Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Poster Abstract - 4.18

MOLECULAR PROFILING OF GM-MAIZE LINES AS RELATED TO THE ISSUE OF ESSENTIAL DERIVATION

BORGO L., SERANI B., CONTI S.

Department of Agroenviromental Science and Technology, University of Bologna, Via Filippo Re 6-8, 40126 Bologna, Italy

maize, essential derivation, AFLP, somaclonal variation, GMO

Essential derivation is an important issue for the protection of breeders’ rights and for defining appropriate levels of royalties. So far, similarity thresholds for declaring essential derivation have not been clearly defined, also due to the difficulty in (I) correctly estimating genetic distances based on morphological and biochemical markers and (II) establishing precise confidence intervals of such estimates. In alternative to morphological and biochemical markers, DNA-based markers provide a more powerful and precise diagnostic tool to verify the degree of genetic similarity for those categories of genetic materials more likely to be classified under the category of essential derived variety (EDV). Genetically modified (GM) plants represent an interesting case for this type of study, due to the presence of novel sequence variation introduced through the gene/s directly manipulated and, more importantly for the EDV issue, for the presence of new genetic variability due to somaclonal variation arisen during the in vitro phase. The objective of our research, conducted within an EU-funded project (MMEDV) investigating the EDV issue in a number of crop species, has been to verify to what extent somaclonal variation affects the genetic similarity between GM maize and the donor lines. Six sets of T1, T2, T3 and T4 GM-seed, kindly provided by B. Gengenbach (University of Minnesota), were considered for this study. T0 plants were regenerated from calli proliferated from immature embryos explanted from a cross between two sister lines (derived from an A188 x B73 genetic background); calli were genetically engineered using the bar gene as selectable marker introduced with the particle gun. Seed of the two sister lines and the F1 hybrid was provided along with the GM seed. For each one of the transgenic groups, the two parental lines and the F1, DNA was extracted from 12 plants. For the parental lines, DNA was extracted from each plant separately. For the F1, T1, T2, T3 and T4 sets, DNA was extracted from a bulk of leaf tissue of all plants. So far, we have tested 13 AFLP primer combinations (PCs) based on EcoRI (rare cutter) and two isoschizomers (HpaI and MspII) recognizing the same restriction site (CCGG) and showing different sensitivity to cytosine methylation, a major cause of somaclonal variation. In consideration that transposon-based activity is also an important cause of somaclonal variation in maize, nine REMAP PCs were also considered. Cluster analysis was conducted with NTSYS and similarity estimates among genotypes were calculated according to the Dice coefficient. The 13 AFLP PCs revealed 109 polymorphisms. A considerable number of these bands (90 in total) were found polymorphic also between and/or within the control (non-GM) genotypes (parental sister lines and their hybrid), while only 19 were truly polymorphic between the non-GM and the GM-derived materials. Therefore, cluster analysis was conducted using the set with only 19 bands. The overall level of diversity shown with this set of 19 AFLPs was low; in fact, the average similarity level was higher than 0.99. The nine REMAP PCs revealed 10 new polymorphisms between the parental lines and the GM-derived progenies. The results obtained with cluster analysis with the REMAP markers were rather similar to those obtained with AFLPs and indicated a low level (ca. 1%) of genetic diversity. Because the level of genetic diversity in the materials examined herein is low, a large number of bands should be examined in order to draw sound conclusions on the precise level of polymorphism present in GM-derived materials due to somaclonal variation. As to the relevance of this work for the issue of essential derivation, the low level of polymorphism attributable to somaclonal variation can be disregarded in EDV considerations concerning GM maize, particularly when the commercialized GM maize hybrids are obtained using GM lines backcrossed with non-GM lines, thus unaffected by somaclonal variation.