Kor. J. Hort. Sci. Technol. 30(5):549-556, 2012 DOI http://dx.doi.org/10.7235/hort.2012.11058 An Efficient Identification of 68 Apple Cultivars Using a Cultivar Identification Diagram (CID) Strategy and RAPD Markers Wenyan Wang1, Kun Wang2, Fengzhi Liu2, and Jinggui Fang1* 1College of Horticulture, Nanjing Agricultural University, Nanjing 210095, P. R. China 2Research Institute of Pomology, Chinese Academy of Agricultural Sciences, Xingcheng 125100, P. R. China Abstract. The study aimed to establish an efficient tool for cultivar identification and characterization being the first steps of apple introduction and improvement program. We utilized a method to efficiently record DNA molecular fingerprints of plant individuals genotyped by RAPD, which could be used as efficient reference information for quick plant identification. Ten of sixty 11-mer primers were screened to identify the 68 apple genotypes which could be distinguished by a combination of several primers. All cultivars were easily identified by the corresponding primers marked on the cultivar identification diagram (CID). The results indicated that the CID strategy developed and employed in the apple cultivar identification could be vital in the utilization of DNA marker in other plants as well as the development of the apple industry. Additional key words: fruit industry, longer primer, molecular marker, polymorphic band Introduction commercial production are facing an awkward situation. The main reason for this awkward situation is dearth of analysis Apple (Malus domestica) is one of the most economically strategies for DNA fingerprints. The currently popular analysis important woody plants cultivated for its value as a fruit techniques for DNA banding patterns known as cluster analyses crop as well as for its ornamental beauty (Young et al., 2007). cannot be applied efficiently in cultivar or species separation. Most apple cultivars are diploid (2n = 34) while some of This has made practical application of DNA marker in crop the main cultivars are triploid (2n = 3x = 51). China is a and seed identification unfeasible. Various DNA-based markers major producer and supplier of apples to Asia and most have recently been developed and used for studies on genetic growth in the apple trade in Asia is attributed to imports diversity, fingerprinting, and cultivar origins (Cheng and Huang, from China. This represents a very important aspect in the 2009; D’Onofrio et al., 2009; Elidemir and Uzun, 2009; fruit industry, particularly since the sale of fruit trees and Melgarejo et al., 2009; Papp et al., 2010). Randomly amplified planting of orchards represent major investments of time and polymorphic DNA (RAPD) markers used in apple before money. Since cultivar identification and characterization are have proven to be a reliable marker system for genetic the first steps of any fruit or other crop introduction and fingerprinting and also in determining the genetic relationships improvement program, rapid identification and characterization among germplasm collections. RAPD markers have the advantage of these cultivars would provide valuable information for of being simple, able to detect relatively small amounts of introduction and genetic improvement of apple cultivars. genetic variation and do not need prior information on the With the role of DNA fingerprinting as a powerful tool genome. However, it should be pointed out that RAPDs do currently applied widely in forensic science, it can also be not give information about the genome. The technique has equally useful in the genotyping of plants. However, the already been successfully applied to estimate genetic relationships powerful DNA markers available for plant identification have in apricot (Marinello et al., 2002), mulberry (Vijayan, 2004), not made fruit crop variety identification efficient, recordable, grape (Benjak et al., 2005), figs (Sadder and Ateyyeh, 2006), and as a easy task as desired in practice, fruits and other litchi (Ding et al., 2000), cherry (Demirsoy et al., 2008), crop scientists owning to the potential of this technique in Indian cashew (Archak et al., 2003), longan (Yonemoto et *Corresponding author: [email protected] ※ Received 21 June 2011; Revised 3 July 2012; Accepted 12 July 2012. We are thankful for funding support from the NCET Program of China (grant no. NCET-08-0796) and the Fundamental Research Funds for the Central Universities (No. KYJ200909). Kor. J. Hort. Sci. Technol. 30(5), October 2012 549 al., 2006), olive (Belaj et al., 2003) and pear (Stark-Urnau, information for 68 apple cultivars identification. The CID 2002b; Lee et al., 2004). Among many markers available, shows the separation of 68 selected apple cultivars, based RAPD markers are useful for cultivar analysis with important on RAPD banding patterns and can definitely be of great advantages like simplicity, efficiency, relative ease of execution service to the apple industry worldwide. and non-requirement of any previous sequence information (Williams et al., 1990). Optimization of the RAPD technique Materials and Methods by choosing 11 mer primers and strict screening PCR annealing temperature before it is employed in fingerprinting plants, Plant Materials can make RAPD a preferable technique for use in plant Young leaves of 68 selected apple cultivars were collected cultivar identification. from the Research Institute of Pomology of Chinese Academy In this study, we use a Cultivar Identification Diagram of Agricultural Sciences at Xingcheng. The additional (CID) which could be utilized as a source of reference information of these cultivars is shown in Table 1. Table 1. Name, origin and source of the materials used in the experiment (Li et al., 1980; Xin and Xiang, 1998). No. Cultivar Origin Source 1 ‘Northern Spy’ America Seedling 2 ‘Mutsu’ Japan ‘Golden Delicious’ × ‘Indo’ 3 ‘New Jonagold’ Japan ‘Jonagold’ bud sport 4 ‘Stark Spur Supreme’ ‘Red Red America ‘Red Delicious’ bud sport 5 ‘Alps Otome’ Japan ‘Kitakami’ × (‘Tsugarall’ × ‘Summer Pearmain’) 6 ‘Sheng Fang Fu’ Japan ‘Fuji’ bud sport 7 ‘Ji Guan’ China, Liaoning Seedling 8 ‘Xiu Shui Guo Guang’ China, Shandong ‘Ralls Janet’ Seedling 9 ‘Shinsekai’ Japan ‘Fuji’ × ‘Akagi’ 10 ‘Hatsuaki’ Japan ‘Jonathan’ × ‘Golden Delicious’ 11 ‘Ralls Janet’ America Seedling 12 ‘Lobo’ Canada ‘McIntosh’ seedling 13 ‘Teng Mu 1’ America * 14 ‘Early McIntosh’ America ‘Yellow Transparent’ × ‘McIntosh’ 15 ‘Cui Qiu’ China, Liaoning ‘Golden Delicious’ × ‘Yellow Transparent’ 16 ‘Chi Cheng’ China, Gansu Seedling 17 ‘Hong Rou Ping Guo’ China, Xinjiang Seedling 18 ‘Aki 5’ Japan ‘Fuji’bud sport 19 ‘Hong Wang Jiang’ China ‘Fuji’ bud sport 20 ‘Miyasaki’ Japan ‘Fuji’ bud sport 21 ‘Summer Pearmain’ America Seedling 22 ‘Senshu’ Japan ‘Toko’ × ‘Fuji’ 23 ‘Vista Bella’ America Hybridization 24 ‘Mac Spur’ England ‘McIntosh’ bud sport 25 ‘Orin’ Japan ‘Golden Delicious’ × ‘Indo’ 26 ‘Jin Hong’ China, Jilin ‘Golden Delicious’ × ‘Hong tai yang’ 27 ‘Ruby Red Spur’ America ‘Starkrimson’ bud sport 28 ‘Well Spur Delicious’ America ‘Fuji’ bud sport 29 ‘Melrose’ America ‘Delicious’ × ‘Jonathan’ 30 ‘Starking Red’ America ‘Delicious’ bud sport 31 ‘Aki 2’ Japan ‘Fuji’bud sport 32 ‘Golden Grimer’ America Seedling 33 ‘Qunfu 1’ Japan ‘Fuji’bud sport 34 ‘Nagafu 7’ Japan ‘Fuji’ bud sport 35 ‘Golden Delicious’ America ‘Golden Grimer’ × ‘Golden Reinette’ 36 ‘Qiu Fu 1 Hao’ China, Liaoning * 37 ‘Qiufu 1’ Japan ‘Fuji’ bud sport 38 ‘Ying Qiu’ China, Liaoning ‘Jonathan’ × ‘Summer Pearmain’ 39 ‘Black Ber Davis’ America ‘Ber Davis’ bud sport 40 ‘Starkrimson’ America ‘Starking Red’ bud sport 41 ‘Giant Jenifon’ China ‘Ralls Janet’ bud sport 42 ‘Close’ America * 43 ‘Cox’s Orange Pippin’ England Hybridization 550 Kor. J. Hort. Sci. Technol. 30(5), October 2012 Table 1. Countinued. No. Cultivar Origin Source 44 ‘Han Fu’ China, Shenyang ‘Tohoku’ × ‘Fuji’ 45 ‘Fu Shuai’ China, Zhengzhou ‘Early McIntosh’ בGolden delicious’ 46 ‘Smith Cider’ America Seedling 47 ‘Jonagold’ America ‘Golden Delicious’ × ‘Jonathan’ 48 ‘Discovery’ England Seedling 49 ‘Yellow Transparent’ Russia * 50 ‘Jerseymac’ America ‘NJ24’ × ‘Julyred’ 51 ‘Megum’ Japan ‘Ralls Janet’ × ‘Jonathan’ 52 ‘Qin Guan’ China, Shanxi ‘Golden Delicious’ × ‘Ji Guan’ 53 ‘Sweet Mcintosh’ America ‘Mcintosh’ × ‘Lawver’ 54 ‘Silver Spur Red’ America ‘Hi Early’ bud sport 55 ‘Jonathan’ America Seedling 56 ‘Ruby Spur’ America ‘Starkrimson’ bud sport 57 ‘Stark Jumbo’ America ‘Spokane Beauty’ bud sport 58 ‘Bi Hong’ China * 59 ‘Hardispur Delicious’ America * 60 ‘Red Spur’ America ‘Starking Red’ bud sport 61 ‘Tian Huang Kui’ China, Liaoning ‘Summer Pearmain’ × ‘Yellow Transparent’ 62 ‘Zao Hong Xing’ China, Shandong ‘Red delicious’ bud sport 63 ‘Liao Fu’ China, Liaoning ‘Lodi’ × ‘Summer Pearmain’ 64 ‘Tsugaru’ Japan ‘Golden Delicious’ hybridization 65 ‘Fu Jin’ China, Liaoning ‘Delicious’ × ‘Yellow Transparent’ 66 ‘McIntosh’ Canada Seedling 67 ‘Gala’ New Zealand ‘Kidd’s orange Red’ × ‘Golden Delicious’ 68 ‘Golden Spur’ America ‘Golden Delicious’ bud sport *Indicates unknown origin and source of the cultivar. Genomic DNA Extraction Table 2. Ten primers were used for the separation of the 68 apple genotypes. Total genomic DNA of each genotype was extracted from Nucleotide Annealing young leaves using the modified cetyl trimethyl ammonium Primer bromide method (Bousquet et al., 1990; Murray and sequence (5’–3’) temperature（°C） Thompson, 1980). The extracted DNA was diluted to a final Y-3 GTTTCGCTCCA 44.7 concentration of 30 ng･μL-1 with1 × TE buffer and stored Y-5 GTTTCGCTCCG 43.7 at -20°C before used. Y-6 GTTTCGCTCCC