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Protoplast Fusion Hybrids:  3 Levels of Difference between Mt Types a, b, g, d, e  and Novel Cp-Mt Configurations


Dihaploid clones used for protoplast fusions, and the complete set of tetraploid German potato cultivars were analysed on their cytoplasms using specific PCR primers and 11 homologous mt DNA-probes, and classified as mt types a, b, g, d, and e according to their RFLP patterns. From the 4x cultivars 79 had the typical mt type b of Solanum tuberosum being different to 46 varieties which had the mt-a type and 19 with mt-g. A dendrogram shows their relationships to other Solanum species. The distantly related mt-e was only found in dihaploids particularly in clones deriving from S. phureja and S.andigena. On this website the different actualized mt types are given. According to this mt type classification 4 different fusion combination classes were selected for analysis:
  I: a (+) b,    IIa (+) g,   III: a (+) d   IVa (+) e
On mitochondrial expression level hybrids from an [a (+) e ] fusion could be distinguished by in-organello translation from [a (+) b] hybrids and other dihaploids by an additional translation product of 15 kDa. Differential expression was detected as well on transcription level.
In fusion parents with mt-a and g an additional atp6 reading frame is detectable in substoichiometric amounts by usage of specific PCR primers.The gene differs from the original 211 bp 3´ from the stop codon. Novel RFLP-patterns in 10% of somatic hybrids are due to a high-rate replication of this preexisting parental genome region.
A second characteristic for somatic hybrids was the partial addition of parental mt subgenomes . The main part of them revealed a new organization in their mt genomes at the mt-type characteristic loci rpl5, rps14, cob, rps10, coxI and rpl2, which contains recombination specific repeats homologous to Petunia ssp. and Nicotiana. A schematic model for the formation of novel mitochondrial genomes in somatic hybrids is given.

A thorough characterization of potato cytoplasms in five main Types a, b, g, d, e was a precondition for the detection of correlations between cytoplasm und phenotypic data. For this purpose we have (following plastid and mitochondrial DNA isolation) developed a set of probes and PCR primers usefull for the quick characterization of breeding material. Novel configurations of cytoplasmic types occurred in fusion hybrids in shape of partial or complete additions of parental fragments and even novel fragments . In fusion hybrids the strict assortment of cp and mt types was lacking. By in-organello translation differences in protein expression was shown. Analysis of cytoplasmic types and its deviations made it possible to trace back variant phenotypes to the influence of distinct cytoplasmic components as are cytoplasmic-male sterility and starch content.

Andreas Lössl
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Acknowledgements: The authors are grateful to Mr. A. Barth, Mrs. E. Gerick, Mr. C. Kornbauer and Mrs. Ammerseder for technical assistance. This work was supported by grants of the BMBF under the number 0310768.