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Kersten B.,Thuenen Institute TI of Forest Genetics | Voss M.M.,Thuenen Institute TI of Forest Genetics | Fladung M.,Thuenen Institute TI of Forest Genetics
Trees - Structure and Function | Year: 2015

Key message: We aim to develop, for the first time, poplar species-specific mitochondrial SNP markers which can be employed as diagnostic tool, e.g., to identify mitochondria belonging to different poplar species used in protoplast fusion experiments and for population genetics approaches. In most higher plant species, the inheritance of chloroplasts and mitochondria is maternal. In contrast to chloroplast DNA, however, the mitochondrial genome, especially for woody plants including poplar, has seldom been the object of molecular biological research. Published studies on mitochondrial DNA variation in poplar have dealt with genetic relationships using RFLP analyses for population genetic purposes, but so far not for species identification. In this study, we for the first time aim to develop poplar species-specific mitochondrial SNP markers which can be employed as a diagnostic tool, e.g., to identify mitochondria belonging to different poplar species used in protoplast fusion experiments. In total, seven mitochondrial regions were PCR amplified, sequenced and screened for SNPs differentiating clones belonging to six different poplar species from three sections. Two regions (Sc438, contig42) revealed to be monomorphic in the six Populus species. For the five other regions, SNPs could be identified that are suitable for distinguishing between Populus species, either by a SNP-related CAPS (cleaved amplified polymorphic site) marker to be employed in robust PCR–RFLPs, or simply on the basis of the sequence to be screened by PCR and subsequent Sanger sequencing. As a proof of concept, the developed mitochondrial SNP markers confirmed results obtained with chloroplast SNP markers in interspecific poplar hybrids, either with respect to the direction of the cross or the reclassification of putative pure lines as hybrid lines. © 2014, Springer-Verlag Berlin Heidelberg.


Fladung M.,Thuenen Institute TI of Forest Genetics | Hoenicka H.,Thuenen Institute TI of Forest Genetics | Raj Ahuja M.,Thuenen Institute TI of Forest Genetics
Transgenic Research | Year: 2013

Stable expression of foreign genes over the entire life span of a plant is important for long-lived organisms such as trees. For transgenic forest trees, very little information is available on long-term transgene expression and genomic stability. Independent transgenic lines obtained directly after transformation are initially screened in respect to T-DNA integration and transgene expression. However, very little consideration has been given to long-term transgene stability in long-lived forest trees. We have investigated possible genome wide changes following T-DNA integration as well as long-term stability of transgene expression in different transgenic lines of hybrid aspen (Populus tremula × Populus tremuloides) that are up to 19 years old. For studies on possible genome wide changes following T-DNA integration, four different independent rolC-transgenic lines were subjected to an extensive AFLP study and compared to the non-transgenic control line. Only minor genomic changes following T-DNA integration could be detected. To study long-term transgene expression, six different independent rolC-transgenic lines produced in 1993 and since that time have been kept continuously under in vitro conditions. In addition, 18 transgenic plants belonging to eight independent rolC-transgenic lines transferred to glasshouse between 1994 and 2004 were chosen to determine the presence and expression of the rolC gene. In all transgenic lines examined, the rolC gene could successfully be amplified by PCR tests. Both, the 19 years old tissue cultures and the up to 18 years old glasshouse-grown trees revealed expression of the rolC transgene, as demonstrated by the rolC-phenotype and/or northern blot experiments confirming long-term transgene expression. © 2013 Springer Science+Business Media Dordrecht.


PubMed | Thuenen Institute TI of Forest Genetics
Type: Journal Article | Journal: Transgenic research | Year: 2013

Stable expression of foreign genes over the entire life span of a plant is important for long-lived organisms such as trees. For transgenic forest trees, very little information is available on long-term transgene expression and genomic stability. Independent transgenic lines obtained directly after transformation are initially screened in respect to T-DNA integration and transgene expression. However, very little consideration has been given to long-term transgene stability in long-lived forest trees. We have investigated possible genome wide changes following T-DNA integration as well as long-term stability of transgene expression in different transgenic lines of hybrid aspen (Populus tremula Populus tremuloides) that are up to 19 years old. For studies on possible genome wide changes following T-DNA integration, four different independent rolC-transgenic lines were subjected to an extensive AFLP study and compared to the non-transgenic control line. Only minor genomic changes following T-DNA integration could be detected. To study long-term transgene expression, six different independent rolC-transgenic lines produced in 1993 and since that time have been kept continuously under in vitro conditions. In addition, 18 transgenic plants belonging to eight independent rolC-transgenic lines transferred to glasshouse between 1994 and 2004 were chosen to determine the presence and expression of the rolC gene. In all transgenic lines examined, the rolC gene could successfully be amplified by PCR tests. Both, the 19 years old tissue cultures and the up to 18 years old glasshouse-grown trees revealed expression of the rolC transgene, as demonstrated by the rolC-phenotype and/or northern blot experiments confirming long-term transgene expression.

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