Identification of major walnut cultivars grown in China based on nut phenotypes and SSR markers

• Thirty-five cultivars of walnut were identifying by 11 nut-shape characters and 4 SSR markers.
• Cultivars with the same parents were easily differentiated based on different nut shape.
• The classification of nut shape is important for nut batch processing and regional cultivar selection.
• The SSR markers we describe here are useful for genetic structure studies, phylogenetic analysis, and cultivar identification of the genus Juglans.
• Regardless of nut phenotype characters, the four SSR loci allowed unambiguous separation of the cultivars studied.

Walnut (Juglans regia L.) is an economically important tree widely cultivated in temperate regions for its nuts and timber. In this study, molecular identification using 12 simple sequence repeat (SSR) markers and 22 nut-shape characters were conducted on 35 cultivars of walnut grown in China. All 35 cultivars were classified into 11 broad categories on the basis of the nut phenotype. The shape characteristic of longitudinal sections to the suture presented a broad elliptic or elliptic shape was the largest category and included 18 cultivars. All accessions in each category were differentiated by no more than two SSR primer pairs. In addition to nut phenotype, 35 cultivars could be identified using four primer pairs, and every cultivar presented unique fingerprints. In particular, cultivars with the same parents were easily differentiated, such as ‘Liaoning 1’ and ‘Liaoning 3’ based on their different nut shape. Regardless of nut phenotype characters, just four loci (WJR031, WJR069, WJR265, and WJR281) also allowed the unambiguous separation of the 35 cultivars studied. This study provides a useful protocol for the characterization, identification, and authentication of any walnut cultivar and also shows the power of SSR markers in cultivar identification and intellectual property protection. Furthermore, the classification of nut shape is important for nut batch processing and for regional cultivar selection, which could be useful for the walnut industry.