|Pedigree and DNA marker analysis of Blast resistance genes in US rice|
Blast disease caused by Pyricularia grisea causes economic losses in most rice growing regions of the world. The number of blast pathotypes that occur in the United States is relatively small due to a limited rice growing region and weather conditions which are unfavorable for disease development. As a result, the seven Pi- genes that have been widely used by U.S. breeders have proven to be adequate for disease control. However, selection for the presence of these genes has relied upon labor-intensive phenotypic screening against individual races of blast.
* Develop genetic markers that are closely associated with Pi-genes that can be used for development of improved rice cultivars in germplasm relevant to U.S. rice breeding programs.
* Determine the value of co-dominant single sequence repeat (SSR) markers closely linked with Pi-genes by analyzing their degree of polymorphism in germplasm relevant to U.S. rice breeding programs.
Materials and Methods
Genetic markers were developed by analysis of various genetic crosses made between cultivars known to possess individual Pi- genes and cultivars lacking these resistance genes (Fjellstrom, et al 2000). Segregating progeny were evaluated for reaction to specific pathotypes, indicative of individual Pi- genes (Fig. 1), and for marker genotype. Dominant markers for Pi-ta (Jia et al. 2002) and Pi-b (Fjellstrom et al. in preparation) were used to verify the presence of these resistance genes. Genetic maps of resistance genes and markers were constructed (Fig. 2). A U.S. rice pedigree tree as described by Ayres et al. (1997) was expanded to include current cultivars. Based upon the pedigree association of these cultivars, their reaction to pathotypes, and results from genetic marker analysis of over 100 cultivars (subset shown in (Table 1), the lineage of specific Pi- genes in U.S. germplasm was determined.
Results and Discussion
DNA markers have been developed that show a strong association with five Pi- genes that are currently used in U.S. breeding programs for the development of rice cultivars having improved resistance to blast disease. A small number of haplotypes, unique marker allele combinations, characterize each of the five Pi- genes (Table 2). The closer an individual marker is to the gene, the stronger the association with the resistance gene (see RM 224 and RM 208, (Fig. 2). In segregating progeny, these markers for Pi- genes have proven to be more accurate indicators of genetic resistance than traditional phenotypic screening methods.
The Pi-k region located on chromosome 11 possesses two alleles (- kh and -ks) that have been widely utilized in U.S. breeding efforts to help control blast disease. The Pi-kh gene is commonly found in many U.S. long grains, whereas the Pi-ks allele is predominantly found in medium grain cultivars. Preliminary results indicate that the Pi-Leah gene is also in this region of chromosome 11. Pi-Leah offers resistance to only three of the four pathotypes that Pi- kh does (Fig. 1), and has had more limited use in cultivar development. The original sources of the Pi-kh, Pi-ks, and Pi-Leah genes appear to have come from CI 5309 (selection from China), Blue Rose (selection from Japan), and an unknown red rice outcross, respectively. According to the haplotype, to confidently select for a particular allele in a breeding population segregating for any two of these genes (Pi-kh, Pi-ks, or Pi-Leah), at least two of the three markers would need to be evaluated.
Three markers closely linked to the Pi-ta2 gene have been identified on chromosome 12 (Fig. 2). The source of Pi-ta2 in U.S. germplasm traces to the cultivar Tetep from Vietnam. The Pi-ta2 gene is found in the Tetep accession PI 280682 used in the development of Katy and is also in the Katy derivatives, Kaybonnet, Drew, and Madison. However, our analysis of other sources of Tetep in the U.S. germplasm collection (i.e., GRIN) indicates that not all possess Pi-ta2. The markers for Pi-ta2 are also found in the indica cultivars Jasmine 85, Khao Dawk Mali 105, IR8, and IR36 although, on the basis of the dominant Pi-ta marker, they appear to lack the Pi-ta2 gene. The DNA markers associated with Pi-ta2 appear to be of indica origin and are associated with the Pi-ta2 gene when introgressed into U.S. (japonica) germplasm, however this may not always be the case in other indica materials. Thus, when considering the use of indica germplasm as a resource for the Pi-ta2 gene, the Pi-ta dominant marker should be used to first verify its presence. Due to the broad spectrum of races in the U.S. that Pi-ta2 provides resistance against, it can mask the presence of other Pi- genes. Our research has demonstrated that Pi-ta2 masks the presence of Pi-ks in Drew and Katy, Pi-kh in Kaybonnet, and Pi-ks and Pi-kh (heterogeneous mixture) in Madison. However, the presence of Pi-ks in Katy cannot be explained by its reported parentage. Previous research has shown that the Waxy allele found in Katy is also different from its parents (data not shown). As a result of the heterogeneity found Madison, it is being purified to have Pi-kh and Pi-ta2. The presence of the two Pi- genes in these cultivars will likely increase the durability of resistance.
Pi-b is a new gene introgressed into U.S. germplasm tracing to the cultivar Te Qing, introduced from China. Our research has determined that this gene provides resistance to a wide spectrum of U.S. pathotypes (Fig. 1). The dominant Pi-b marker was used to verify the presence of the Pi-b gene in Bolivar, Saber, and LSBR33. LSBR33 and LSBR5 are registered as somaclonal variants of Labelle, which does not possess Pi-b nor other DNA markers found in these LSBR lines (Table 2). The indica cultivars, Tadukan, Tetep, and Taichung Native 1 do not have the dominant Pi-b marker but have two of the three DNA markers associated with this gene. Thus, like the markers near Pi-ta2, these markers are likely of indica origin and the presence of the Pi-b gene may need to be verified by the dominant marker.
DNA markers have been developed that show a strong association with the important Pi- genes currently used in U.S. cultivar development programs. Use of these markers will improve the efficiency and effectiveness of developing rice cultivars having improved resistance to blast disease. These markers more accurately identify genetic resistance than traditional phenotypic methods and will result in savings of time and labor, as well as allow selections independent of field seasons. The cost of marker analysis will decrease as additional markers are used for other traits; e.g., markers for amylose content, aroma, grain elongation, and semidwarfism that we currently have and new ones as they become available. Research is continuing to develop DNA markers for the Pi-i gene as well as identifying new genetic sources of resistance to blast.
Financial support provided by The U.S. Rice Foundation, the Texas Grass and Grain Initiative, The Texas Rice Research Foundation, and the Texas Advanced Technology Program.
Ayres, N.M., A.M. McClung, P.D. Larkin, H.F.J. Bligh, C.A. Jones, and W.D. Park. 1997. Microsatellite and a single-nucleotide polymorphism differentiate apparent amylose classes in an extended pedigree of US rice germplasm Theor. Appl. Genet. 94:773-781.
Fjellstrom, R.G., C. Conaway, W.D. Park, M.A. Marchetti, and A.M. McClung. 2000. Utilization of molecular markers for selection of blast resistance in USA rice breeding lines. In Proc. Fourth Intl. Rice Genet. Symp. IRRI, Manila, Philippines.
Jia, Y., Z. Wang, and P. Singh. 2002. Development of dominant rice blast Pi-ta resistance gene markers. Crop Sci. 4: 21452149.