Somatic and Meiotic Chromosomal Recombination between Inverted Duplications in Transgenic Tobacco Plants.

Abstract

Homologous recombination has been extensively studied in bacteria, yeast, and more recently in animal cells, but little is known about this process in plants. We present here an analysis of meiotic and somatic chromosomal recombination between closely linked inverted duplications located on a single chromosomal region in tobacco. Transgenic tobacco lines were constructed by Agrobacterium transformation with plasmid vectors containing a functional hygromycin phosphotransferase (hyg) selectable marker flanked by a pair of defective neomycin phosphotransferase (neo) genes positioned as inverted repeats. As each neo gene is mutated in a different site, recombination between the two defective genes can be detected following selection for kanamycin-resistant plant cells. The recombination substrates were designed to allow investigation into the nature of molecular events underlying homologous recombination by restriction endonuclease analysis. Chromosomal recombination was studied in mitotically dividing cells (cultured leaf mesophyll cells) and after meiosis (germinated seedlings). Spontaneous somatic recombinants were recovered at frequencies between ~3 x 10-5 to 10-6 events per cell. Low dose y irradiation of somatic cells resulted in a threefold maximum increase in the recovery of recombinants. Recombinants were also detected at low frequency when transgenic T3 seeds were germinated under kanamycin selection. DNA gel blot analyses demonstrated that homologous recombination occurred mainly as gene conversion unassociated with reciprocal exchange, although a variety of other events including gene coconversion were also observed.

The effects of trans modifiers and tandem duplications on meiotic recombination in maize

“…Similar to homologous recombination between single-copy sequences, unequal interhomolog and interchromatid recombination between duplicated sequences ( Figure 2) has been observed in yeast (JACKSON and FINK 1981;JACKSON and FINK 1985;KLEIN 1984;KLEIN and PETES 1981;MOTOVALI-BASHI et al 2004;SZOSTAK and Wu 1980;THOMPSON and STAHL 1999), tobacco (TOVAR and LICHTENSTEIN 1992) and Arabidopsis (ASSAAD and SIGNER 1992;JELESKO et al 1999;MOLINIER et al 2004;OPPERMAN et al 2004). Also similar to equal recombination, unequal interchromatid recombination predominated during mitosis in a study conducted in yeast (JACKSON and FINK 1981), whereas unequal interhomolog recombination was preferred in meiosis (JACKSON and FINK 1985).…”

“…Although several studies in plants (ASSAAD and SIGNER 1992;MOLINIER et al 2004;TOVAR and LICHTENSTEIN 1992) have identified the mechanism by which unequal recombinants were generated (e.g., gene conversion, interchromosomal or intrachromosomal unequal recombination), the recombination breakpoints associated with unequal recombination events have been analyzed in only two related studies. Unequal recombination breakpoints (n=25) were distributed non-randomly in recombinants isolated from hemizygotes (i.e., unequal recombination between sister chromatids) in a synthetic tandemly arrayed cluster of RBCSB genes JELESKO et al 1999).…”

“…Even so, the findings that 1) rates of unequal recombination at Al-b were higher in the presence of a homolog than in its absence (i.e., hemizygotes) and 2) recombinants isolated from marked hétérozygotes most often showed an exchange of flanking phenotypic markers (LAUGHNAN 1949;LAUGHNAN 1952), were interpreted to suggest that the homolog is the preferred recombination template (LAUGHNAN 1952). More recently, molecular characterizations in plants have documented both meiotic interhomolog (ASSAAD and SIGNER 1992;MOLINIER et al 2004;TOVAR and LICHTENSTEIN 1992) and interchromatid (ASSAAD and SIGNER 1992;JELESKO et al 1999;MOLINIER et al 2004;TOVAR and LICHTENSTEIN 1992) unequal recombination events. Meiotic unequal recombination events isolated from synthetic direct repeats in Arabidopsis (MOLINIER et al 2004) and synthetic inverted repeats in tobacco (TOVAR and LICHTENSTEIN 1992) were conducted using both homozygotes (in which recombination can occur either between homo logs or sister chromatids) and hemizygotes (in which recombination can occur only between sister chromatids).…”

“…More recently, molecular characterizations in plants have documented both meiotic interhomolog (ASSAAD and SIGNER 1992;MOLINIER et al 2004;TOVAR and LICHTENSTEIN 1992) and interchromatid (ASSAAD and SIGNER 1992;JELESKO et al 1999;MOLINIER et al 2004;TOVAR and LICHTENSTEIN 1992) unequal recombination events. Meiotic unequal recombination events isolated from synthetic direct repeats in Arabidopsis (MOLINIER et al 2004) and synthetic inverted repeats in tobacco (TOVAR and LICHTENSTEIN 1992) were conducted using both homozygotes (in which recombination can occur either between homo logs or sister chromatids) and hemizygotes (in which recombination can occur only between sister chromatids). Although the recombination template (i.e., homolog vs. sister chromatid) can not be directly determined in homozygotes, the finding that unequal recombination occurred more than two times as frequently in the homozygotes than in hemizygotes has been interpreted to suggest that in plants meiotic unequal interhomolog recombination occurs more often than interchromatid recombination.…”

“…At Al-b, meiotic unequal recombination occurred more frequently when Al-b was paired with a homolog than when it was not (i.e., hemizygotes). Likewise, in Arabidopsis (ASSAAD and SIGNER 1992) and tobacco (TOVAR and LICHTENSTEIN 1992) unequal recombination between synthetic repeats occurred more than two fold more frequently in homozygotes than in hemizygotes. These studies indirectly suggest that the homolog is the preferred unequal recombination template during meiosis.…”