SUMMARY ZUSAMMENFASSUNG INTRODUCTION IDENTIFICATION OF APPLE CULTIVARS USING RAPD MARKERS DNAMARKERS LINKED TO MALUS FLORIBUNDA

Eleven apple cultivars were differentiated using RAPD markers obtained by PCR. Variability of the technique and of the origin of the DNA extract was analyzed. A set of bands consistent in presence or absence was chosen to create a differentiating band pat¬ tern. A key to differentiate apple cultivars using a commercially available primer is pro¬ posed. Introduction Discrimination of apple cultivars as they are being multiplied and grown is extreme¬ ly important, as correct identification is usually not possible conventionally until fruit are produced [7]. The characterization of cultivars requires a large set of phenotypic data which is often difficult to assess and sometimes variable due to environmental influen¬ ces. The term cultivar means today that all trees with the particular cultivar name are phenotypically equal and originate from the same ancestor by vegetative reproduction. This implies basically the same genome for all trees of a certain cultivar. Although isoenzyme systems have been useful in cultivar identification [7], they are limited by the number of informative markers and give no direct assessment of the po¬ tential variation present in the genome. In addition, certain systems are prone to environ¬ mental or developmental variation. Direct assessment of genetic variation at the DNA level avoids such difficulties. Restriction fragment length polymorphisms (RFLPs) have been used to identify apple clones and seedlings [5, 6], but the technique is laborious and not suited for studies of a large number of samples [10]. Randomly amplified poly¬ morphic DNA (RAPD) markers generated by Polymerase Chain Reaction (PCR) can be used to differentiate between morphologically indistinguishable strains and varieties [8, 9, 3]. DNA profiles based on arbitrarily primed PCR is both time and cost effective [2]. Furthermore, the availability of markers will aid in mapping genes coding for agronomically important characters. Such a molecular aid will increase efficiency and reduce the Published as: B. Koller, A. Lehmann, J. McDermott and C.Gessler. 1993. Identification of apple cultivars using RAPD markers. Theoretical and Applied Genetics 85:901-904. 10 Identification ofapple cultivars time-scale of plant breeding [4, 11]. In this work we tested the reliability of the RAPDPCR as a tool for the identification of apple cultivars. Material and Methods The following apple cultivars were used in this study: Arlet, Cox's Orange Pippin, Fiorina, Gala, Glockenapfel, Golden Delicious, Idared, Ingold, Ontario, Red Delicious and Spartan. DNA isolation: Apple leaves of one tree of each cultivar were frozen immediately in liquid nitrogen and stored at -80 °C. Leave samples were taken from trees grown at Fe¬ deral Research Station Wadenswil and Ingenieurschule Wadenswil, Switzerland. From cultivar Golden Delicious, five samples of leaves were taken from five scions (one sam¬ ple/scion). The Golden Delicious trees were grown on M26 rootstocks. DNA was extrac¬ ted as described by Dellaporta et al. [1] with the following modifications: after RNAse treatment 1 Volume of Chloroform-Isoamylalcohol (24:1) was added, mixed and centrifuged at 6500 x g for 10 min. The upper aqueous phase was transferred to a new tube and 5 M NaCl was added to a final concentration of 0.2 M. Two volumes of cold ethanol (-20 °C) was added, mixed and allowed to stand for 30 min at 4 °C. After centrifugation at 6500 x g, the supernatant was discarded and the DNA pellet was rinsed in 500 pi of 70 % ethanol. After centrifuging, the ethanol was discarded and the pellet air-dried. The DNA was dissolved in 100 ul of TE buffer pH 7.4. Amplification conditions: Amplification reaction volumes were 25 ul containing 10 mM Tris-HCl pH 8.3, 50 mM KC1, 2.5 mM MgCl2, 100 M each of dATP, dCTP, dGTP and TTP (Boehringer), 0.28 uM Primer, 5 ng of genomic DNA and 1 U Taq DNAPoly¬ merase (Boehringer). Amplification was performed in a Perkin Elmer Cetus Gene Amp PCR System 9600 programmed as follows: 2 cycles of 30 sec at 94 °C, 30 sec at 36 °C, 120 sec at 72 °C; 20 cycles of 20 sec at 94 °C, 15 sec at 36 °C, 15 sec at 45 °C, 90 sec at 72 °C; 19 cycles of 20 sec at 94 °C (increased 1 sec/cycle), 15 sec at 36 °C, 15 sec at 45 °C, 120 sec at 72 °C (increased 3 sec/cycle), followed by 10 min at 72 °C. Amplification products were electrophoresed in 1.5 % agarose (Biorad) gels with 1 x TPE (0.09 M Tris-phosphate, 0.002M EDTA) and stained with ethidium bromide (0.5 ug/ml). The following primers were used: Primer P2 5'ACGAGGGACT; E6 5'AA-