Development of SNP-based identification method of hop varieties

Technical Session 13: Hops III Session
Hiromasa Yamauchi, Suntory Business Expert Ltd., 57 Imaikami-cho, Nakahara-ku, Kawasaki, Japan
Co-author(s): Yuri Mukouzaka, Susumu Furukubo, Kazuhiko Nakashima, and Takayuki Taniguchi, Suntory Business Expert Ltd., Kawasaki, Japan; Masami Harada, Suntory Holdings Ltd., Tokyo, Japan

ABSTRACT: Hop is one of the key raw materials affecting beer quality, and the correct identification of hop varieties is very important. Generally, hop varieties are identified by differences in cone structures, sensory analysis, and the content of substances such as alpha-acids and essential oils. However, these methods have limitations because the content of the substances in hop can be variable depending on cultivation conditions and pelletized hop cannot be identified by observation of external appearance. Several DNA analysis techniques have been developed for the identification of hop varieties, e.g., SSR method, RAPD method, RFLP method, AFLP method, etc., which generally utilize the polymorphism of PCR-amplified products or restriction enzyme-digested fragments of hop DNA. These methods are generally complicated and have limitations to detection of mixing of other varieties. Analysis of SNPs (single nucleotide polymorphisms) in genome DNA can be a powerful tool for the identification of varieties. However, in order to obtain sufficient SNP positions for the identification of many varieties, large amounts of DNA sequences should be needed. In recent years, high throughput DNA sequencing technology has been developed using a so-called “next generation sequencer.” Using this technique, we tried to develop SNP-based identification method for hop varieties. Large amounts of DNA sequence data in several European hop varieties were obtained using the next generation sequencer. By comparing DNA sequences between the varieties, several SNP-rich DNA regions in hop genome were selected as candidates for identification markers. DNA sequences of these regions in other European hop varieties were also determined using the traditional Sanger method, and it was evaluated whether these regions could be DNA markers for the identification of all varieties tested. As a result, 14 hop varieties could be identified by using four SNP-rich DNA regions. Moreover, it was studied whether a mixture of two varieties could be correctly evaluated by this method. A hop pellet sample of one variety was mixed with that of another variety at various ratios (0, 5, 10, 50, and 100%), and their DNA was extracted to sequence the DNA marker regions. By observing the electropherogram of SNP positions, it was suggested that the mixture of with the other variety at a 5% level could be detected. A quantitative determination method of mixture rate can be expected using DNA techniques, such as quantitative real-time PCR, etc. Because this method utilizes the DNA sequence itself, it could be a simple and reproducible tool for the identification of hope varieties.

Hiromasa Yamauchi received his doctor of agriculture degree from the University of Tokyo in 1991. In 1978, he began employment with Suntory Ltd. as a researcher in the Institute for Alcoholic Beverages, and later, in the Institute for Fundamental Research. He conducted research on bacteria, yeast, fungi, and plant genetics and biochemistry. In 1996, he attended the 62nd ASBC Annual Meeting and made a presentation on “Rapid Methods of Detecting Beer Spoilage Yeasts by Using Polymerase Chain Reaction.” Since April 2001, he has served in the Quality Assurance Division, in which he has developed several identification techniques for plant and living organisms using DNA analysis.