M.T. Mansour et al/Journal of Genetics&Breeding 63: 17-22 (2011) 17 M.T. Mansour 1 , D.S. Hassawi 1 *, H.M. Migdadi 2 and M. Brake 3 1 Al-Balqa Applied University, Biotechnology Department, Al-Salt 19117, Jordan, 2 King Saud University, College of Food and Agricultural Sciences. Riyadh, Saudi Arabia, 3 Jarash Private University, College of Sciences, Biology Department, Jarash, 26150, Jordan. ABSTRACT Identification of date palm genotypes is important for farmers and date palm industries. Correct identification is usually dependent on fruit analysis, however the generation time is long and genotype identification requires phenotypic data that may be variable due to environmental conditions. Traditionally, date palm is propagated by off shoots, but currently it is also propagated by tissue culture which may result in mutations and genetic instability. Ten date palm genotypes propagated by off shoots were analyzed using Inter Simple Sequence Repeat markers (ISSRs) to identify, fingerprint, and study the genetic relationship among them. Genotypes were clearly identified using eight of the twelve primers tested. Total number of 428 bands with fragment sizes between 400 and 5000 bp were identified. The primer [(AG)10T] produced the largest number of bands (92), while primer [(CT)10T] revealed the highest degree of polymorphism (100%) among genotypes. These primers were also used to compare four genotypes that were propagated by off shoots to those propagated by tissue culture. Genotype Medgoul showed the highest similarity (0.93) followed by Hayyani (0.92), Zehdi (0.86) and Bream (0.82). Method of propagation did not show clear separation among the genotypes tested. The similarity ranged from 0.82 to 0.93 according to the method of propagation. INTRODUCTION Date palm (Phoenix dactylifera L.) belongs to the Arecaceae family, which comprises 200 genera and more than 2500 species (Diaz et al 2003). It is a diploid (2n=36) dioecious perennial monocotyledon fruit trees with long generation time (Al-Bekr 2002). Date palm has traditionally been vegetativelly propagated from off shoots; tissue culture is another way to propagate date palm (Ben-Abdallah 2000) but mutation often appears during callus formation (Pierik 1987), which means this technique is genetically unstable. Correct identification of date palm trees is usually possible if the fruits are produced. However, identification is difficult because of the long generation time and because it requires a large set of phenotypic data that are often hard to assess and sometime variable due to environmental conditions (Sedra et al 1993 and 1996). Since the certification of varieties based upon phenotypes characterization, the recognition of the genotypes by farmers has been a real problem. The genetic identification of genotypes is important for certified plant materials, but requires fast and reliable techniques (Bianchi et al 2002). The last few years have revealed a widespread use of molecular markers for fingerprinting, selection, and breeding of fruit crops. Inter Simple Sequence Repeats (ISSRs), also called microsatellites, have been used as primers in polymerase chain reaction. The resultant PCR reaction amplifies of this sequence, yielding a multilocus marker system useful for fingerprinting, diversity analysis and genome mapping (Godwin et al 1997). In addition, ISSR can be targeted towards particular sequences, which are reported to be abundant in the genome and can overcome the technical difficulties of RFLP and RAPD (Rajesh et al 2002, Barth et al 2002). Since high level of polymorphism with ISSR has been observed in plants, this technique was used for fingerprinting of many fruit trees such as: oil palm (Billotte et al 2005), date palm (Zehdi et al 2004), fig (Salhi-Hannachi et al 2004), olive (Gemas et al 2004; Bandelj et al 2002), Indian cashew (Archak et al 2003). ISSR also used in field crops like rice (Saini et al 2004); Cicer (Mehmet 2004, Rajesh et al 2002), barley (Fernandez et al 2002, Bahattin 2003), ryegrass (Ghariani et al 2003), bean (Galvan et al 2003), wheat (Ammiraju et al 2001). This study aimed to identify different genotypes of date palm (Phoenix dactylifera L.) that grown in Jordan at molecular level using ISSR markers. It also aimed to show if date palm genotypes that are produced by tissue culture technique have some genetic changes comparing with the same genotypes that propagated by offshoot. MATERIALS AND METHODS Plant materials Fourteen date palm genotypes were used in this study; 10 genotypes represent offshoot propagation and 4 genotypes represent tissue culture propagation (TABLE 1). These genotypes were obtained from a collection maintained in the experimental orchard of the National Center for Agricultural Research and Extension (NCARE), in Jordan. For each genotype, samples (about 50 gm) from the tips of young leaves were collected, labeled, and kept in ice box; the samples were then stored at -70 °C until using. DNA extraction Genomic DNA from each sample was extracted according to CTAB method as described by Jubrael et al. (2005). RNA that can interfere with PCR was removed by digesting each sample with 4µl of Dnase-free Rnase (10 mg/ml) at 37°C for 20 min (Sambrook et al 1989). Polymerase Chain Reaction (PCR) The method described by Zehdi et al. (2004) was followed with some modifications in the annealing temperature. 25µl reaction mixture was used For PCR amplifications; it contained 25 ng of genomic DNA, 0.2 mM of primer, 1X PCR buffer [100 mM Tris- HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl2, 0.01% Difco Gelatin], 1.5 unit of Taq DNA polymerase, 0.2 mM of each dNTPs and one drop of mineral oil to prevent evaporation during PCR cycles. Fingerprinting date palm genotypes (Phoenix dactylifera L.) using Inter Simple Sequence Repeat (ISSR) markers ARTICLE INFO Received July 15, 2009 Received in revised form July 5, 2010 Accepted September 10, 2010 *Corresponding author. E-mail: dhassawi@yahoo.com