Campus Units
Genetics, Development and Cell Biology, Agronomy, Bioinformatics and Computational Biology, Plant Biology
Document Type
Article
Publication Version
Published Version
Publication Date
6-2009
Journal or Book Title
The Plant Journal
Volume
58
Issue
5
First Page
883
Last Page
892
DOI
10.1111/j.1365-313X.2009.03821.x
Abstract
Insertional mutagenesis is a cornerstone of functional genomics. High-copy transposable element systems such as Mutator (Mu) in maize (Zea mays) 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 (thermal asymmetrically interlaced) and amplification of insertion mutagenized sites (AIMS) 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 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 co-segregates 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%.
Rights
Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The content of this document is not copyrighted.
Language
en
File Format
application/pdf
Recommended Citation
Yi, Gibum; Luth, Diane; Goodman, Timothy D.; Lawrence, Carolyn J.; and Becraft, Philip W., "High-throughput linkage analysis of Mutator insertion sites in maize" (2009). Genetics, Development and Cell Biology Publications. 27.
https://lib.dr.iastate.edu/gdcb_las_pubs/27
Included in
Agricultural Science Commons, Agriculture Commons, Agronomy and Crop Sciences Commons, Bioinformatics Commons, Computational Biology Commons, Plant Biology Commons
Comments
This article is from The Plant Journal 58 (2009): 883–892, doi:10.1111/j.1365-313X.2009.03821.x.