Micro-methods for genotypic screening of hazelnut (Corylus Avellana) seeds to accelerate breeding for seed metabolite improvement

The expression of the taste and aroma of the hazelnut seed is related to the content, amount, and relative proportion of metabolite components in the cotyledons. Therefore, for breeding purposes, it is desirable to detect those compounds at a single seed level and establish the possible association of the content of those compounds with the embryo genotype and with the alleles transmitted by the pollen, i.e. male parent. We used hazelnut full-sib (FS) seed progenies from controlled pollination for efficiency testing of a multi-micro-method procedure comprising: a generalized low reagent-cost and low amount of extracting-tissue protocol for DNA purification and SSR seed genotyping, nuclear magnetic resonance (NMR) detection of analyte spectra for phenotyping metabolite content, and in vitro culture for clonal propagation of the embryo genotype. NMR was extremely efficient to measure the amount of multiple primary and secondary cotyledonary metabolites; SSR-PCR produced neat and polymorphic DNA fragments for the tested primer set and in vitro culture gave each embryo a probability of 0.85 to germinate and to be cloned in 3-4 ramets through seedling micro-cutting propagation. These micro-methods and the results obtained by their application are now available to be integrated into breeding programs for hazelnut fruit metabolite improvement.