15th International Conference on Arabidopsis Research - TAIR

T10-051
Development of a novel reporter to monitor
homoeologous recombination events in Arabidopsis.
Liang Liang Li(1), Martine Jean(1), Samuel Santerre-Ayotte(1), Francois Belzile(1)
1-Department of Plant Sciences, 1243 Marchand Building, Laval University, Quebec, Canada G1K
7P4
Homoeologous recombination involves DNA sequences that are very similar
but not identical. It has been shown in model eukaryotes such as yeast that
sequence divergence can significantly decrease the frequency of recombination
between homoeologous sequences (Datta et al., 1997). In plants, it
has been proposed that the low recombination rates often encountered when
attempting to introgress traits of interest from wild relatives of crop species
are attributable to this sequence divergence. To facilitate the identification
and characterization of genes involved in limiting recombination between
homoeologous sequences, we have developed a novel reporter system based
on the GUS gene. The construct consists of a disrupted GUS gene containing
two copies of a large intron (626bp). Upon recombination between the two
copies of the intron, a functional gene containing a single copy of the intron
is produced. Recombination events are visualized as blue sectors on a white
background following histochemical staining. As introns are non-coding, mutations
can be made in one copy of the intron to study their impact on the recombination
frequency between the two introns. The introduction of as few as
3 mutations (0.5% divergence) decreased the recombination frequency more
than four-fold relative to a homologous construct with two identical copies
of the intron. A maximum of 53 mutations (9% divergence) were engineered
and resulted in a 20-fold decrease in the frequency of recombination. This
reporter system was used to investigate the role of a key component of the
DNA mismatch repair system, MSH2, a gene known to restrict homoeologous
recombination in other model eukaryotes (Hunter et al., 1996). An Arabidopsis
insertional mutant (SALK line 2708; Atmsh2::T-DNA) was crossed to two
reporter lines: one with 0% and the other with 2% sequence divergence. F3
families homozygous for the reporter and for either the MSH2 or msh2 allele
were stained. Loss of MSH2 activity had no significant impact on recombination
between homologous substrates, whereas it led to a 10-fold increase
between homoeologous substrates.
Datta et al., 1997. PNAS 94:9757-62.
Hunter et al., 1996. EMBO J. 15(7):1726-33.
T10 Novel Tools, Techniques and Resources
T10-052
Mapping LUX ARRHYTHMO, a novel myb
transcription factor essential for circadian rhythms,
and other circadian clock mutants by oligonucleotide
array genotyping
Samuel P Hazen(1), Justin O Borevitz(2), Thomas F Schultz(1), Frank G Harmon(1),
Jose L Pruneda-Paz(1), Joseph R Ecker(2), Steve A Kay(1)
1-The Scripps Research Institute, 10550 North Torrey Pines, La Jolla, California 92037 USA
2-Plant Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037 USA
Many components of the circadian clock remain undiscovered. In an effort to
increase throughput for mutant screens, we performed a novel screen that
relies on hypocotyl growth alterations followed by circadian phenotyping as
a tool to isolate circadian mutants. Mutations were then mapped using highdensity
oligonucleotide arrays as a tool to assay several hundred thousand
loci in a single assay. The Affymetrix ATH1 GeneChip can detect approximately
8,000 hybridization differences between the Columbia and Landsberg
erecta accessions at the DNA level. Using bulk segregant analysis in the F2
generation and treating these polymorphisms as genetic markers, we routinely
map mutations down to regions of 6cM or less. This approach assisted
in the identification of a mutation in LUX ARRHYTHMO (LUX), a gene that
encodes a myb family transcription factor essential for circadian rhythms.
Expression of LUX and the core clock component TOC1 are tightly correlated
in constant light, short and long day conditions as well as in multiple mutant
backgrounds. In constant light, lux mutants lack normal rhythmic expression
of TOC1, PRR3, 5, 7, and 9, CCA1, LHY, and ten other clock regulated genes.
However, in constant darkness, lux mutants exhibit normal rhythmic CCR2::
LUC expression suggesting it is involved in mediating light signaling into the
clock. Using this approach, we have also identified several new alleles of
known clock genes such as ELF3 and ELF4. Another method to identify a
mutant locus is by mapping large deletions caused by fast neutrons bombardment.
Without having to make a mapping cross, array genotyping can
identify a deleted locus that is either homozygous or heterozygous. We have
successfully demonstrated this technique with two well-studied deletions:
the 77 kb fkf1 deletion and the 7kb cry2-1 deletion. A new circadian reporter
line (TOC1::LUC) bombarded with fast neutrons has yielded several mutants
showing multiple types of circadian aberrations. Both duplications and deletions
have been detected in these lines. Candidate genes in those regions are
currently under investigation.
15 th <strong>International</strong> <strong>Conference</strong> on Arabidopsis Research 2004 · Berlin