Mishchenko A. Development and use of DNA markers for identification of hazelnut varieties of Ukrainian breeding

European hazelnut (Corylus avellana L.) is an important nut crop with high commercial potential, the industrial production of which is impossible without precise control of variety composition in plantations. Errors in propagation of planting material lead to significant economic losses for producers and breeders. For a long time, the only tool for variety identification was morphological analysis based on UPOV descriptors. However, the considerable phenotypic variability of hazelnut morphological traits under the influence of growing conditions and age-related changes substantially limits its reliability, particularly when identifying closely related genotypes. In modern practice, the solution to this problem is associated with the use of DNA markers, which produce stable results regardless of the growth conditions and developmental state of a plant. Despite advances in molecular identification of hazelnut in global practice, the genetic characterisation and certification of Ukrainian hazelnut varieties had not been carried out until this study, which determined the relevance and practical significance of the work. The dissertation research corresponds to the research plan of the Department of Cell Population Genetics of the Institute of Molecular Biology and Genetics of the NAS of Ukraine and was carried out within the framework of state budget funding projects of the department: " Genetic, physiological, and biochemical mechanisms for plant adaptation to extreme environmental conditions" (state registration number 0120U105249, 2021–2025), "Genomic variability and peculiarities of accumulation of specific biologically active compounds in tissue culture of medicinal plants" (state registration number 0125U002734, 2026–2030), as well as in co-execution of the R&D project "Theoretical fundamentals of hazelnut breeding using molecular genetic markers for identification of species belonging of initial materials and hybrids generated" (state registration number 0120U103133, 2026–2030). Object of the study – Ukrainian-bred varieties of hazelnut and Corylus species. Subject of the study – DNA markers for the characterisation and identification of Ukrainian hazelnut varieties. Aim of the study – to identify DNA markers suitable for genetic analysis of Ukrainian hazelnut varieties, to evaluate their informativeness, and to apply them for genetic characterisation of hazelnut varieties. Research objectives: 1. To optimise the protocol for extraction and purification of DNA from hazelnut leaf tissue that is suitable for PCR analysis. 2. To determine the informativeness of existing SSR markers for identification and differentiation of Ukrainian-bred hazelnut varieties. 3. To characterise the allele composition and analyse the molecular structure of the selected microsatellite loci. 4. To investigate the allelic profiles of Ukrainian-bred hazelnut varieties using the selected SSR markers. 5. To screen for polymorphic iPBS markers suitable for molecular-genetic characterisation of Ukrainian hazelnut varieties. 6. To assess the informativeness of the selected iPBS primers for identification and differentiation of Ukrainian hazelnut varieties. 7. To compare the efficiency of iPBS- and SSR markers for differentiation of Ukrainian-bred hazelnut varieties. Methods: DNA extraction from dried hazelnut leaves using a modified CTAB protocol; multiplex PCR with fluorescently labelled primers and fragment analysis of SSR loci; PCR with iPBS primers and agarose gel electrophoresis; cloning and Sanger sequencing; bioinformatic and statistical methods. Scientific novelty of the results: For the first time, a CTAB-based DNA extraction and purification protocol was optimised to obtain PCR-grade DNA preparations from dried hazelnut leaf specimens. For the first time, the genetic diversity of 25 Ukrainian hazelnut varieties was assessed using 9 SSR loci, and individual allelic profiles were compiled for each variety. The informativeness of the markers used and the minimum marker set sufficient for genotyping of large samples of up to 1,000 specimens were determined. The molecular structure of the microsatellite loci used was examined in Corylus species; the relationship between molecular structure and allele size was established, and species-specific variants of certain microsatellite loci were identified. iPBS markers were applied for hazelnut variety differentiation for the first time. It was demonstrated that one of the selected iPBS primers (2402) is sufficient to differentiate all 25 studied varieties. For the first time, dendrograms of genetic similarity based on SSR- and iPBS markers were constructed for Ukrainian-bred hazelnut varieties.