High-throughput linkage analysis of Mutator insertion sites in maize

dc.contributor.author Yi, Gibum
dc.contributor.author Luth, Diane
dc.contributor.author Goodman, Timothy
dc.contributor.author Lawrence, Carolyn
dc.contributor.author Becraft, Philip
dc.contributor.department Agronomy
dc.contributor.department Genetics, Development and Cell Biology
dc.contributor.department Bioinformatics and Computational Biology
dc.date 2018-02-17T08:43:30.000
dc.date.accessioned 2020-06-30T04:02:22Z
dc.date.available 2020-06-30T04:02:22Z
dc.date.issued 2009-06-01
dc.description.abstract <p>Insertional mutagenesis is a cornerstone of functional genomics. High-copy transposable element systems such as <em>Mutator</em> (<em>Mu</em>) in maize (<em>Zea mays</em>) 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 <em>Mu</em>-active genetic stocks, but current methods limit the ability to rapidly identify the causal <em>Mu</em> insertions. Here we present a method to rapidly assay <em>Mu</em> 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 <em>Mu</em> primer, which results in the preferential amplification and labeling of <em>Mu</em>-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 <em>Mu</em> insertion sites linked to seed-lethal mutations with a preliminary success rate of nearly 50%.</p>
dc.description.comments <p>This article is from <em>The Plant Journal</em> 58 (2009): 883–892, doi:<a href="http://dx.doi.org/10.1111/j.1365-313X.2009.03821.x" target="_blank">10.1111/j.1365-313X.2009.03821.x</a>.</p>
dc.format.mimetype application/pdf
dc.identifier archive/lib.dr.iastate.edu/gdcb_las_pubs/27/
dc.identifier.articleid 1026
dc.identifier.contextkey 7946614
dc.identifier.s3bucket isulib-bepress-aws-west
dc.identifier.submissionpath gdcb_las_pubs/27
dc.identifier.uri https://dr.lib.iastate.edu/handle/20.500.12876/37937
dc.language.iso en
dc.source.bitstream archive/lib.dr.iastate.edu/gdcb_las_pubs/27/2009_Yi_HighthroughputLinkage.pdf|||Fri Jan 14 23:05:23 UTC 2022
dc.source.uri 10.1111/j.1365-313X.2009.03821.x
dc.subject.disciplines Agricultural Science
dc.subject.disciplines Agriculture
dc.subject.disciplines Agronomy and Crop Sciences
dc.subject.disciplines Bioinformatics
dc.subject.disciplines Computational Biology
dc.subject.disciplines Plant Biology
dc.subject.keywords Transposon
dc.subject.keywords Tagging
dc.subject.keywords Mutant
dc.subject.keywords Transposon display
dc.subject.keywords Gene cloning
dc.subject.keywords Method
dc.title High-throughput linkage analysis of Mutator insertion sites in maize
dc.type article
dc.type.genre article
dspace.entity.type Publication
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relation.isOrgUnitOfPublication 9e603b30-6443-4b8e-aff5-57de4a7e4cb2
relation.isOrgUnitOfPublication c331f825-0643-499a-9eeb-592c7b43b1f5
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