High-throughput linkage analysis ofMutatorinsertion sites in maize

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Insertional mutagenesis is a cornerstone of functional genomics. High-copy transposable element systems such asMutator(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 inMu-active genetic stocks, but current methods limit the ability to rapidly identify the causalMuinsertions. Here we present a method to rapidly assayMuinsertions 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 labeledMuprimer, which results in the preferential amplification and labeling ofMu-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 identifyingMuinsertion sites linked to seed-lethal mutations with a preliminary success rate of nearly 50%.

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