Molecular karyotyping and aneuploidy detection in Arabidopsis thaliana using quantitative fluorescent polymerase chain reaction.
Certain cellular processes are sensitive to changes in gene dosage. Aneuploidy is deleterious because of an imbalance of gene dosage on a chromosomal scale. Identification, classification and characterization of aneuploidy are therefore important for molecular, population and medical genetics and for a deeper understanding of the mechanisms underlying dosage sensitivity. Notwithstanding recent progress in genomic technologies, limited means are available for detecting and classifying changes in chromosome dose. The development of an inexpensive and scalable karyotyping method would allow rapid detection and characterization of both simple and complex aneuploid types. In addition to the problem of karyotyping, genomic and molecular genetic studies of aneuploids and polyploids are complicated by multiple heterozygous combinations possible at loci present in more than two copies. Quantitative scoring of allele genotypes would enable large-scale population genetic experiments in polyploids, and permit genetic analyses on bulked populations in diploid species. Here, we demonstrate that quantitative fluorescent-polymerase chain reaction (QF-PCR) can be used to simultaneously genotype and karyotype aneuploid and polyploid Arabidopsis thaliana. Comparison of QF-PCR with flow cytometric determination of nuclear DNA content indicated near perfect agreement between the methods, but complete karyotype resolution was only possible using QF-PCR. A complex karyotype, determined by QF-PCR, was validated by comparative genomic hybridization to microarrays. Finally, we screened the progeny of tetraploid individuals and found that more than 25% were aneuploid and that our artificially induced tetraploid strain produced fewer aneuploid individuals than a tetraploid strain isolated from nature.
Henry
Isabelle M
IM
Department of Biology, University of Washington, Box 355325, Seattle, WA 98195-5325, USA.
Dilkes
Brian P
BP
Comai
Luca
L
D000483:Alleles; D000782:Aneuploidy; D017360:Arabidopsis / Q000235:genetics*; D004247:DNA / Q000032:analysis; D005434:Flow Cytometry; D005838:Genotype; D017404:In Situ Hybridization, Fluorescence; D007621:Karyotyping / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011123:Polyploidy
D004247:DNA