced) and AIMS (Amplification of Insertion Mutagenized Sites) protocols and is applicable to the analysis of single mutants or to high-throughput analyses of mutant collections. Briefly, genomic DNA is digested with a restriction enzyme and adapters are ligated. Polymerase Chain Reaction (PCR) is performed with TAIL cycling parameters, using a fluorescently labeled Mu primer, which results in the preferential amplification and labeling of Mu-containing genomic fragments. Products from a segregating line are analyzed on a capillary sequencer. To recover a fragment of interest, PCR products are cloned and sequenced. Sequences with lengths matching the size of a band that cosegregates with the mutant phenotype represent candidate linked insertion sites, which are then confirmed by PCR. We demonstrate the utility of the method by identifying Mu insertion sites linked to seed-lethal mutations with a preliminary success rate of nearly 50%.