Crop Science 42:2145-2149 (2002)
© 2002 Crop Science Society of America
CELL BIOLOGY & MOLECULAR GENETICS
Development of Dominant Rice Blast Pi-ta Resistance Gene Markers
Yulin Jia*,a,
Zhonghua Wangb and
Pratibha Singha
a USDA-ARS, Dale Bumpers National Rice Research Center, P. O. Box 1090, Stuttgart, AR 72160-0287
b Institute of Nuclear Agricultural Sciences, Zhejiang Univ., Hangzhou, P. R. China 310029
* Corresponding author (yjia{at}spa.ars.usda.gov)
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ABSTRACT
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Incorporation of resistance genes into existing rice (Oryza sativa L.) cultivars is a powerful strategy and is commonly applied in breeding rice resistance to blast disease [caused by Pyricularia grisea Sacc. = P. oryzae Cavara (teleomorph: Magnaporthe grisea (Hebert) Barr)]. The rice blast resistance gene, Pi-ta, originally introgressed into japonica from indica rice is important in breeding for rice blast resistance worldwide. In the southern USA, the rice cultivar Katy contains Pi-ta and is resistant to the predominant blast M. grisea races IB-49 and IC-17 and has been used as the blast resistant breeding parent. Three pairs of DNA primers specific to the dominant indica Pi-ta gene were designed to amplify the Pi-ta DNA fragments by polymerase chain reaction (PCR). PCR products amplified by these Pi-ta specific primers were cloned and sequenced. Sequence analysis confirmed the presence of the dominant indica Pi-ta allele. These Pi-ta primers were used to examine the presence of Pi-ta alleles in advanced Arkansas rice breeding lines. The Pi-ta containing rice lines, as determined by PCR analysis, were resistant to both IB-49 and IC-17 in standard pathogenicity assays. In contrast, lines lacking the Pi-ta genes failed to protect rice plants against both races IB-49 and IC-17. The presence of Pi-ta markers correlated with the Pi-ta resistance spectrum. Thus, the Pi-ta gene markers provide a basis for stacking other blast resistance genes into high yielding and good quality advanced breeding rice lines.
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INTRODUCTION
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RICE BLAST DISEASE caused by the fungus is one of the most devastating diseases worldwide (Zeigler et al., 1994). Resistance to the pathogen is a classic gene-for-gene system, where a major resistance gene is effective against M. grisea strains containing the corresponding avirulence gene (Silue et al., 1992). Twenty resistance genes have been identified by extensive genetic studies (Chao et al., 1999; Mackill and Bonman, 1992; Yu et al., 1996). Pi-b and Pi-ta, two major resistance genes, introgressed from indica cultivars, have recently been molecularly characterized (Bryan et al., 2000; Inukai et al., 1994; Wang et al., 1999). Both Pi-b and Pi-ta encode predicted nucleotide binding site type proteins that are characteristics of products of major resistance genes (Wang et al., 1999; Bryan et al., 2000; Wise, 2000). Intriguingly, a single amino acid difference between resistant and susceptible indica alleles of pi-ta was identified from an indica cultivar C101A51 (Bryan et al., 2000). Further DNA sequence analysis of a japonica susceptible pi-ta allele revealed unusually low DNA polymorphism to indica resistant Pi-ta allele (Bryan et al., 2000; Jia et al., 2001b).
Molecular markers linked to resistance genes have been used for selection at the early seedling stages, and genotypes can be easily identified (Huang et al., 1997; Hittalmani et al., 2000). A PCR-based Pi-ta gene marker is useful in marker-assisted selection breeding since it is the part of resistance gene, and is simple, rapid and inexpensive and can be used for analyzing large numbers of samples. The Pi-ta gene marker is important for rice breeding program worldwide (Bryan et al., 2000; Hittalmani et al., 2000; Inukai et al., 1994). In the southern USA breeding programs, Katy, a japonica rice cultivar containing a tightly linked cluster of at least seven resistance genes near the Pi-ta locus, has been used as a blast resistant parent (Chao et al., 1999; Moldenhauer et al., 1990). Resulting progenies, such as ‘Drew’ and ‘Kaybonnet’, have been successfully released as U.S. blast-resistant cultivars (Gravois et al., 1995; Moldenhauer et al., 1998). More breeding lines based on Katy, Drew, and Kaybonnet as parents are still in the early trials (K. Moldenhauer and J. Gibbons, per. commun.).
The objectives of this research were to develop Pi-ta gene-specific primers to distinguish the dominant indica Pi-ta allele from the japonica pi-ta allele, to examine the presence of Pi-ta in 10 advanced Arkansas rice breeding lines, and to determine disease reactions of these lines to confirm the reliability of the Pi-ta gene marker.
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MATERIALS AND METHODS
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Plant Materials and Growth
Rice cultivars Katy (Moldenhauer et al., 1990), Drew (Moldenhauer et al., 1998), Kaybonnet (Gravois et al., 1995), ‘Nipponbare’, ‘M-202’ (Johnson et al., 1986), and 10 advanced rice breeding lines (experimental seeds) used in this study (Table 1)
were provided by Karen Moldenhauer and James Gibbons. Seed was pregerminated on moistened filter paper for 3 d at 30°C. Seedlings were transplanted to 12.5-cm pots with a media mixture of one part sterilized local soil to one part RediEarth potting mix (Hummert, Earth City, MO). RediEarth potting is a porous lightweight, essentially sterile growing medium. It contains Vermiculite and Canadian sphagnum peat moss, the soil conditioners that help retain moisture and add aeration for plant roots. Plants were grown in a greenhouse 24 to 30°C with 16 h light for 2 to 4 wk until plants were at the 4-leaf stage for disease reaction testing and for DNA preparation.
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Table 1. The presence and absence of the Pi-ta genes in named cultivars and selected advanced Arkansas rice breeding lines.
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DNA Isolation
Rice leaves were rapidly frozen in liquid nitrogen and stored at -80°C. Rice genomic DNAs were prepared from frozen leaves using DNeasy Plant Mini Kit (Qiagen, Valencia, CA) 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 and purified by DNeasy columns.
PCR Amplification
Primers (Table 2)
synthesized by Operon Technologies, Inc. (Alameda, CA) were used for the amplification of the genomic DNA from the rice cultivars and advanced breeding lines by PCR. Each PCR reaction consisted of 10 to 20 ng of total DNA, 10 mL of Taq PCR Master Mix [(2x concentrated) containing 0.5 unit Taq DNA Polymerase, Qiagen PCR Buffer (with 3 mmol MgCl2)] and 400 mmol of each dNTP (Taq PCR Master Mix Kit, Qiagen), 2 µL of MgCl2 (25 mmol), 1mL of primer 1 (10 mmol), and primer 2 (10 mmol) each in a final volume of 20 mL. The PCR reactions were performed in a Peltier Thermal Cycler (PTC-20, MJ Research, Waltham, MA) with the following program: 3 min at 95°C for initial denaturation followed by 29 cycles of 30 s at 95°C, 30 s at 55°C for YL 153/YL154 and YL155/YL87 and at 64.5°C for YL100/YL102, 30 s each at 72°C; and final extension at 72°C for 7 min. Aliquots (10 µL) of each of the PCR products were separated by electrophoresis on 1.5% (w/v) agarose gels in 1x TBE buffer, then stained in ethidium bromide and visualized by means of an ultraviolet transilluminator.
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Table 2. Sequences of 19- or 20-mer oligonucleotide primers for the dominant Pi-ta specific markers and optimized annealing temperatures.
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Cloning and Sequence of PCR Products
PCR products amplified from rice cultivars Katy, Kaybonnet, and Drew were cloned into pDrive Cloning Vector modified on the basis of the manufacturer's recommendation (Qiagen). Briefly, 1 µL pDrive vector was mixed with 1 to 4 µL of the PCR product and 5 µL of the ligation master mix for 1 h at 4°C and transformed by electroporation. Resulting plasmids were identified by blue-white selection and plasmid DNA was purified by means of Qiagen Plasmid Mini Preparation Kits. The sequences of inserts were determined by means of ABI-PRISM BigDye Terminator Cycle Sequencing on ABI 377 (Applied Biosystems, Foster City, CA). Sequence analysis was performed with Vector NTI suite (InforMax).
Disease Reactions
Magnaporthe grisea race IB-49 (ZN52) and IC-17 (ZN57) (Correll et al., 2000) were provided by Fleet N. Lee (Professor of Plant Pathology, University of Arkansas, Rice Research and Extension Center). Disease reactions of breeding lines were performed with standard pathogenicity assays (Valent et al., 1991). Briefly, plants were grown in a greenhouse, to the three to four-leaf stage, and then inoculated with 2 mL of spore suspensions (2.5 x 105 spores/mL) with an airbrush. Plants were inoculated inside a plastic bag that was then sealed to maintain high humidity. After 24 h, the plants were removed from the bags and returned to the greenhouse. Disease reactions were determined 7 d after inoculation. Resistant reaction was based on no visible infection and no conidia produced from affected tissue. Susceptible reaction was based on a lesion size greater than 3 cm in length, visible infection, and conidia evident in affected tissue (Valent, 1997). The resistance and susceptibility of each cultivar and line were determined on the basis of obtaining the same disease reactions using three plants in each pot with three repeats.
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RESULTS
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Development of the Indica Pi-ta Gene Markers
To develop a Pi-ta gene specific primer, the nucleotide that distinguishes the dominant indica Pi-ta allele from susceptible japonica pi-ta alleles was included as the last base (Jia et al., 2001b). Two pairs of primers, YL153/YL154 and YL100/YL102 (Table 2, Fig. 1)
, were designed to amplify specifically regions of the Pi-ta gene encoding the translation start site (YL153/YL154) and the carboxyl terminus (YL100/YL102). The specificity of PCR reaction can be affected by the annealing temperature. The optimal annealing temperature for each set of primers is listed in Table 2. Pi-ta containing rice cultivars Katy and Drew (Jia et al., 2001b) were used as positive controls. Rice cultivar Nipponbare that does not contain Pi-ta (Bryan et al., 2000) was used as the negative control. As shown in Fig. 1B, DNA fragments of 403 (YL100/YL102) and 440 base pairs (bp) (YL153/YL154) were amplified from Katy and Drew, respectively, and the same size fragments were also amplified from Kaybonnet suggesting that Pi-ta is also present in Kaybonnet. The absence of PCR product from M-202 indicates that it does not contain Pi-ta. These results suggest that both primer pairs were specific for the dominant indica Pi-ta allele.
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Fig. 1. A schematic representation of the double strands of the 6927 bp of the Pi-ta gene (A) and PCR products from genomic DNA prepared from Katy (1), Drew (2), Kaybonnet (3), Nipponbare (4), and M-202 (5) amplified by the dominant Pi-ta-specific primers. Line 6 is the water control (B). Names and locations of primers and region amplified by PCR using primers YL153 and YL154 (i), YL155/YL87 (ii), and YL100/YL102 (iii) are shown in A. The sizes of fragments were estimated by mean of kilobase markers. ATG indicates the translation start site and TGA indicates the termination site.
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To verify the presence of the entire Pi-ta gene, DNA primers YL155/YL87 specific to the middle region were designed for the PCR amplification. Consistent with results obtained by primers YL100/YL102 and YL153/YL154, a predicted fragment of 1042 bp was also specifically amplified from Katy, Drew, and Kaybonnet. Similarly, no amplification was obtained from Nipponbare or M-202 (Fig. 1B).
To verify the amplification of the dominant indica Pi-ta allele, PCR products were cloned into pDrive vector, and sequenced. Searching the current GenBank database (http://www.ncbi.nlm.nih.gov/blast; verified May 22, 2002) for sequences of all PCR products revealed 100% identity with DNA fragments from nucleotide positions 2021-2442 (YL153 and YL154), 4409-5450 (YL155 and YL87), and 6257 to 6659 (YL100 and YL102) of the Pi-ta gene (Table 2, GenBank accession no. AF207842). These results indicate that portions of the indica dominant Pi-ta alleles were amplified from Katy, Drew, and Kaybonnet.
Detecting the Presence of Pi-ta in Advanced Arkansas Breeding Lines by PCR
To demonstrate the utilization of Pi-ta gene markers in breeding, 10 advanced breeding lines based on Katy, Drew, and Kaybonnet as parents were used for PCR amplification. Katy, Drew, and Kaybonnet were used as positive controls and Nipponbare and M-202 were used as negative controls. The presence and absence of the Pi-ta genes were all consistent with all three dominant primer pairs (YL153/YL154, YL155/YL87, and YL100/YL102). As shown in Table 1, five of 10 breeding lines contain the Pi-ta gene and the other lines do not contain the Pi-ta gene.
Disease Reactions to Predominant Arkansas M. grisea Races IB-49 and IC-17
Katy, Drew, and Kaybonnet, which are resistant to IB-49 and IC-17 (Jia et al., 2001a; Moldenhauer et al., 1992), were used as positive controls. M-202, which is susceptible to IB-49 and IC-17, was used as a negative control (Jia et al., 2001a). The controls, Nipponbare, and advanced breeding lines were inoculated with avirulent M. grisea races IB-49 and IC-17 to confirm the spectrum of the Pi-ta resistance. As shown in Table 1, Nipponbare, lacking Pi-ta, was susceptible to both IB-49 and IC-17. All Pi-ta containing lines were resistant to both IB-49 and IC-17 (Table 1). On the other hand, those rice lines that do not contain Pi-ta, as determined by PCR analysis, were susceptible to IB-49 and IC-17 (Table 1). Thus, the presence of the Pi-ta marker correlated well with the spectrum of Pi-ta resistance.
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DISCUSSION
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Two major challenges to rice breeders are the selection of appropriate resistance genes and prediction of the stability of resistance in rice cultivars with such gene combinations. A simple but efficient method to analyze blast resistance genes is necessary for rice breeding programs where many cultivars with many resistance genes are used for crossing and many breeding lines are maintained. The genotypes of major genes for blast resistance can be deduced through their reaction patterns following inoculations with blast isolates that attack known resistance genes. Rice blast follows a gene-for-gene recognition where a major resistance gene specifically interacts with the corresponding fungal avirulence gene (Silue et al., 1992). The instability of avirulence genes in the rice blast fungus (Valent, 1997) and overlapping spectra of race-specific resistance are major limiting factors. For rapid development of resistant cultivars, resistance-gene-mediated selection for gene stacking is one of the more reliable approaches. Cloning resistance genes have made it possible to develop resistance gene markers.
We have developed optimal condition for utilizing a dominant Pi-ta gene marker. DNA polymorphisms of nucleotides between resistant indica Pi-ta allele and susceptible japonica pi-ta alleles (Jia et al., 2001b) allowed us to develop the Pi-ta gene markers. DNA primers were designed to perform in PCR conditions as defined above (Table 2), and only the resistant indica Pi-ta allele were amplified. Only two nucleotides in each pair of the primers (YL100/YL102 and YL153/YL154) distinguish them from the recessive japonica pi-ta allele. Resulting PCR products distinguish the dominant indica Pi-ta allele from the recessive japonica pi-ta allele. Precautions were taken throughout this work to avoid the introduction of contaminating DNA. High quality DNA polymerase, MgCl2 concentration, and annealing temperatures for each primer set were all critical factors that contributed to the successful determination of Pi-ta gene presence. Primers that have been thawed repeatedly are not recommended for the amplification. Primers YL100/YL102 distinguishing dominant indica Pi-ta allele with a single nucleotide from recessive indica pi-ta allele (Bryan et al., 2000) amplify both dominant and recessive indica Pi-ta alleles (Wang and Jia, unpublished data). Determination of the dominant Pi-ta alleles in indica breeding lines still awaits the sequence confirmation of PCR products amplified byYL100/YL102 primers.
In the southern USA, blast resistance provided by Katy has lasted over the decade since its release. Katy contains a cluster of resistance genes at the Pi-ta region near the centromere of chromosome 12 (Bryan et al., 2000; Chao et al., 1999; Moldenhauer et al., 1992). Decreased recombination near the centromere (Bryan et al., 2000) may facilitate the incorporation of tightly linked resistance genes into new breeding lines by means of these Pi-ta gene markers. Thus, the Pi-ta gene markers may serve as an indicator for a cluster of resistance genes. Selection of breeding parents for a broader-spectrum blast resistance may be achieved with these Pi-ta gene markers.
Using Pi-ta gene specific PCR not only assists plant breeders in making parental selection but also can facilitate stacking resistance genes into advanced breeding lines. If advanced breeding lines occur that are resistant to IB-49 and IC-17 but do not contain Pi-ta, then these lines may contain other resistance genes against IB-49 and IC-17. Subsequent incorporation of Pi-ta and other resistance genes will lead to broader spectra of the resistance. However, if advanced breeding lines occur that are resistant to IB-49 and IC-17 and contain Pi-ta, the presence of other blast resistance genes can be determined by virulent fungal isolates that infect Pi-ta containing lines. Subsequent resistant reactions indicate that these lines also contain other blast resistance genes. Thus, Pi-ta is stacked with other resistance genes into advanced breeding lines. Alternatively, selection for other resistance genes can be achieved through marker-assisted breeding (Huang et al., 1997; Hittalmani et al., 2000).
In this study, we demonstrated the development of the first dominant resistance gene markers using the DNA sequence of the cloned gene in the rice blast system. Increasing efforts to clone more resistance genes (Wang et al., 1999; Wise, 2000) worldwide will accelerate the development of more dominant resistance gene markers for molecular breeding, thereby accelerating introduction of durable, broad-spectrum disease resistance into high yield, good quality rice cultivars.
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ACKNOWLEDGMENTS
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We thank Lori Imboden and Andrea L. Blas for technical support. For critical reading we thank Drs. J. Neil Rutger, Yinong Yang, Hong Li Wang, and members of Molecular Plant Pathology Program at DB NRRC. We thank Dr. Barbara Valent for sharing unpublished data of DNA polymorphisms of dominant and recessive Pi-ta alleles. We thank Drs. James Gibbon and Karen Moldenhauer for providing rice cultivars and advanced breeding lines. We thank Drs. Fleet N. Lee and James Correll for providing fungal isolates. This research was funded in part by grants from the Arkansas Rice Research and Promotion Board.
Received for publication February 8, 2002.
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ABSTRACT
INTRODUCTION
