In order to understand the evolutionary trend of the genus Pistacia and genetic variation among Iranian pistachio genotypes, two DNA libraries enriched for dinucleotide (AG) n and trinucleotide (ATG) n microsatellite motifs were developed from Pistacia khinjuk genome. Total genomic DNA was extracted from Khinjuk leaves and digested with a mixture of four restriction enzymes ( Nhe I, Rsa I, Alu I and Hae III). Overhanging ends of the obtained DNA fragments were removed using mung bean nuclease, dephosphorylated and ligated with phosphorylated SNX linkers. The linker-ligated fragments were selected through selective hybridization to biotinylated oligonucleotides (AG) 15 , (ATG) 10 and capturing with streptavidin coated magnetic beads. The selected fragments, containing AG and ATG repeats, were inserted in pBluescript vector, transferred into Escherichia coli (MC1061 strain) and then enriched libraries were screened by colony PCR technique. The plasmids of 44 positive clones were extracted and sequenced. For two clones the sequences were not clear enough, however, the repeat sequences were obvious in 35 clones (83.3%) among the 42 clones showed high quality sequences. The comparison of enrichment and sequencing results showed that AG microsatellite motifs are more frequent comparing to ATG motifs and they have more repeated number. For clones contained the repeats, 27 pair of primers were designed from flanking regions of the repeats using Primer3 software. After optimization of their PCR amplifications, the primer sets were used to assay the polymorphism of Pistachio species and Iranian genotypes of P. vera . The amplification patterns of the fragments on polyacrylamid gel were analyzed using PowerMarker V3.25 and MEGA3 softwares and the dendrogram were then represented. These microsatellites were found to be polymorphic in different related species and the dendrogram showed that all wild species of pistachio were derived from P. vera . The dendogram also revealed an interesting evolutionary trend from P. vera to P. khinjuk , followed by P. integerrima , P. palaestina , and then to P. atlantica . The genotypes of domestic pistachio were dir=rtl In order to understand the evolutionary trend of the genus Pistacia and genetic variation among Iranian pistachio genotypes, two DNA libraries enriched for dinucleotide (AG) n and trinucleotide (ATG) n microsatellite motifs were developed from Pistacia khinjuk genome. Total genomic DNA was extracted from Khinjuk leaves and digested with a mixture of four restriction enzymes ( Nhe I, Rsa I, Alu I and Hae III). Overhanging ends of t
