Plant Materials: Arabidopsis Polyploids

Arabidopsis thaliana has become the system of choice for many basic studies in plant biology due to its small genome and its amenability to genetic and molecular studies (see http://www.arabidopsis.org/home.html ). It is closely related to a number of crucifer species and has undergone allopolyploid hybridization with at least one of these relatives. Arabidopsis thaliana is the maternal parent of the allotetraploid A. suecica , a selfing species native to northern Europe. The 26 chromosomes of A. suecica (2n=26, set a'a'c'c') comprise two sets: 10 from the diploid selfing species A. thaliana (2n=2x=10, set aa) and 16 from the tetraploid outcrossing species Cardaminopsis arenosa (2n=4x=32, set cccc). The parental origin of the A. suecica chromosomes was demonstrated by DNA sequence analyses and by in situ hybridization to the repeat sequences of either A. thaliana or C. arenosa , which paint 10 and 13 centromeres, respectively (Kamm et al., 1995, Plant Mol Biol 27, 853-62; O'Kane et al, 1996, Sys Bot 21:559-566; Price et al., 1994, In: Arabidopsis, Meyerowitz and Somerville, eds). Although very closely related in some aspects, these two taxa nonetheless exhibit 5-8% nucleotide sequence divergence in protein-coding genes and 30-40% divergence in the 180 bp centromeric repeats.

We have produced synthetic lineages of allotetraploids by crossing an autotetraploid A. thaliana to the autotetraploid C. arenosa . The resulting F1s are allotetraploids, having two sets of each parental genome as determined by in situ hybridization. F1s and following generations are self-compatible (even though the parent C. arenosa is not), but vary in fertility and viability. Advanced inbreeding generations of four lineages have been produced.

	PDF file of image Schematic karyotypes of parents, F1 hybrid and backcrosses. The five Arabidopsis thaliana chromosomes are shown in red. The eight Cardaminopsis arenosa chromosomes are shown in green.
	PDF file of image Lineages of allotetraploids. The presumed cytoplasmic origin is indicated in the first letter. Lower case letters define the nuclear genomes. The straight arrows. Allopolyploids self spontaneously (or can be manually selfed) and progressive inbreeding is expected in advanced generation. Empty boxes below a box with number correspond to generations for which seed is usually available.
C. arenosa and A. thaliana	Allotetraploid F1 plant 49-2B
	The synthetic allotetraploids have the expected chromosome complement. Fluorescent In Situ Hybridization (FISH) of species specific centromeric repeats to a cytological preparation of metaphase I chromosomes from the F4 allotetraploid 745. The five pairs of A. thaliana chromosomes are evident. Some of the eight C. arenosa pairs are overlapping, but could be distinguished in other preparations (data not shown). The bottom panels display single hybridization color channels. Red: A. thaliana CEN; Green: C. arenosa CEN; Blue: DAPI stained DNA

We have demonstrated the occurrence of gene silencing in synthetic (Comai etal., 2000, Plant Cell 12: 1551-68) and natural Arabidopsis allotetraploids (Lee and Chen, 2001, PNAS 98:6753-8). Silencing affected both repeats and single copy genes and was correlated to DNA methylation changes. We are now using multiple approaches to define the genetic and epigenetic basis of this phenomenon (see Approaches).

We are producing autopolyploids of several Arabidopsis ecotypes to study the effect of genome dosage (diploid, triploid, and tetraploid) on gene regulation. The approach used for generation of autopolyploids is detailed in the Figure below.

PDF file of image

Derivation of matched autotetraploids, autotriploids and diploids of A. thaliana Schematic drawing of colchicine induction of polyploids displaying the C0, C1, and C2 generations. Note that the production efficiency of autotetraploids by colchicine is not 100%. Therefore, matched tetraploids and diploids can be identified in the C2 generation and maintained as lines. Each line in a ploidy class is derived from a different C1 parent.

Arabidopsis lines are available through the Arabidopsis Biological Resource Center .