33. Selection of bacterial blight resistant rice plants in the F2 generation via their linkage to molecular markers


International Rice Research Institute, P.O. Box 933, Manila, Philippines

Tagging of genes of economic importance with DNA markers provides a potentially powerful tool to increase rice breeding efficiency through marker-aided selection (MAS). Many publications have discussed the theoretical considerations, but reports on the actual application of MAS in plant breeding programs are very limited. We report here our preliminary results on the use of DNA markers to predict the genotype of F2 plants for bacterial blight resistance.

In the IRRI breeding program, genes for intermediate amylose content, brown planthopper resistance (Bph-3) and bacterial blight resistance (Xa-21) are being combined into one breeding line. A cross between near isogenic lines of IR24 with genes for intermediate amylose and Xa-21 on one hand and intermediate amylose and Bph-3 on the other was made. F2 populations derived from this cross were homozygous for intermediate amylose content but were segregating for Bph-3 and Xa-21. Seven F2 populations were screened for brown planthopper (BPH) resistance at the seedling stage. Two hundred-eighty-eight BPH resistant seedlings were then transplanted and inoculated with Race 6 of Xanthomonas campestris pv. oryzae (Xco) through the leaf clipping technique (Kauffmann et al. 1973). Since both Bph-3 and Xa-21 are dominant, individuals homozygous and heterozygous for these genes showed the same phenotypic reaction. We were interested in identifying the plants homozygous for Xa-21 using a DNA marker closely linked to this gene. Since this gene has been mapped recently (Ronald et al. 1992) and suitable primers for allele specific PCR (polymerase chain reaction) amplification are available (Chunwongse et al. 1992), we used the rapid PCR procedure to analyze the F2 population.

A single leaf sample was collected from each bacterial blight resistant F2 plant. The leaves were kept in water in separate tubes until testing. The leaves were either boiled in 200 μl 5% Chelex (modified from Chunwongse et al. 1992) or cut to about 0.5 mm2 thin size and directly incorporated in the PCR reaction mix (modified from Berthomieu et al. 1991). Cuts were made by using an emasculation scissor dipped in 70% ethanol and wiped dry before each cut. PCR amplification was carried out using DNA templates from either 5 μl of liquid from boiled leaf cuts or tiny leaf cuts. A set of allele-specific primers for Xa-21 (PB7 and PB8, provided by Susan McCouch), were used in a PCR reaction mix containing H2O, dNTP's, PCR buffer, MgCl2 and Taq polymerase. A drop of light white mineral oil was overland on each well. Denaturation of template DNA was carried out at 93°C for 1 min, annealing of primers to the template was done at 55°C for 1 min, and primer extension was at 72°C for 2 min. This profile was repeated for 35 cycles, followed by a 5 min soak at 72°C on a Techne thermal cycler. Samples were loaded on a 1% agarose + 1% NuSieve gel made with 1 x TAE and electrophoresed at 70V. PCR genotypes were assigned to plants based on the banding pattern of the amplified products. Figure 1 shows the genotypes of IR24 (susceptible check), IRBB21 (isoline with Xa-21) and Oryza longistaminata (donor of Xa-21). F2 plants were designated RR if they showed a band similar to IRBB21, and Rr if the plants showed both IR24 and IRBB21 bands (Fig. 1). Since PB7/8 markers are located within 1.2 cM of Xa-21 (Ronald et al. 1992), we predicted that all 34 individuals found to be homozygous for DNA marker will be homozygous for the Xa-21. The other 28 F2 individuals showing heterozygosity for the DNA marker were predicted to be heterozygous for the Xa-21.

To examine the effectiveness of this classification procedure, PCR-based genotypes of F2 individuals were compared with the genotypes determined by usual inoculation and progeny tests of F3 families (Table 1). All F2 individuals predicted by PCR-based genotyping to have Xa-21 gene were indeed carrying at least one Xa-21 allele as determined by progeny tests, giving a 100% accuracy of prediction. Of the 34 F2 individuals classified by PCR markers to be homozygous (RR) for Xa-21, 31 were indeed homozygous in progeny tests. The accuracy of this prediction is 91.2%. Of the 28 plants heterozygous at DNA marker locus, 24 were found to be segregating for resistance to Xco Race 6 in progeny tests. This represents a predictive accuracy of 85.7%. Thus there was about 10% discrepancy between the genotypes predicted on the basis of PCR analysis and progeny tests. This might be due to the recombination between the DNA marker and Xa-21 as the parents used in this study are not identical to thoseused by Ronald et al. (1992). It is also possible that the discrepancy is due to the inherent problem in the use of rapid PCR procedure which amplifies DNA from leaf tissue instead of purified DNA. The potential effect of environment on phenotyping of Xco resistance might be the other reason for the discrepancy. In spite of this discrepancy, results of this study show that homozygotes for Xa-21 can be identified with high probability (90%) in a segregating population using specific PCR amplification, thus opening the way for marker-aided selection for this gene. Experiments are underway to use flanking markers to predict Xa-21 genotypes with enhanced accuracy. In conventional screening techniques, the F2 phenotype has to be confirmed by F3 analysis in order to select homozygous resistant genotypes. Because specific primer PCR amplification can distinguish homozygous and heterozygous individuals in the F2 generation, the selection efficiency is increased and breeding cycle is shortened by one growing season.

Table 1. Genotypes of F2 plants for Xa-21 as determined by PCR analysis and progeny tests

       PCR analysis           F3 progeny test           Accuracy
========================  ===========================    (%)
Genotype    No of plants  Genotype       No of plants
RR               34          RR               31
                             Rr                3          91.2
Rr               28          RR                4
                             Rr               24          85.7


Berthomieu, P. and C. Meyer, 1991. Direct amplification of plant genomic DNA from leaf and root pieces using PCR. Plant Mol. Bio. 17: 555-557.

Chunwongse, J., G.B. Martin and S.D. Tanksley, 1993. Pre-germination genotypic screening using PCR amplification of half-seeds. Theor. Appl. Genet. 86: 694-698.

Kauffman, H.E., A.P.K. Reddy, S.P.Y. Hsien and S.D. Merca, 1973. An improved technique for evaluating resistance of rice varieties to Xanthomonas oryzae. Plant Dis. Rep. 57: 537-541.

Ronald, P.C., B. Albano, R. Tabien, L. Abebes, K. Wu, S. McCouch and S. Tanksley, 1992. Genetic and physical analysis of rice bacterial blight disease resistance locus, Xa-21. Mol. Gen. Genet. 236: 113-120.