Abstract—The cultivated barley (Hordeum vulgare subsp. vulgare) is one of the major crops in the world. In this study the genetic diversity of 32 individuals of two-rowed and six-rowed Iranian landraces barley evaluated using 17 microsatellite markers. A high level of polymorphism information content (PIC; average = 0.651) and an average of 8.117 allele per locus were observed. In dendrograms constructed based on the SSR data, the two group of cultivars (var. distichon and var. hexastichon) were separated. Based on the results of this study, it can be concluded that there is a high level of genetic diversity between the barely landraces in Iran and that the barely Iranian gene pool is valuable source to search for new useful alleles for crop improvement. Index Terms— Genetic diversity, barley, Iran, microsatellite. I. INTRODUCTION Cultivated barley (Hordeum vulgare subsp. vulgare) is one of the most important crop cereals in the tribe Triticeae (Poaceae) that cultivated over the temperate regions [1]. Based on several reports it has been originated from Fertile Crescent in Near East or from Tibetan in the west China [2], [3]. Many studies have demonstrated that Tibetan wild barley populations were clearly different from the Fertile Crescent wild barley in respect to their distribution, ecology; morphology, archaeology, cytogenetics and isozyme complement [4], [5]. Iran is placed in the southeastern edge of Fertile Crescent from where based on several evidences; the cultivation processes of barley have taken placed [3]. Morphological characters are insufficient for the discrimination of barley varieties, and recognition of barley cultivars on the basis of kernel morphology is very hard [6]. In Hordeum species molecular markers, such as RFLP, AFLP, STS and microsatellites have made potential the description of different cultivars, the understanding of phylogenetic relations, and genetic mapping [7]. SSRs have been increasingly used as molecular markers. Molecular markers, such as RFLP, AFLP, STS and microsatellites, provide the mainly powerful methods to assay genetic diversity, to construct genetic mapping and to categorize different varieties [7]. In Hordeum species molecular markers, such as RFLP, AFLP, STS and microsatellites have made potential the description of different cultivars, the understanding of phylogenetic relations, and genetic mapping [7]. SSRs have been increasingly used as molecular markers. Regarding the high variable geographical and ecological conditions in Iran, the Iranian landraces can be considered as valuable gene sources for modern cultivar improvement. The study of genetic diversity in a germplasm such as barley landraces is fundamental to perform a strategy for landrace conservation and to find germplasem with higher priority. The aims of the present study were: (1) to determine variability a set of microsatellite markers, and (2) to use them for genome analysis, distinguishing between varieties, genotypes, and the estimation of genetic diversity in Iranian barley landraces germplasm collection. II. METHODOLOGY A total of 32 individuals of barley landraces were collected from various regions of Iran (from the altitude of 43m to 2051m) by two of the authors (Khodayari and Saeidi) and these were identified morphologically according to Bothmer et al. [1] (Table I). The accession numbers and geographic origins of samples are shown in Table I. In order to assessing genetic diversity, 17 SSR markers derived from wild barley were used [8]. Seeds from each individual plant were grown in experimental field and DNA was isolated from fresh leaves according to Komatsuda et al. [9]. For SSR analysis at individual level, the DNA was isolated from individual plants. PCR amplifications were carried out in 10 μL, containing approximately 50-100 ng template genomic DNA, 250 nM of each primer, 0.2 mM of each dNTP, 1.5 mM Mgcl2, 1.2 U EX-Taq Polymerase. PCR amplifications procedure of SSR markers was performed by an initial denaturation step of 5 min at 94 °C followed by 30 cycles of three steps: denaturation for 30 s at 94°C, annealing for 30 s at 55–60 °C, extension for 30 s at 72 °C with a final extension for 7 min at 72 °C [8]. Along with size marker tracks (100bp DNA ladder, Promega), PCR products were mixed with loading buffer (2:1) and loaded on 12% non-denaturing polyacrylamide gels (Fig. 1). Genetic Diversity of Cultivated Barley Landraces in Iran Measured Using Microsatellites Hamed Khodayari, Hojjatolah Saeidi, Azadeh Akhavan Roofigar, Mohammad Reza Rahiminejad, Mohammad Pourkheirandish, and Takao Komatsuda International Journal of Bioscience, Biochemistry and Bioinformatics, Vol. 2, No. 4, July 2012 287 Manuscript received June 25, 2012; revised July 27, 2012. Hamed Khodayari is with the Dep. of Biology, Faculty of Basic Science, Lorestan University, Khorramabad, Iran (Tel.: +989132042734; e-mail: hamedkhodayari75@yahoo.com). Hojatollah Saeidi, Azadeh Akhavan Roofigar, and Mohammad Reza Rahiminejad were with Dept. of Biology, Faculty of Science, University of Isfahan, Isfahan, Iran (e-mail: ho.saeidi@sci.ui.ac.ir; Tel.: +98-311-7932467) Mohammad Pourkheirandish and Takao Komatsuda are with Crop Genome Research Unit, National Institute of Agrobiological Sciences (NIAS), 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan. DOI: 10.7763/IJBBB.2012.V2.118