Application of SSR markers and real-time PCR for variety identification in azuki-bean [Vigna angularis (Willd.) Ohwi and Ohashi]

Abstract

The azuki bean in Korea consists of seven domestic varieties which have been developed and registered for the public during last 25 years. Here, we present a simple but reliable method to screen and identify Korean azuki bean varieties. A method based on simple sequence repeat (SSR) markers is widely used for prominent gene identification and variety discrimination. In molecular biology, real-time polymerase chain reaction (PCR) is a laboratory technique based on the polymerase chain reaction that is used to amplify and simultaneously quantify a targeted DNA molecule. It enables easy detection of a specific sequence in a DNA sample without performing electrophoresis and further processes. For separation of seven Korean azuki bean varieties, 110 unique azuki bean SSR markers from an (AG)n-enriched library were selected, synthesized and used for polymerase chain reaction (PCR). Data were taken through acrylamide gel electrophoresis and automated multi-capillary electrophoresis system for selection of specific markers and then changed into proper formats for data mining analysis. Ten primer pairs that showed high polymorphism were chosen for the indepth study. These ten primers were re-amplified with real-time PCR and checked the cycle threshold (Ct) and temperature (Tm) for comparison of amplification sequence in seven varieties. Consequently, a total of 20 alleles and 6 SSR primers were detected from the standard PCR amplification. Within these 6 primers, 7 alleles of 3 SSR primers were isolated for variety identification. From real-time PCR results, 3 SSR primers were selected as efficient markers for discrimination of seven Korean azuki bean varieties. The approach described here could be applied in monitoring our varieties and can be adapted in the azuki bean breeding program.