Introduction Juglans regia L. (walnut) is a monoecious tree crop with a haploid chromosome number of n = 16 and it belongs to the family Juglandaceae (Forde and Griggs, 1972). Although about 20 Juglans species are included in the genus, J. regia is considered most important owing to its edible nuts (Manning, 1978). Walnut is pollinated by wind (Rom and Carlos, 1987) and its origin is believed to be in Central Asia and neighboring regions (Browicz, 1976). Juglans species are also widely distributed in North and South America (Aradhya et al. 2007). In Europe, J. regia is the only commonly cultivated species of the genus. Turkey is considered as one of the origins of the species with a high production of nuts (212,140 t/year), which is fourth after China, the United States, and Iran (http://www.fao.org). Walnuts have high nutritional value and contain proteins, fats, vitamins, and minerals and thus they are considered important for human nutrition (Gandev, 2007). Germplasm characterization, phylogenetic analysis, and genetic diversity studies in walnut have been conducted using a few molecular markers such as restriction fragment length polymorphisms (Fjellstrom et al., 1994), randomly amplified polymorphic DNAs (Nicese et al., 1998), intersimple sequence repeats (Potter et al., 2002), and amplified length polymorphisms and the selective amplification of microsatellite polymorphic loci (Kafkas et al., 2005). However, most of them were dominant with a lower level of allelic variation and less reproducible and thus have limited application in marker-assisted breeding programs (Ikhsan et al., 2016). Therefore, developing a codominant marker system in this species is necessary. Among the codominant markers, simple sequence repeats (SSRs) have been widely used across studies, including genotyping, parental identification, population genetics, and linkage map construction. In addition to the codominant nature, SSR markers are highly polymorphic, multiallelic, highly reproducible, and have good genome coverage. SSR marker development in Juglans was first attempted by Woeste et al. (2002) by using J. nigra genomic DNA. Hoban et al. (2008) reported 13 polymorphic microsatellite markers in J. cinerea, and Chen et al. (2013) developed 20 SSRs in J. mandshurica. Furthermore, several studies have Abstract: Walnut (Juglans regia L.) is the most important species in the genus Juglans due to the high commercial value of its nuts and timber. Simple sequence repeats (SSRs) are considered as the markers of choice owing to their codominant nature. Since very few SSRs have been developed in walnut, this study aimed to develop numerous polymorphic SSRs from Class I repeats by using DNA sequences of the Chandler cultivar. In all, 800 SSRs were designed and tested in 8 Turkish, 3 French, and 5 US walnut cultivars, of which 88 (11%) did not produce bands, 161 (20.1%) were monomorphic, and 551 (68.9%) were polymorphic. In all, 2696 alleles were produced by the 551 polymorphic SSR loci in the 16 walnut cultivars, ranging from 2 to 14 alleles ...

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