Development of Dominant Rice Blast Pi-ta Resistance Gene Markers

genotypes can be easily identified (Huang et al., 1997; Hittalmani et al., 2000). A PCR-based Pi-ta gene marker Incorporation of resistance genes into existing rice (Oryza sativa is useful in marker-assisted selection breeding since it L.) cultivars is a powerful strategy and is commonly applied in breeding rice resistance to blast disease [caused by Pyricularia grisea Sacc. is the part of resistance gene, and is simple, rapid and P. oryzae Cavara (teleomorph: Magnaporthe grisea (Hebert) Barr)]. inexpensive and can be used for analyzing large numbers The rice blast resistance gene, Pi-ta, originally introgressed into japonof samples. The Pi-ta gene marker is important for rice ica from indica rice is important in breeding for rice blast resistance breeding program worldwide (Bryan et al., 2000; Hittalworldwide. In the southern USA, the rice cultivar Katy contains Pimani et al., 2000; Inukai et al., 1994). In the southern ta and is resistant to the predominant blast M. grisea races IB-49 and USA breeding programs, Katy, a japonica rice cultivar IC-17 and has been used as the blast resistant breeding parent. Three containing a tightly linked cluster of at least seven resispairs of DNA primers specific to the dominant indica Pi-ta gene were tance genes near the Pi-ta locus, has been used as a designed to amplify the Pi-ta DNA fragments by polymerase chain blast resistant parent (Chao et al., 1999; Moldenhauer reaction (PCR). PCR products amplified by these Pi-ta specific primet al., 1990). Resulting progenies, such as ‘Drew’ and ers were cloned and sequenced. Sequence analysis confirmed the presence of the dominant indica Pi-ta allele. These Pi-ta primers ‘Kaybonnet’, have been successfully released as U.S. were used to examine the presence of Pi-ta alleles in advanced Arkanblast-resistant cultivars (Gravois et al., 1995; Moldensas rice breeding lines. The Pi-ta containing rice lines, as determined hauer et al., 1998). More breeding lines based on Katy, by PCR analysis, were resistant to both IB-49 and IC-17 in standard Drew, and Kaybonnet as parents are still in the early pathogenicity assays. In contrast, lines lacking the Pi-ta genes failed trials (K. Moldenhauer and J. Gibbons, per. commun.). to protect rice plants against both races IB-49 and IC-17. The presence The objectives of this research were to develop Pi-ta of Pi-ta markers correlated with the Pi-ta resistance spectrum. Thus, gene-specific primers to distinguish the dominant indica the Pi-ta gene markers provide a basis for stacking other blast resisPi-ta allele from the japonica pi-ta allele, to examine the tance genes into high yielding and good quality advanced breeding presence of Pi-ta in 10 advanced Arkansas rice breeding rice lines. lines, and to determine disease reactions of these lines to confirm the reliability of the Pi-ta gene marker. R blast disease caused by the fungus is one of the most devastating diseases worldwide (Zeigler MATERIALS AND METHODS et al., 1994). Resistance to the pathogen is a classic Plant Materials and Growth gene-for-gene system, where a major resistance gene is effective against M. grisea strains containing the correRice cultivars Katy (Moldenhauer et al., 1990), Drew (Moldenhauer et al., 1998), Kaybonnet (Gravois et al., 1995), ‘Nipsponding avirulence gene (Silue et al., 1992). Twenty ponbare’, ‘M-202’ (Johnson et al., 1986), and 10 advanced rice resistance genes have been identified by extensive gebreeding lines (experimental seeds) used in this study (Table netic studies (Chao et al., 1999; Mackill and Bonman, 1) were provided by Karen Moldenhauer and James Gibbons. 1992; Yu et al., 1996). Pi-b and Pi-ta, two major resisSeed was pregerminated on moistened filter paper for 3 d at tance genes, introgressed from indica cultivars, have 30 C. Seedlings were transplanted to 12.5-cm pots with a media recently been molecularly characterized (Bryan et al., mixture of one part sterilized local soil to one part RediEarth 2000; Inukai et al., 1994; Wang et al., 1999). Both Pi-b potting mix (Hummert, Earth City, MO). RediEarth potting and Pi-ta encode predicted nucleotide binding site type is a porous lightweight, essentially sterile growing medium. It proteins that are characteristics of products of major contains Vermiculite and Canadian sphagnum peat moss, the resistance genes (Wang et al., 1999; Bryan et al., 2000; soil conditioners that help retain moisture and add aeration for plant roots. Plants were grown in a greenhouse 24 to 30 C Wise, 2000). Intriguingly, a single amino acid difference with 16 h light for 2 to 4 wk until plants were at the 4-leaf between resistant and susceptible indica alleles of pi-ta stage for disease reaction testing and for DNA preparation. was identified from an indica cultivar C101A51 (Bryan et al., 2000). Further DNA sequence analysis of a japonDNA Isolation ica susceptible pi-ta allele revealed unusually low DNA polymorphism to indica resistant Pi-ta allele (Bryan et Rice leaves were rapidly frozen in liquid nitrogen and stored al., 2000; Jia et al., 2001b). at 80 C. Rice genomic DNAs were prepared from frozen Molecular markers linked to resistance genes have leaves using DNeasy Plant Mini Kit (Qiagen, Valencia, CA) been used for selection at the early seedling stages, and according to the manufacturer’s protocol. Rice leaves were ground in liquid nitrogen to a fine powder using a mortar and pestle. DNAs were extracted using manufacturer’s solutions Yulin Jia and Pratibha Singh, USDA-ARS, Dale Bumpers National and purified by DNeasy columns. Rice Research Center, P. O. Box 1090, Stuttgart, AR 72160-0287; Zhonghua Wang, Institute of Nuclear Agricultural Sciences, Zhejiang Univ., Hangzhou, P. R. China 310029. Received 8 Feb. 2002. *CorrePCR Amplification sponding author ([email protected]). Primers (Table 2) synthesized by Operon Technologies, Inc. (Alameda, CA) were used for the amplification of the Published in Crop Sci. 42:2145–2149 (2002).