A report of the project "Polymorphism survey among hexaploid wheat and its relatives by DNA markers" granted by the National Bioresource Project-Wheat, Japan

A report of the project “Polymorphism survey among hexaploid wheat and its relatives by DNA markers” granted by the National Bioresource Project-Wheat, Japan

Miyuki Nitta and Shuhei Nasuda

Laboratory of Plant Genetics, Graduate School of Agriculture, Kyoto University, Kitashirakawaoiwake-cho, Sakyo-ku, Kyoto 606-8502 Japan

Corresponding author: Shuhei Nasuda

E-mail: nasushu@kais.kyoto-u.ac.jp

The National Bioresource Project-Wheat (NBRP-Wheat), launched by the Japanese government in 2002, is aimed to maintain and distribute seed stocks and DNA clones of wheat. Additionally to its primary roles in handling seed stocks and DNA clones, the second-term NBRP-Wheat, started in 2007, features the collection and characterization of DNA markers. The objectives of the project are, (1) to make the resources of the NBRP-Wheat more valuable for molecular studies by addition of genotype information, and (2) to find a set of DNA markers that is suitable for detecting polymorphisms among wheat samples. The outline of the current project is illustrated in Figure 1.

Simple sequence repeat (SSR) is a PCR-based method to detect polymorphisms. SSR is widely used in wheat sciences such as linkage mapping, QTL mapping, marker-assisted selection, and phylogenetic studies. We obtained primer information of more than 3000 SSR markers from the publications and public databases. Avoiding duplications, we synthesized a total of 2545 primer sets (1861 SSR primers and 684 STM primers). The markers consisted primary of barc (Song et al. 2005), cfa and cfd (Guyomarc’h et al. 2002; Sourdille et al. 2004), gdm (Pestsove et al. 2000), gwm (Röder et al. 1995, 1998), hbg, hbe, and hbd (Torada et al. 2006), and wmc (Gupta et al. 2002; Somers et al. 2004) markers. Primers for the STM markers were synthesized according to Hayden et al. (2002, 2004).

The 48 plant lines subjected to polymorphism survey are listed in Table 1. The lines to be tested includes; eight Aegilops species with representative diploid genomes, Triticum monococcum, T. boeoticum, T. urartu, T. durum, T. spelta and 31 hexaploid wheat accessions. We also took samples of Hordeum vulgare, H. spontaneum, and Secale cereale as outgroup species.

Plant DNA was isolated from young fresh leaves with the DNeasy Plant Mini Kit (QIAGEN) and quantified by a spectrophotometer. PCR reaction was carried out with the iCycler (BioRad). On our hands, the optimum PCR condition was achieved by addition of betaine and DMSO into the reaction mixture at the final concentrations of 1 M and 3%, respectively. The PCR products were subjected to electrophoresis in a capillary gel-electrophoresis apparatus HAD-GT12 (eGene, now available from QIAGEN) with a gel cartridge GCK-5000. Electrophoretic patterns were analyzed using the software Biocalculator supplied with the electrophoresis apparatus.

We started our survey with those SSR markers mapped by Somers et al. (2004). So far, we obtained amplification profiles of 717 SSR primers for the 36 lines, and 222 SSR markers for the rest 12 lines (in total, we obtained 28476 amplification profiles). Due to the technical limitations of the capillary gel-electrophoresis, we encounter difficulties in detecting polymorphism with small differences in fragment lengths. We are planning to test at least 2000 markers on the 48 samples by the end of year 2010, resulting in 96000 PCR profiles. We are in discussion with Dr. Yukiko Yamazaki at National Institute of Genetics, Japan for the method of presentation of our data through the KOMUGI web site (http://www.shigen.nig.ac.jp/wheat/komugi/top/top.jsp). We welcome your valuable comments for establishing a user-friendly database.

Acknowledgements

We thank the following wheat researchers for their providing plant materials; Drs. T. Ban, T. R. Endo, M. Fujita, B. Friebe, S. Ikeguchi, K. Kato, T. Kawahara, H. Matsunaga, Y. Matsuoka, H. Miura, K. Murai, K. Nakamura, Z. Nishio, K. Sato, T. Terachi, H. Tsujimoto, and Y. Yoshimura. We especially thank Dr. Masaya Fujita for his generous coordination of the lines used in Japanese breeding programs. We express our gratitude to Drs. G. Ishikawa, K. Kato, Y. Matsuoka, and S. Takumi for their valuable advices. This work is supported by the National Bioresorce Project-Wheat, the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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