pubs.acs.org/JAFC Published on Web 05/18/2009 © 2009 American Chemical Society 4620 J. Agric. Food Chem. 2009, 57, 4620–4625 DOI:10.1021/jf900394t Cross-Fertilization for Enhancing Tocotrienol Biosynthesis in Rice Plants and QTL Analysis of Their F 2 Progenies PHUMON SOOKWONG, †,^ KAZUMASA MURATA, ‡,^ KIYOTAKA NAKAGAWA, † AKIRA SHIBATA, † TOSHIYUKI KIMURA, § MASAYUKI YAMAGUCHI, § YOICHIRO KOJIMA, ) AND TERUO MIYAZAWA* ,† † Food & Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan, ‡ Toyama Prefectural Agricultural, Forestry and Fisheries Research Center, Toyama 939-8153, Japan, § Japan National Agricultural Research Center for Tohoku Region, Fukushima 960-2156, Japan, and ) Takaoka Agriculture Forestry Promotion Center, Takaoka 933-0806, Japan. ^ These authors made equal contributions to this work. As rice bran tocotrienol (T3) has been known to have a wide range of physiological functions (e.g., antiangiogenesis), we aimed at developing a T3-rich rice variety for nutraceutical purposes. T3 content in more than 250 kinds of rice bran samples were investigated, and Milyang23 was found as the best variety rich in T3. The variety was therefore chosen for cross-fertilization with Koshihikari. Among obtained F 2 progenies, some of them became improved in T3 content (up to 2-fold of reference Koshihikari). QTL analysis of the F 2 progenies revealed five putative loci corresponding to T3 biosynthesis, in which the main loci were located near a marker RM3827 on chromosome 6. The results show that cross-breeding is effective in improving rice bran T3 and provides more genetic understanding on T3 biosynthesis in rice plants. KEYWORDS: Oryza sativa L.; quantitative trait locus; tocotrienol; tocopherol INTRODUCTION Rice bran, the combined part of pericarp, seed coat, nucellus, and aleurone layer or rice seeds, has been known to contain functional compounds such as tocotrienol (T3) and tocopherol (Toc). Among these compounds, T3, an unsaturated form of vitamin E with three double bonds in its isoprenoid side chain (Figure 1), has recently been receiving considerable attention for its several biological properties (1 ). T3 shows better antioxidative (2 ), antihypercholesterolemic (3 ), anticancer (4 ), and neuroprotective activities (5 ) than Toc. In addition, we have found that T3 suppresses pathological angiogenesis (6-8), which is the important stage in the progression of some disorders (i.e., diabetic retino- pathy, rheumatoid arthritis, and cancers). These findings suggest that T3 has a wide range of physiological functions, and develop- ing a rice variety that can biosynthesize high amounts of T3 would be useful for nutraceutical applications. Considering the biosynthesis of T3 in plants (e.g., rice and barley), T3 is synthesized together with Toc in plastids from precursors derived from the shikimate and methylerythritol phosphate pathways (9, 10), and homogentisic acid geranylger- anyl transferase (HGGT, belonging to plant prenyltransferases) has been believed to work as the key enzyme for regulating T3 production. In support of this, it was reported that transgenic expression of the barley HGGT in Arabidopsis thaliana leaves resulted in the accumulation of T3, which was absent in its nontransgenic leaves (11 ). Therefore, biosynthesis of T3 in plants would be genetically controlled, but the genetic regulation of activity of HGGT as well as other enzymes for T3 production in rice has been poorly understood. In our previous study, we found a wide variation of T3 content among several rice bran samples (12 ), which may be an outcome of their difference in genetic characteristics for T3 production. The findings hypothe- size that cross-breeding would be effective in improving T3 content in rice cultivars by genetically increasing the activity of the enzymes (e.g., HGGT) for T3 biosynthesis. Accordingly, as we aim at developing a rice variety that can synthesize a high level of T3, a series of studies was conducted. A number of rice bran samples were determined for their T3 and Toc contents by using our previously developed method (12 ), and the best rice cultivar that was able to produce a reliable high content of T3 was then chosen. The chosen variety was crossed with japonica Koshihikari, the most popular rice variety in Japan, resulting in a wide distribution of T3 content in their F 2 progenies. Some of the F 2 individuals showed markedly high T3 levels, and the F 2 progenies were useful in evaluating the T3 biosynthesis in rice plants by using a quantitative trait locus (QTL) analysis. MATERIALS AND METHODS Chemicals. Four isomers of T3 (R-, β-, γ-, and δ-T3) were gifts from Eisai (Tokyo, Japan). Four isomers of Toc (R-, β-, γ-, and δ-Toc) and 2- propanol were obtained from Wako (Osaka, Japan). All reagents used were of analytical grade. Rice Bran Samples and Vitamin E Analysis. The seeds of more than 250 kinds of rice varieties including the rice diversity research set of germplasm (RDRS) provided by National Institute of Agrobiological *Corresponding author. Tel: +81-22-717-8904. Fax: +81-22-717- 8905. E-mail: miyazawa@biochem.tohoku.ac.jp.