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 F\2\ 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. F\2\ populations derived from this cross were homozygous for intermediate amylose content but were segregating for Bph-3 and Xa-21. Seven F\2\ 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 F\2\ population.
A single leaf sample was collected from each bacterial blight resistant F\2\ plant. The leaves were kept in water in separate tubes until testing. The leaves were either boiled in 200 micro-l 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 micro-l 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 H\2\O, dNTP's, PCR buffer, MgCl\2\ and Taq polymerase. A drop of light white mineral oil was overland on each well. Denaturation of template DNA was carried out at 93degC for 1 min, annealing of primers to the template was done at 55degC for 1 min, and primer extension was at 72degC for 2 min. This profile was repeated for 35 cycles, followed by a 5 min soak at 72degC 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). F\2\ 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 F\2\ 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 F\2\ individuals were compared with the genotypes determined by usual inoculation and progeny tests of F\3\ families (Table 1). All F\2\ individuals predicted by PCR-based genotyping to have Xa-21 gene were indeed carrying at
Fig. 1. Analysis of PCR products amplified from leaf tissues of IR24, IRBB21,
O. lotigistaminata and representative F\2\ individuals. The genotype of Xa2l
for each F\2\ plant is indicated based on the marker bands. RR=F\2\
individuals carry IRBB21 band only, Rr=F\2\ individuals carry both IRBB21 and
IR24 bands.
Table 1. Genotypes of F\2\ 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 ====================================================================
References
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.