241 NUCLEIC ACID EXTRACTION FROM CORYLUS AVELLANA TISSUES N.V. Bassil, J. Davis, A.N. Azarenko and S.A. Mehlenbacher Dept. of Horticulture Oregon State University 4017 Agricultural & Life Sciences Bldg. Corvallis, OR 97331-7304 USA Keywords : DNA, RNA, PCR, RAPD Abstract A protocol for large-scale DNA extraction from young hazelnut leaves and catkins was developed. It is suitable for daily extractions of 200 to 300 samples and requires 2-5 days to complete. About 97% of the DNA samples obtained are amenable to amplification by the polymerase chain reaction. Six methods were compared for RNA isolation. Good quality RNA was successfully isolated from young hazelnut leaves and styles using the RNeasy Plant Kit (Qiagen) with modifications to the manufacturer's protocol. Yet a CTAB (hexadecyltrimethylammonium bromide) method originally used to isolate RNA from pine needles was superior to all five RNeasy Plant Kit-based protocols. 1. Introduction Extraction of good quality DNA and RNA is essential for conducting various molecular studies. Every spring, we screen 6000-8000 hazelnut seedlings for eastern filbert blight (EFB) resistance using the Random Amplified Polymorphic DNA (RAPD) marker UBC 152 800 (Davis and Mehlenbacher, 1997). Early selection of resistant seedlings is valuable to the breeding program at Oregon State University (OSU) because it reduces costs associated with planting and caring for large numbers of trees susceptible to the disease. Hazelnut DNA has also been used to develop molecular markers for incompatibility alleles (Pomper et al., 1998). The DNA has to be extracted from very young actively growing tissue such as the apical meristem or young leaves of seedlings shortly after germination or of field-planted trees shortly after budbreak. Therefore, DNA extraction is limited to spring and has to be adapted to high throughput. Furthermore, the quality of the DNA should be adequate for amplification by the polymerase chain reaction (PCR) used in marker-assisted selection. A DNA extraction protocol originally used for avocado was previously adapted to identify RAPD markers linked to EFB resistance in hazelnut (Davis, 1998). We made further modifications to accommodate large numbers of samples. Protocols for RNA extraction from hazelnut tissue do not exist in the literature. Hazelnut tissue is rich in polysaccharides and polyphenolics that can hinder RNA isolation. Developing a reliable method for extracting good quality RNA from various hazelnut tissues is a necessary prerequisite for many downstream applications. 2. Materials and Methods 2.1. DNA Extraction The DNA extraction protocol is based on that of Davis et al. (1998) for avocado. Young leaf tissue in the spring is generally used, but the protocol can also be used on catkins in the fall. DNA is extracted over the course of 2-5 days depending on time constraints. Up to 3 new sets of 96 samples can be processed every day. In the morning of day 1, one young leaf is collected from seedlings in the greenhouse or field and Proc. V Int. Congress on Hazelnut Ed. S.A. Mehlenbacher Acta Hort. 556, ISHS 2001