Molecular karyotyping and aneuploidy detection in Arabidopsis thaliana using quantitative fluorescent polymerase chain reaction

Abstract

SummaryCertain 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.

The BOY NAMED SUE Quantitative Trait Locus Confers Increased Meiotic Stability to an Adapted Natural Allopolyploid of Arabidopsis

“…Aneuploidy is very common in polyploids (Ramsey and Schemske, 1998), including in A. thaliana (Henry et al, 2006), and the types and frequency of aneuploidy in a population can be diagnostic of meiotic irregularity and pairing preferences. Different types of irregularities were observed in our populations, such as whole chromosome aneuploidy (Figure 2, top), segmental aneuploidy (Figure 2, second plot), as well as more complex dosage configurations in which select portions of the genomes were present in more than one extra copy or exhibited both increased and decreased copy number within a single chromosome (Figure 2, bottom plot, black arrow).…”

“…To characterize meiotic stability, FISH of developing microspores was performed on each F2 individual, as well as the parental genotypes, as described previously (Henry et al, 2006). Briefly, fluorescent probes specific to the A. thaliana and A. arenosa centromeric repeats were used to distinguish between the two types of chromosomes, allowing us to record chromosome numbers in developing microspores at different stages of meiosis, as well as observations consistent with meiosis instability, such as chromosomal bridges or laggards (Figure 4).…”

Dosage and parent-of-origin effects shaping aneuploid swarms in A. thaliana

“…Aneuploidy is very frequent in plant polyploid populations (Khush, 1973;Ramsey and Schemske, 2002;Henry et al, 2006). This frequency is even higher in areas where diploid and tetraploid populations coexist and can intercross to produce triploid individuals (Ramsey and Schemske, 1998;Henry et al, 2005).…”

“…Microspores obtained from developing flower buds were harvested and used for chromosome counts by FISH (fluorescent in situ hybridization) as described in Henry et al (2006). Briefly, FITC (fluorescein isothiocyanate)-labeled A. thaliana centromeric DNA probes (Comai et al, 2003) were hybridized to slides prepared as follows: The slides were first incubated in 100 mg ml À1 ribonuclease A (cat.…”

“…Furthermore, we showed that the progenies of genotypically different triploids varied in the distribution of GC that was present in the aneuploid swarm, suggesting a genetic basis to the mechanisms shaping them. To facilitate further study, we have developed a PCR-based highthroughput method allowing rapid karyotyping of aneuploid individuals and showed that it could be used to characterize the chromosomal composition of aneuploid populations derived from triploid or tetraploid individuals (Henry et al, 2006). Using this method, we have shown that the progeny of the tetraploid A. thaliana comprised 25% of aneuploid individuals.…”