Autor: Juliane Würdig

This thesis studies the development of methods to generate cisgenic apple plants. As classical apple breeding is a time consuming process, the concept of cisgenesis, recently presented, is a promising opportunity to develop resistant apple cultivars in a relatively short time frame. The major objectives of this study are (1) to develop a cisgenic approach via the Clean Vector Technology exploiting a heat inducible Flp/FRT recombinase system, (2) to identify a further apple specific scab resistant gene Rvi15 of GMAL 2473 to be used in transformations, and (3) to utilize the apple specific transcription factor coding gene MYB10 as a potential morphological marker. In the first two parts, the apple cultivars 'Brookfield Baigent', 'Mitchgla', 'Novajo' and 'Pinova' were genetically engineered in a cisgenic approach using the apple own scab resistance gene Rvi6 of the wild apple accession Malus floribunda No. 821. In Advance, three new methods to activate the heat inducible Flp/FRT recombinase system were tested by means of a monitoring vector, and a molecular characterisation of the system followed by analysing the expression of transferred genes. During the new methods, heat induction treatment of leaves/leaf explants was conducted at 42 °C for 4 h at 100 % humidity (method “wet chamber“), on solid media (method “plate surface”) and in liquid media (method “ddH2O”/”MSO”). Two cisgenic apple lines were generated, namely iM879-68 and iM946-193. Cisgenic line iM879-68 of the cultivar 'Pinova' was produced using the method “wet chamber” and cisgenic line iM946-193 of the cultivar 'Brookfield Baigent' was produced using the method “MSO”. Both cisgenic lines showed one T-DNA integration site. The correct excision of the recombinase cassette out of the T-DNA located in the apple genome was confirmed by sequencing. Both cisgenic lines were proofed to be resistant against Venturia inaequalis isolate 104 (race 1) in greenhouse tests and showed Rvi6 expression levels comparable to traditional bred Rvi6 harbouring cultivars. This study reports for the first time about generation of cisgenic apple plants based on a Flp/FRT recombinase system. In the third part, the candidate genes Vr2-A, Vr2-B and Vr2-C at the Rvi15 locus were investigated by PCR in selected scab resistant, respectively scab 13 Abstract susceptible descendants of a mapping population GMAL 2473 × M. ×domestica 'Golden Delicious'. Five investigated seedlings of the mapping population showed a recombination event within the Rvi15 locus. Due to the presence of the three candidate genes in all investigated resistant descendants and the absence of the three candidate genes in all investigated susceptible descendants, none of the candidate genes was identified as the resistance mediating gene. In the fourth part, the MYB10 gene of apple, exhibiting a natural allele (MYB10 (R6)) that causes a visible anthocyanin accumulation in all tissues of apple, was used. The MYB10 (R6) allele has an insertion in the promoter region of the MYB10 gene, responsible for autoinduction of the MYB10 gene expression. The incidence of the MYB10 (R6) allele was studied in genetic resources of the genus Malus. These molecular data were compared to phenotypic data for fruit flesh colour and red pigmentation. A defined promoter region of MYB10 was sequenced for numerous apple accessions and cultivars and revealed three main types of the MYB10 promoter: the most frequent MYB10 (R1) allele, the MYB10 (R6) allele and the previously unknown ~1 kb MYB10 allele. All red fleshed accessions of the Malus genebank collection carried the MYB10 (R6) allele and belong to Malus species native to Asia. A regeneration experiment with MYB10 (R6) transgenic apple lines showed a restricted usability of the MYB10 (R6) allele as a morphological marker for the cultivar 'Pinova'.