Browsing by Subject "Mikrosatelliten Marker"

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Study of the natural resistance towards apple proliferation disease and establishment of an in vitro resistance screening system in view of the development of resistant apple rootstocks
(2007) Bisognin, Claudia; Reustle, Götz
Apple proliferation (AP) is an economically important disease of apple which occurs in all countries of central and southern Europe. All currently grown cultivars and rootstocks are susceptible to the disease and no curative treatments are applicable. AP is caused by a phytoplasma, Candidatus Phytoplasma mali, which is restricted to the phloematic tissue of the plant. Ca. P. mali is naturally spread by psyllid vectors and by root bridges as well as by man through infected planting material. An efficient control of the disease is hampered by these different ways of transmission. The objective of the thesis was therefore to evaluate a strategy for a long-term solution to AP based on natural resistance. This resistance has been detected in the wild apomictic species Malus sieboldii and in first and second generation hybrids of M. sieboldii. Although the obtained progeny turned out to be too vigorous for modern apple culture, a certain number of genotypes remained resistant to AP. While phytoplasmas colonise constantly the roots of infected trees, infections in the susceptible cultivar are eliminated each year during the renewal of the phloem in early spring. A resistance strategy towards AP can therefore be solely based on AP-resistant rootstocks in order to prevent the re-colonisation of the canopy in spring. The first part of the thesis was concentrated on the re-evaluation of the AP resistance in M. sieboldii and its hybrids in a 12-years field trial under natural infection pressure at BBA Dossenheim. The annual data for symptom recording and fruit size were analysed as cumulative disease index and cumulative undersized fruit index, respectively. By the end of the trial the phytoplasma concentration in roots and shoots was analysed by quantitative real-time PCR. The results confirmed previous data that M. sieboldii and its hybrids exhibit resistance towards AP. Infected trees of these genotypes had low concentrations of phytoplasmas in the roots and showed almost no symptoms and no undersized fruits. Contrary, previously as resistant classified genotypes derived from M. sargentii reacted highly susceptible. As individual trees of the resistant genotypes showed an altered behaviour, molecular analyses were performed to verify if the seed propagated, apomictic material used for the trial was really true-to-type. For this analysis, co-dominant microsatellite (SSR) markers were used which were derived from published work. However, for each M. sieboldii genotype suitable, polymorphic markers had to be selected. This analysis revealed that apomixis was not complete and that a varying percentage of progeny of the different genotypes was recombinant due to open pollination. As the resistant M. sieboldii genotypes were too vigorous for modern apple culture, new breedings were carried out with these genotypes in combination with dwarfing rootstock genotypes such as M9. More than 3.000 seedlings have been produced in 17 cross combinations. All seedlings were examined by microsatellite analysis in order to distinguish recombinant from non-recombinant, apomictic progeny. SSR markers were also useful in determining the ploidy level of the parents and their progenies. These results were confirmed by flow cytometric analysis. The recombinant progeny is currently being evaluated in the field for its AP resistance. As the screening of resistance towards AP in the field necessitates several years of observation, an alternative in vitro method was developed. This method is based on in vitro graft-inoculation of the genotype to test. As a prerequisite in vitro cultures of all parental genotypes of the breeding program were established. For each genotype the optimal culture medium was defined. Thus, efficient micropropagation and in vitro rooting protocols were established for the M. sieboldii genotypes which are difficult to root under normal nursery conditions. The established protocols enable a large scale production of these genotypes on a commercial scale. The in vitro resistance screening method allows the evaluation of a given genotype under standardized conditions by using repetitions of micro-grafts. The quality of the grafts, the mortality, the transmission rates of the phytoplasmas as well as the concentration of the phytoplasmas in the inoculated genotypes was determined. The results obtained by qPCR showed that the phytoplasma concentration in resistant genotypes was significantly lower than in susceptible ones. Whereas susceptible genotypes exhibited stunted growth and proliferation symptoms in vitro the resistant genotypes had a phenotype almost comparable to the healthy control. Interestingly, significant differences in phytoplasma concentration could be found between two different Ca. P. mali subtypes used in the experiments. The results demonstrated that the method enables a reliable result 3 months p.i. and can be used to evaluate the virulence of different Ca. P. mali strains.