QTL Analysis for Plant and Grain Characters of Sake-Brewing Rice Using a Doubled Haploid Population

Breeding Science 52 : 309-317 (2002) QTL Analysis for Plant and Grain Characters of Sake-brewing Rice Using a Doubled Haploid Population Shinya Yoshida*1), Masaru Ikegami1), Junko Kuze2), Keiko Sawada2), Zentaro Hashimoto3), Takashige Ishii2), Chiharu Nakamura3) and Osamu Kamijima2) 1) Hyogo Prefectural Research Center for Agriculture, Forestry and Fisheries, 1533 Minamino-oka, Befu, Kasai, Hyogo 679-0198, Japan 2) Laboratory of Plant Breeding, Faculty of Agriculture, Kobe University, 1-1 Rokkodai, Nada, Kobe, Hyogo 657-8501, Japan 3) Laboratory of Plant Genetics, Faculty of Agriculture, Kobe University, 1-1 Rokkodai, Nada, Kobe, Hyogo 657-8501, Japan Rice (Oryza sativa L.) varieties used for brewing sake Introduction are commonly characterized by traits such as large grain size with white-core (an opaque structure inside Rice (Oryza sativa L.) is one of the most important sta- the rice grain). A linkage map was constructed using ple food crops in the world. In Japan, rice is also used for the doubled haploid lines derived from the cross of Reiho (a production of Japanese rice wine, sake. Sake is brewed from cooking variety) and Yamada-nishiki (a sake-brewing steamed rice through fermentation by koji (Aspergillus variety). Random amplified polymorphic DNA (RAPD), oryzae) and yeast. Rice varieties used strictly for sake- amplified fragment length polymorphism (AFLP) and brewing are characterized by a larger grain size than that of simple sequence repeat (SSR) marker systems were em- ordinary cooking rice, and by the presence of an opaque ployed in QTL analysis. A total of 145 markers were structure named white-core located in the center of rice identified and mapped on rice chromosomes. QTLs for grains. These characters are considered to be suitable for plant and grain characters were detected by interval sake-brewing processes. Contents of proteins, amylose and mapping and single point analysis. Several QTLs with a fatty acids are also known to markedly affect the taste and significant contribution were identified for important flavor of sake. However, the large grain size tends to be as- sake-brewing characters including grain size, grain sociated with several undesirable characters such as white- shape, white-core grain rate and protein content. Sever- belly and cracking of grain. White-belly resembles white- al QTLs simultaneously affected the grain weight, width core as an opaque structure in rice grain, but differs in that it and thickness, while QTLs for the grain length independ- is located on the belly side of grains. Grain size is usually ently affected the grain size. QTLs for the white-core evaluated by the grain weight, but the grain weight may be grain rate did not affect the grain size, although one correlated with sevral characters including grain length, QTL for the white-belly grain rate simultaneously af- grain width and grain thickness. However, the predominant fected the grain weight, width and thickness. Several components in determining the grain size remain largely un- QTLs were detected for the protein content in both known. Genetic studies on the grain size and/or shape have brown and polished rice. One QTL on chromosome 4 been conducted, and in many cases polygenic gene systems that was effective for the decrease of the protein content were implicated. Takita (1985) reported that grain length in polished rice showed a positive relation with the and width were controlled by four to five genes. Kato (1989) grain length. One QTL with the largest effect on the observed additive effects on grain length and width, and grain length on chromosome 11 did not contribute to considered that both traits were controlled by different ge- the decrease of the protein content in polished rice. netic systems. A case of major gene control was reported in Therefore, it is suggested that the grain length QTL on that an incomplete dominant major gene Lk-ƒ controled the chromosome 4 might control not only the grain shape long kernel of the variety Fusayoshi (Takeda and Saito but also the internal structure related to the milling ef- 1980). The grain protein content is also known to be con- ficiency and/or location of the storage protein. trolled by polygenes, although Okamoto (1994) identified one or two major genes affecting the nitrogen content in the Key Words: QTL analysis, sake-brewing rice, grain endosperm. Therefore, it is necessary for the breeding of quality, RAPD, AFLP, SSR. sake-brewing rice to identify the genes involved in the deter- mination of grain characters that are required for sake- brewing, and to define interactions between plant and grain characters. For studying many important agronomic traits in rice, Communicated by C. Kaneda QTL analyses have been applied mainly using restriction Received March 28, 2002. Accepted July 20, 2002. fragment length polymorphism (RFLP) (for a review, see *Corresponding author (e-mail: [email protected]) Yano and Sasaki 1997). Recently, amplified fragment length 310 Yoshida, Ikegami, Kuze, Sawada, Hashimoto, Ishii, Nakamura and Kamijima 2 2 2 2 polymorphism (AFLP) markers (Vos et al. 1995) and micro- according to the following formula: h = σ G ⁄ ()σ G + σ E , 2 2 satellite or simple sequence repeat (SSR) markers have be- where σ G is the genotypic variance and σ E is the environ- come alternative choices for a variety of genetic studies. In mental variance. rice, an SSR map of genome-wide coverage has been con- Rice flour was prepared from brown rice and also from structed and marker information is open to the public 70 % polished rice of each line using CYCLOTEC 1093 (Panaud et al. 1996, Chen et al. 1997, Temnykh et al. 2000). Sample Mill (Tecator, Höganäs, Sweden) with 0.5 mm mesh Here, we constructed a linkage map based on RAPD, or the measurement of the amylose (AM) and protein con- AFLP and SSR markers using anther culture-derived dou- tents (BP for brown rice and PP for polished rice). The amy- bled haploid lines. QTL analysis was conducted to detect lose content was measured using AutoAnalyzer™II significant chromosomal regions controlling plant and grain (BRAN+LUEBBE, Hamburg, Germany) after de- characters important for sake-brewing. granulation of 100 mg polished rice flour by 5 ml of 0.5 N NaOH, according to the method of Inatsu (1988). BP and PP Materials and Methods were measured using InfraAlyzer® 500 (BRAN+LUEBBE). Plant materials RAPD analysis We developed 91 doubled haploid lines (DHLs) Total DNA samples of the 91 DHLs (A1 generation) through anther culture of F1 plants from the cross between and their parental varieties were prepared from leaves at the Reiho (an ordinary cooking rice variety) and Yamada- seedling stage by the CTAB method (Murray and Thompson nishiki (a sake-brewing rice variety). Anthers with micro- 1980). A total of 520 random 10-mer primers (QIAGEN spores of the uninucleate stage were excised from spikes, and Operon, Alameda, CA, USA) were used for RAPD analysis. plated on N6 agar medium (Chu 1975) containing 2 mg/l For the amplification reaction, 20 ng genomic DNA, 1 × 2,4-D, 0.1 mg/l BA and 50 g/l sucrose. Induced calli were buffer (Applied Biosystems, Foster City, CA, USA), 4 pmole plated on MS basal agar medium (Murashige and Skoog arbitrary primer, 2 nmole each of dNTPs and 0.5 unit 1962) containing 1 mg/l NAA, 4 mg/l BA and 50 g/l sucrose. AmpliTaq GOLD (Applied Biosystems) were mixed in a The cultures were incubated at 27°C under a 16-h photoperi- total volume of 20 µl. DNA fragments were amplified using od with a light intensity of ca. 38 µEm−2s−1 provided by cool a thermal cycler GeneAmp9600 (Applied Biosystems) with white fluorescent lamps. Regenerated plants (A0 generation) the following cycling conditions: 1 cycle at 95°C for 5 min, were planted in pots with soil and grown in a greenhouse. 45 cycles at 95°C for 1 min, 37°C for 1 min, 72°C for 1.5 min, The ploidy level of the regenerants (A0) was estimated by and post-extension at 72°C for 5 min. Amplified products the observation of the morphological characters and fertility were separated by electrophoresis through a 1.5 % agarose (selfed seed set) of the panicles, according to the method of gel and stained with ethidium bromide. Nakamura et al. (1994). One panicle from each self- fertilized A0 plant was harvested and maintained as one- AFLP analysis panicle-to-one line (A1 generation). A1 and A2 generations AFLP analysis was conducted by using a high- were cultivated in 1998 and 2000, respectively, in an exper- efficiency AFLP genome-scanning system (Mano et al. imental field. For each line, twenty-five seedlings were 2001), which is a modification of the original AFLP method planted in a single-row plot with a distance of 20 cm between (Vos et al. 1995). Total DNA (150 ng) was digested with plants and a row space of 30 cm. EcoRI and MseI, and EcoRI and MseI adaptors were ligated to the ends of the restriction fragments. Twenty cycles of Data collection and analysis PCR were performed for pre-amplification: 30 sec at 94°C, Five plants per line were randomly selected from the A1 1 min at 56°C and 1 min at 72°C. EcoRI and MseI primers, and A2 generations of DHLs, and culm length (CL), panicle which did not contain additional nucleotides at the 3′ ends, length (PL) and panicle number (PN) were measured. The were used for the pre-amplification. Selective amplification data on the A1 generation were used to evaluate the level of was performed using EcoRI and MseI primers that contained genetic fixation of each line.

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