Table 1.

Genetic alterations and the proposed classification of organisms in the context of GMO regulatory oversight.

	Organism	Description of the alteration	Does it occur in nature?	Likeliness of occurrence	Alteration beyond what does occur naturally by mating and/or natural recombination*	Motivation
1	Any plant	Single point mutation	YES	Very likely	NO	The mutation rate has been estimated in different plants to be on average 1 mutation per genome per generation, meaning that every single plant will contain, at least, one new mutation. See also the Appendix in supplementary material.
2	Any plant	2,000 point mutations	Perhaps only in a nuclear fall-out	Extremely unlikely	YES	This is the example of classical mutagenesis using a radioactive source to induce random mutations. The organisms resulting from this intervention are exempted from the scope of the EU GMO directive on the condition that no recombinant nucleic acid molecules or GMOs have been used.
3	Any plant	Short deletions (a few bp)	YES	Very likely	NO	Such one base pair deletions occur on a regular basis. Mostly after the occurrence of a double-strand break.
4	Any plant	Larger deletions (a few kbp)	YES	It does happen	NO	Larger deletions also do occur, but with a lower frequency. In viruses such deletions occur rather frequently during processes of attenuation.
5	Any plant	Short insertions (a few bp)	YES	Very likely	NO	Small insertions also occur on a regular basis. Similar as with small deletions such insertions may occur, for example after a double-strand break.
6	Maize	50 kb exogenous DNA insertion	YES	Very unlikely	YES	Such size of insertions do occur in very rare occasions, but the larger the insertion the more unlikely they become.
7	Any plant	2 kb exogenous DNA insertion from a non-related source (a transgene)	YES	Very unlikely	YES	Even though there are rare events of horizontal gene transfer, it is very unlikely that such an event occurs. This is also what the legislation truly intended to have covered by the GMO legislation.
8	Any plant	A cisgene flanked by the T-DNA left and right borders	NO	Extremely unlikely	YES?	There are no known examples of such types of alterations occurring in nature. Where T-DNA has been introduced spontaneously, it was with sequences stemming from A.tumefaciens in between.
9	Any plant	An allele swap	YES	It does happen	NO	Allele swaps do occur, especially as a result of recombination during meiosis.
10	Any plant	Cisgene addition	YES	It does happen	NO/YES	The difference with an allele swap is that the cisgene may be present anywhere in the genome, not necessarily on its 'natural' location. It is known however that in plants genes on a regular basis get duplicated, resulting in an extra copy of the gene being inserted at another location in the genome, away from its original ‘natural' location.
11	Any plant	Intragene addition	?	Extremely unlikely	YES	The rearrangement of regulatory sequences does occur in nature and is actually important in evolutionary terms. But it is extremely unlikely to occur within a few generations in the context of plant breeding.
12	Plants that cannot naturally be infected by A. tumefaciens	T-DNA insertion	NO	Extremely unlikely	YES	When a plant cannot naturally be infected by A. tumefaciens, then the insertion of T-DNA cannot occur.
13	Plants that can be naturally infected by A. tumefaciens such as Nicotiana spp., sweet potato and Linaria spp	T-DNA insertion	YES	Unlikely, but it has happened	NO	A. tumefaciens can naturally infect a wide variety of plants, but this usually is a local infection resulting in the formation of crown galls, without the T-DNA being inserted into the reproductive DNA. However, this has happened during evolution of for example sweet potato.
14	Any plant	Genome duplication	YES	It does happen	NO	Genome duplications do spontaneously happen in nature but have even more been exploited by man to strengthen certain characteristics of crops.
15	Any plant	Transposon insertion	YES	It does happen	NO	Transposons mobile genetic elements that account for an important fraction of all plant genomes. Its movement generates mutations that are at the origin of many traits selected by humans to domesticate wild plants of breed new varieties.

^*As proposed in the article organisms should only be considered as GMO subject to specific regulatory oversight when they fulfill two criteria: (1) the alteration must have been achieved by means of a technical intervention that does not occur naturally, and (2) the genetic combination formed must be beyond what does occur naturally by mating and/or natural recombination.