Genetic Diversity of Iraqi Date Palms Revealed By Microsatellite Polymorphism

Genetic diversity in 30 date palm (Phoenix dactylifera L.) cultivars in Iraq representing 24 female and six male cultivars was investigated using 22 microsatellite [simple sequence repeat (SSR)] primers. The tested SSR markers showed a high level of polymorphism. A total of 188 alleles were detected at the 22 loci ranging from three to 21 with an average of 8.54 alleles per locus. The average of heterozygosity for all cultivars was 0.503; genetic distance among cultivars varied from 0.171 to 0.938 indicating diverse relationships. The cultivar Ghanami Akhder was highly divergent from ‘Ghnami Ahmer’, whereas ‘Jamal Al-Dean’ was very closely related to ‘Qitaz’. Unweighted pair group method arithmetic average ordered date palm cultivars into two main clusters. Principal coordinate analysis exhibited the similar clusters of cultivars as in the dendrogram. Date palm (2n = 2x = 36) is considered of great socioeconomic importance in the Arabian region. The tree has been, and is still, at the center of comprehensive agricultural development. It is believed to have originated from Mesopotamia (Wrigley, 1995). The numbers of known date palm cultivars that are distributed all over the world are 5000 of which 600 are found in Iraq. Iraq is one of the largest date-producing countries in the world. Before 1991, Iraq was the largest producer of dates in the world (Food and Agriculture Organization of the United Nations, 2008) and had the largest date forest in the world (MacFarquhar, 2003). However, during the Gulf and Iran–Iraq wars, large numbers of date palm trees were destroyed. Wars and sanctions imposed on Iraq have negatively affected both the production and natural genetic diversity of the crop in Iraq and inhibited the much-needed impetus to rebuild the date palm industry. Development of suitable DNA molecular markers for this crop may allow researchers to estimate genetic diversity, which will ultimately lead to the genetic conservation of date palm. The success of particular genetic conservation or breeding programs is dependent on understanding the amount and distribution of the genetic variation already present in the genetic pool (Jubrael et al., 2005). Morphological traits have been used to describe such genetic variation in date palm cultivars (Barreveld, 1993), which are mainly related to the fruit and influenced by the environment (Askari and Al-Khalifah, 2003; Sedra et al., 1993, 1998). In addition, biochemical studies, including isozyme and activity analyses of peroxidases, have been used to characterize date palms in Morocco and Tunisia (Baaziz, 1988; Baaziz and Saaidi, 1988; Bendiab et al., 1998; Majourhat et al., 2002; Ould Salem Mohamed et al., 2001). As such, analysis does not reflect precisely that polymorphisms occurred (Al-Jibouri and Adham, 1990). DNA marker analysis in date palm is recently in the development stage. Askari and Al-Khalifah (2003) analyzed the genetic diversity among 13 different cultivars using random amplified polymorphic DNA (RAPD) markers. Similar reports are published for North African cultivars (Hussein et al., 2002; Sedra et al., 1998). Amplified fragment length polymorphism (AFLP) markers (Vos et al., 1995) were also been used for genetic fingerprinting and mapping for this crop. Jubrael et al., (2005) and Khierallah et al. (2011) used AFLP fingerprinting to characterize a number of Iraqi date palm cultivars. Microsatellites, also known as SSR, are ideal DNA markers for population studies and genetic mapping as a result of their abundance, high level of polymorphism, dispersion throughout diverse genomes, ease to assay by polymerase chain reaction (PCR), and ease to disseminate among laboratories (Udupa and Baum, 2001). Microsatellite markers were used for investigation of genetic diversity in date palm (Billotte et al., 2004). Zehdi et al. (2004) applied these markers to characterize Tunisian cultivars, whereas Al-Ruqaishi et al. (2008) used these primers to screen and analyze the genetic diversity among clonal genotypes of Omani cultivars. Recently, many researchers in date palm-producing countries have used SSR markers to genotype cultivars. Elshibli and Korpelainen (2008) investigated genetic diversity in Sudan germplasm representing 37 female and 23 male accessions using 16 SSR primers. In Qatar, Ahmed and Al-Qaradawi (2009) used 10 primers to analyze genetic Received for publication 27 Jan. 2011. Accepted for publication 24 June 2011. This project was supported by a grant from Tottori University and funds from BMZ (Bundesministerium fur Wirtschaftliche Zusammenarbeit und Entwicklung), Germany, and the State Board of the Agriculture Research, Ministry of Agriculture, Iraq. We acknowledge the technical support of A. Sabbagh (International Center for Agricultural Research in the Dry Areas, Aleppo, Syria). Corresponding author. E-mail: khierallah70@yahoo.com. 282 J. AMER. SOC. HORT. SCI. 136(4):282–287. 2011. diversity among 15 cultivars. Akkak et al. (2009) developed 17 additional microsatellite markers. Hamwieh et al. (2010) developed 1000 SSR markers in date palm, mining genome sequencing data for this vital crop. The aim of this study is intended to characterize the genotype for 30 Iraqi important date palm cultivars using SSR markers. Materials and Methods PLANT MATERIALS. Leaves were collected from 30 welldefined reference Iraqi date palm cultivars grown in date palm stations belong to the Ministry of Agriculture (Table 1). Twenty-two female cultivar samples were collected from AlMahaweel Date Palm Station, Hilla Governorate, 80 km south of Baghdad. The remaining eight cultivar samples were collected from Al-Za’afarania Date Palm Station, Baghdad (Fig. 1). Total genomic DNA was extracted from young and healthy leaves according to the procedure mentioned by Benito et al. (1993) with minor modifications. After purification, the resultant DNA was quantified on 1% agarose gel electrophoresis as described by Sambrook et al. (1989). MICROSATELLITE AMPLIFICATION. A total of 33 date palmspecific primer pairs were tested; 16 of them were developed by Billotte et al. (2004) and 17 were developed by Akkak et al. (2009) as indicated in Table 2. PCR was performed in a total mixture of 20 mL containing: 13.9 mL H2O, 50 ng of total cellular DNA (2 mL) as template, 1 mL/PCR buffer (Roche, Mannheim, Germany), 1 mL of 0.2 mM of dNTP PCR mix (Roche), 0.1 mL (0.625 U) of Taq DNA polymerase (Roche) and1 mL of 0.2 mM of each primer using forward primer end labeled (6FAM, NED, or TET). Amplifications were performed in a thermocycler (Applied Biosystems, Carlsbad, CA) under the following conditions: a denaturation step of 5 min at 95 C followed by 35 cycles of 30 s at 95 C, 1 min at 52 C and 1 min at 72 C, and a final extension step at 72 C for 7 min. At the testing stage, amplification products were separated by electrophoresis according to their molecular weight in 1.4% agarose gels and then detected by staining with ethidium bromide as described by Sambrook et al. (1989). The DNA profiles were visualized on an ultraviolet transilluminator and documented by using a gel documentation system (Alpha Innotech Imaging Station; Cell Biosciences, Santa Clara, CA). For final analyses, 2 mL of amplified DNA (diluted to 1/10) and 8 mL of (Rox) DNA standard size (Applied Biosystems) were loaded into 96-well plates. Genotyping was carried out using an automatic DNA analyzer (Model 3100; Applied Biosystems). Amplifications were performed at least twice and only reproducible products were taken into account for further data analysis. SIMPLE SEQUENCE REPEAT GENOTYPING. Electropherogram data were analyzed by using Foundation Data Collection software (Genetic Analyser Data Collection Version 2.0; Applied Biosystems). Allele size scoring was performed by Gene Mapper software (Version 3.7; Applied Biosystems). The Jaccard similarity matrix (Jaccard, 1908) was used for cluster analysis using the unweighted pair group method arithmetic average to study the genetic relationships among the cultivars. Jaccard similarity index, major allele frequency, heterozygosity, gene diversity, and polymorphism information content estimation were done using a software package (PowerMarker Version 1.31; Liu and Muse 2005). The phylogenetic diagram was drawn by PAST software [Version 1.91 (Hammer et al., 2001)] on the basis of Hamming similarity index with 100 bootstrap. Principal coordinate analysis (PCA) was performed according to Euclidean similarity index using the PAST software.