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Apples are produced using a high-density plantation system using M.9 rootstock in Korea. However, the virus infection rate of apples tends to be high because the rootstocks produced from virus-infected mother stocks were widely used and vegetative propagation based on the grafting is common with scion cultivars. The most effective way to control plant virus and viroid depends on conducting a massively rapid and precise diagnostic inspection. Therefore, this study aims to develop a real-time PCR assay that can be precisely and inexpensively tested for ACLSV and ASSVd. To expand the detection range, we developed a singleplex real-time PCR method targeting the conserved region of ACLSV and ASSVd, respectively. The detection methods using two kinds of fluorescent chemical dye, SYBR Green I and TaqMan probe, have been developed in this study. Comparing these methods developed with two dyes, the assay using SYBR Green I was efficient for detection as much as the method using TaqMan probe and faster. The field test demonstrated real-time detecting methods developed using SYBR Green I dye were applicable to reliable diagnosis and quantification for virus and viroid in quarantine fields.

Apple trees are perennial plant and have a characteristic of requiring long period from seed germination to flowering and fruiting. Most Korean apple cultivars are derived from closely related varieties genetically; consequently, they are distributed into grafted seedlings at juvenile stage. Therefore, it is difficult for them to distinguish by morphological traits due to the high similarities without fruit characteristics. We have integrated the high-resolution melting (HRM) analysis with simple sequence repeat (SSR) marker genotyping to identify thirteen apple cultivars originated from South Korea. The patterns of temperature-shifted melting curves were investigated from HRM analysis. The SSR loci used in this study generated a unique melting curve profile for each cultivar allowing their comparison and classification. The combination of the allelic patterns obtained with the just three SSR markers was able to identify thirteen apple cultivars. These results suggest that SSR markers used in combination with HRM analysis can be a fast and accurate method for high-throughput cultivar identification and biodiversity assessment.