Meiotic stability of transgene expression is unaffected by flanking matrix-associated regions

Abstract

DNA repeats are associated with gene instability and silencing phenomena in plants. Therefore, the presence of a direct repeat of matrix-associated region (MAR) DNA, that considerably reduced position effects between independent transformants, may increase (epi)genetic instability. To investigate the influence of such a repeat on the stability of the expression of embedded transgenes, the meiotic stability of transgene expression was assessed in eighteen homozygous 1-locus transgenic tobacco lines carrying the kanamycin resistance (NPTII) and the β-glucuronidase (GUS) gene. Half of the lines carry a 3 kb direct repeat of MAR DNA flanking the transgenes. Large progeny populations, totalling over a million seedlings, were screened for kanamycin resistance with the help of a newly developed high-density seedling screen. Kanamycin-sensitive seedlings were detected in selfed progeny at a frequency of 0.5-5.9×10-4. The frequency became as high as 2×10-2 when embryo development occurred under heat and/or drought stress. In backcrossed progeny only, a joint loss of NPTII and GUS gene expression was observed at an average frequency of 2.9×10-5. In selfed and backcrossed progeny we observed similar frequencies of reversion to kanamycin sensitivity, indicating that epigenetic silencing mechanisms rather than MAR repeat-related homologous recombination underlie the reversal to kanamycin sensitivity. Different lines, hence different areas of the tobacco genome, differed in their genetic stability. No significant differences in reversal frequencies were apparent between lines with or without the MAR elements. The use of the MAR repeat is, therefore, not compromised by any increased (epi)genetic instability.