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International audience; Complete Populus genome sequences are available for the nucleus (P. trichocarpa; section Tacamahaca) and for chloroplasts (seven species), but not for mitochondria. Here, we provide the complete genome sequences of the chloroplast and the mitochondrion for the clones P. tremula W52 and P. tremula x P. alba 717-1B4 (section Populus). The organization of the chloroplast genomes of both Populus clones is described. A phylogenetic tree constructed from all available complete chloroplast DNA sequences of Populus was not congruent with the assignment of the related species to different Populus sections. In total, 3,024 variable nucleotide positions were identified among all compared Populus chloroplast DNA sequences. The 5-prime part of the LSC from trnH to atpA showed the highest frequency of variations. The variable positions included 163 positions with SNPs allowing for differentiating the two clones with P. tremula chloroplast genomes (W52, 717-1B4) from the other seven Populus individuals. These potential P. tremula-specific SNPs were displayed as a whole-plastome barcode on the P. tremula W52 chloroplast DNA sequence. Three of these SNPs and one InDel in the trnH-psbA linker were successfully validated by Sanger sequencing in an extended set of Populus individuals. The complete mitochondrial genome sequence of P. tremula is the first in the family of Salicaceae. The mitochondrial genomes of the two clones are 783,442 bp (W52) and 783,513 bp (717-1B4) in size, structurally very similar and organized as single circles. DNA sequence regions with high similarity to the W52 chloroplast sequence account for about 2% of the W52 mitochondrial genome. The mean SNP frequency was found to be nearly six fold higher in the chloroplast than in the mitochondrial genome when comparing 717-1B4 with W52. The availability of the genomic information of all three DNA-containing cell organelles will allow a holistic approach in poplar molecular breeding in the future.

Problems, such as the impact on biodiversity and the potential escapes of the trans-genes into wild populations can be amplified in the case of long-lived forest species. Therefore, studies of possible environmental impacts of transgenic forest plants (poplar) in the forest environment are important. Few studies have been published considering the alteration of the transgenic host plant genome for the possibility that transgenes can also cause pleiotropic, often undesirable alterations in plant metabolism and physiology. Other studies carried out so far, concerned the use of transgenesis to obtain poplar mutants for genomic studies or poplar breeding just for an economic purpose. The screening of the transgenic plants carrying the cry gene is in course, and the evaluation of the copy number as well as expression of the inserted gene is under evaluation. The final aim is to unravel possible pleiotropic metabolic effects in the transgenic trees following cry gene expression in P. alba and P. tremula x P. tremuloides transgenic lines.

Preliminary results of different cry transgenic poplar lines.

Problems, such as the impact on biodiversity and the potential escapes of the trans-genes into wild populations can be amplified in the case of long-lived forest species. Thus, studies of possible environmental impacts of transgenic forest plants (poplar) in the forest environment are important. Although a number of studies have been carried out so far considering the use of transgenesis for genomic studies, only little is known about biosafety issues of transgenic trees foreseen for breeding purposes. As a model transgenic tree system, we have produced Populus alba and P. tremula transgenic lines carrying the cry gene from Bacillus thuringiensis. These transgenic are under screening to unravel possible pleiotropic metabolic effects in the transgenic trees following cry gene expression. It is also not known how the use of cry-transgenic poplars affects composition and activities of soil microbial communities which affect soil health. In addition to foreign DNA, proteins from crytransgenic poplars can be released into the soil via plant residues and their persistence can constitute not only a selective pressure towards the target organisms, but also a possible threat for non-target ones with unpredictable consequences on the food chain sustained by these organisms. The work is carried out on an integrated and multidisciplinary approach (physiology, soil microbiology and biochemistry, genomic, proteomic, and microscopy), involving different scientific groups with different and specific expertise, to assess the mentioned biosafety issues important for forest trees. The screening of the transgenic plants is in course, and the evaluation of the number copy of the inserted gene as well as expression of the inserted gene is under evaluation.

Sine 1989, sprays of commercial preparations of Bacillus thuringiensis Berliner subsp. kurstaki (Btk) (usually a mixture of cells, spores, and parasporal crystals) have been used to defend the cork oak forests in Sardinia (Italy) against the defoliation by gypsy moth (Lymantria dispar L.). Up to now, the efficacy of the treatment rather than the possible effect on the environment has been evaluated. In a previous work we have demonstrated that Btk and its toxin introduced into soils in sprays can persist for long periods (at least 88 months for Btk and at least 28 months for its toxin), and there was no correlation between numbers of Btk and toxin detected in the Calangianus soil. In order to investigate the potential for genetic exchange of Btk with autochthonous bacteria, we have done the screening of 36 soil samples for each area treated with Bacillus thuringiensis subsp. kurstaki (Btk) in 1993, 1997, and 1998. Colonies of autochthonous bacteria, identified as B. mycoides, have been isolated from the soil treated five years after spraying, and the molecular analysis has indicated that they have acquired part of the cry1Ab gene from the released Btk. These results suggest that the gene transfer between introduced Btk and native bacteria can occur under natural field conditions and that the persistence of the Btk favours this genetic exchange.

Transgenic Organisms and their Impact on The Environment