Instability of Plastid DNA in the Nuclear Genome

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Abstract
Functional gene transfer from the plastid (chloroplast) and mitochondrial genomes to the nucleus has been an important driving force in eukaryotic evolution. Non-functional DNA transfer is far more frequent, and the frequency of such transfers from the plastid to the nucleus has been determined experimentally in tobacco using transplastomic lines containing, in their plastid genome, a kanamycin resistance gene (neo) readymade for nuclear expression. Contrary to expectations, non-Mendelian segregation of the kanamycin resistance phenotype is seen in progeny of some lines in which neo has been transferred to the nuclear genome. Here, we provide a detailed analysis of the instability of kanamycin resistance in nine of these lines, and we show that it is due to deletion of neo. Four lines showed instability with variation between progeny derived from different areas of the same plant, suggesting a loss of neo during somatic cell division. One line showed a consistent reduction in the proportion of kanamycin-resistant progeny, suggesting a loss of neo during meiosis, and the remaining four lines were relatively stable. To avoid genomic enlargement, the high frequency of plastid DNA integration into the nuclear genome necessitates a counterbalancing removal process. This is the first demonstration of such loss involving a high proportion of recent nuclear integrants. We propose that insertion, deletion, and rearrangement of plastid sequences in the nuclear genome are important evolutionary processes in the generation of novel nuclear genes. This work is also relevant in the context of transgenic plant research and crop production, because similar processes to those described here may be involved in the loss of plant transgenes. In eukaryotes, mitochondria and plastids are the descendents of once free-living prokaryotic ancestors. Over time, these organelles have donated a great deal of genetic material to the nuclear genome. Although usually non-functional, these DNA transfer events have, over evolutionary time, resulted in a large pool of functional nuclear genes and therefore the process of DNA transfer has been an important driving force in eukaryotic evolution. Previous studies showed that DNA transfer of a specific marker gene (neo) from the plastid to the nucleus occurred in one in every 11,000 to 16,000 male gametes. Because of this high frequency of transfer and the large size of integrants, this process would be expected to result in a cumulative increase in genome size, unless there are counterbalancing deletion events. In this study, we analysed the stability of the neo gene after integration into the nuclear genome. We found that the gene is highly unstable, with deletion often occurring within a single generation. These results indicate that plastid DNA insertion into and removal from the nuclear genome are in dynamic equilibrium, thus providing a mechanism by which the chances of functional DNA insertion are maximised without compromising the nuclear genome as a whole.