A novel reporter for intrachromosomal homoeologous recombination in Arabidopsis thaliana

Abstract

A reporter system using engineered introns as recombination substrates in the uidA (GUS) gene has been developed and characterized in Arabidopsis thaliana. The non-coding nature of the recombination substrate has allowed us to monitor recombination events between duplicated copies of the intron that are either identical (homologous recombination) or harbour sequence polymorphisms (homoeologous recombination). The effects of substrate length and divergence on the frequency of recombination events were examined. A positive correlation between substrate length and somatic recombination frequency was found as the frequency of recombination increased 183-fold when the recombination substrate was lengthened from 153 to 589 bp. The existence of 11 polymorphisms in a 589-bp recombination substrate (1.9% sequence divergence) led to an almost 10-fold reduction in the frequency of recombination. This result demonstrates that relatively modest levels of sequence divergence can substantially reduce the frequency of recombination in plants. A molecular analysis of recombination products revealed that the recombination junctions are more frequent in the central segment of the recombination substrate.

Parental Age Affects Somatic Mutation Rates in the Progeny of Flowering Plants

“…To study the effect of parental reproductive age on somatic mutation rates in the progeny, we made use of various Arabidopsis transgenic lines carrying mutated or fragmented versions of the uidA reporter gene encoding GUS (Liu and Crawford, 1998;Kovalchuk et al, 2000;Li et al, 2004;Azaiez et al, 2006;Van der Auwera et al, 2008). These mutation reporter lines carrying a nonfunctional uidA gene enabled us to score somatic frameshift mutations, base substitutions, intrachromosomal recombination (ICR), and transpositions events, which led to the formation of a functional uidA gene.…”

“…7). It was shown that ICR rates increase with the length of the recombination intron substrate present in the uidA gene (Li et al, 2004). ICR rates are lower in transgenic line R2L1 carrying a 418-bp intron compared with line R3L30 having a 589-bp intron.…”

“…A recombination event within the identical sequences of the catalase introns generates a functional uidA gene, resulting in GUS activity that can be visualized as blue spots upon histochemical staining (Li et al, 2004).…”

“…Transgenic ICR lines (R2L1 and R3L30) carrying inverted catalase introns in the uidA gene and the frameshift detector line (G10) were a gift from Francois Belzile (Li et al, 2004;Azaiez et al, 2006). The transposition detection line harboring the transposable Tag1 element was a gift from Nigel M. Crawford (Liu and Crawford, 1998).…”

In Planta Somatic Homologous Recombination Assay Revisited: A Successful and Versatile, but Delicate Tool

“…To test whether small heterologies within homologous sequences might influence the efficiency of recombination (homoeologous recombination), GUSbased recombination substrates that contained defined numbers of mismatches within the overlap were set up. The presence of mismatches indeed reduced recombination frequencies (Li et al, 2004;Opperman et al, 2004). It became clear that the sister chromatids play an important role in SHR; therefore, a system was established that permitted the analysis of interchromatid recombination .…”