Sequence-characterized amplified region markers and multiplex-polymerase chain reaction assays for kiwifruit cultivar identification

Abstract

A simple and reliable multiplex polymerase chain reaction (PCR) method was developed using sequence-characterized amplified region (SCAR) markers to identify kiwifruit (Actinidia spp.) cultivars. We observed 183 different polymorphic random amplified polymorphic DNA (RAPD) markers using 23 arbitrary primers in 30 kiwifruit cultivars. The number of polymorphic bands per primer varied from five (OPM-15, OPN-05, and OPN-19) to 12 (OPG-01), with an average of eight. Fifty-eight cultivar-specific polymorphic RAPD fragments were converted into 16 SCAR markers after validating their specificity. Among them, 15 SCAR markers retained the dominant behavior of the original RAPD fragment, whereas the KA11_774 SCAR marker showed a co-dominant polymorphism. Based on RAPD and SCAR data sets, cluster analyses were performed with the unweighted pair-group method of arithmetic averages. The mean genetic similarities were estimated as 0.703 and 0.677 using the RAPD and SCAR markers, respectively. Thirty kiwifruit cultivars were divided into three clusters for RAPD analysis and into two clusters for SCAR analysis, based on a similarity value of 0.67. Overall, the kiwifruit cultivars with a similar breeding background tended to cluster together. Therefore, RAPD and SCAR markers could be successfully applied to assay genetic relationships for kiwifruit cultivar identification. Cluster analysis of combined RAPD and SCAR markers showed very similar results when compared to the RAPD analysis in dendrogram form. The kiwifruit cultivars tested in this study could be identified using only 10 different SCAR markers (KB08_182, KK08_287, KG19_295, KN15_316, KG01_411, KF07_433, KG09_468, KG09_477, KL01_764, and KA11_774). Furthermore, we optimized SCAR markers for simultaneous identification of the 30 kiwifruit cultivars by multiplex PCR, using two sets of five SCAR primer pairs. The developed SCAR markers and multiplex PCR approach facilitate efficient identification of kiwifruit cultivars.