Genotyp-Identifizierung und Wechselwirkungen an zwei Populus-Chima?ren

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Two Populus graft chimeras (MA and AI) produced of P. x canadensis ‘Marilandica’ (M), P. maximowizcii x P. trichocarpa ‘Androscoggin’ (A) and P. nigra L. ’Italica’ (I) were used for investigations of leaf and flower development. In MA the exogenous layer (L1) forms the epidermis and is derived from M while inner layers (L2, L3 etc.) descend from A whereas in AI L1 is formed by A while L2, L3 etc. descend from I. The exogenous epidermis of the periclinal chimeras imposes morphological effects such as an extra female sex in some of the MA flowers. The morphological characteristics of different plant tissues of parents and chimera were compared to determine how they were modified or altered by the tissue transplantation and possibly identify co-existing or interacting genotypes in one tissue. RAPD-PCR was tested for its usefulness to amplify polymorphic fingerprints including donor specific DNA fragments. One random 10mer primer (GGAGTGGACA) out of 20 tested revealed the amplification of patterns including donor specific DNA bands using extracts from leaf tissues of the M and A parents that were combined using extract from leaf tissue of the MA chimera. This indicates that the leaves of the MA chimera are formed by tissues of M and A. However, the inherent disadvantage of RAPD-PCR is the reproducibility of PCR product generation. Therefore the discriminative potential of the ITS region located between the rRNA genes was investigated. The application of specific 16S ribosomal DNA (rDNA) primers for amplification and sequencing of PCR products revealed a closely phylogenetic relationship between I, M and A. Consequently the ITS1 and ITS2 of nuclear rDNA between 18S and 25S were used. The amplified fragments were purified, cloned in E. coli and sequenced. Analyses of multiple clones demonstrated extensive paralogy within and between I, M and A ITS operons. For each parent were at least two rDNA operons as well as multiple paralogous sequences within operons identified. The use of PCR and sequence analyses showed that one of the operons encodes a putative expressed (functional) rDNA whereas the second encodes a pseudogen (truncated) in low copy number. We also characterized the ITS regions of I, M and A to gain insights into structure and phylogeny of the Salicacaee family. Based on sequence divergence primers were designed for A and I and used for the identification of A in MA carpels. Schlagwörter: Populus-Pfropfchimären, RAPD-PCR, 16s-rDNA, kernkodierten rDNA (ITS-Regionen)