37. Identification of blast resistance gene, Pi-2(t) in rice plants by flanking DNA markers

37. Identification of blast resistance gene, Pi-2(t) in rice plants by flanking DNA markers

Shailaja HITTALMANI¹, Majid FOOLAD², Tita MEW¹, Raymond RODRIGUEZ² and Ning HUANG¹

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

2) Section of Molecular and Cellular Biology, University of California-Davis, Davis, CA 95616, USA

Rice blast disease caused by Magnaportha grisea is the most devastating disease of rice and the most economical and effective approach to reduce the rice yield loss is to breed varieties that are resistant to the disease. However, the resistance often breaks down within a few years of cultivar release. To breed rice varieties with durable resistance to rice blast, new approaches need to be exploited. Recent developments in DNA marker technology and mapping of the major genes in rice has helped to develop the concepts of marker-aided identification of plants carrying the target genes and might well serve to improve the conventional breeding efficiency, as DNA markers are not affected by environmental variation and therefore can be selected accurately.

RG64, a rice genomic RFLP marker on chromosome 6 is tightly linked (2.8 cM) to Pi-2(t), a major gene for blast resistance (Yu et al. 1991). In this study we explored the possibility of identifying the resistant genotypes carrying Pi-2(t) by a quick and reliable method based on linked DNA markers. The RG64 rice genomic clone was sequenced and four primers were synthesized based on the known DNA sequences. Two of the four primers were found useful in producing polymorphism between the susceptible and the resistant varieties after the monomorphic PCR product was digested with restriction enzymes (Williams et al. 1991). Of the 16 restriction enzymes used for digestion one enzyme HaeIII produced polymorphism (Specific Amplicon Polymorphism, or SAP) between the susceptible CO39 and the resistant isoline, C101A51 carrying the Pi-2(t). The polymorphic marker was then used to identify rice plants carrying Pi-2(t) from an F₂ population derived from the cross between CO39 and C101A51 (Fig. 1). The effectiveness of the selection for resistant plants based on linked DNA markers, SAP, RFLP, and another RFLP marker RG456 linked to Pi-2(t) gene (approx. 5.0 cM distance) was then compared with phenotyping for blast resistance through progeny testing in the F₃ families by blast inoculation.

Fig. 1. The segregation of RG64 locus as determined by SAP analysis. PCR products of the entire segregating population were generated by DNA amplification with primers #431 and #432. With reference to the banding patterns of the parents, CO39 (S, susceptible) and C101A51 (R, resistant), the genotypes of each F₂ individual was given the disignation S, R, or Rr based on the banding pattern of the RG64 locus. M=molecular weight marker (kb ladder).

Results indicated that identification of plants carrying Pi-2(t) in a large segregating population is possible using one linked marker as well as flanking markers. The accuracy of identification of homozygous resistant genotypes was 96% when RG64 marker (SAP and RFLP) alone was scored. The accuracy of selection increased to 100% (Table 1) when both the markers flanking the Pi-2(t) were scored simultaneously. These results illustrate that marker-assisted identification of linked target gene in a segregating population is possible and efficient in identifying resistant genotypes using flanking markers and also demonstrate the utility of SAP markers as simple and yet reliable landmarks of use in marker-assisted selection and breeding.

     
Table 1.  Prediction of genotypes of F₂ plants based on the flanking RFLP
          markers (RG456 and RG64) for the Pi-2(t) blast resistance locus
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Prediction                  Progeny testing           Accuracy of
____________________    __________________________
RG456     RG64            RR     Rr     rr            prediction
_______________________________________________________________________________
RR (31^a)   RR(25^a)        25                            100%
           Rr (6)          2     4
_______________________________________________________________________________
           RR (4)          4
Rr(53)     Rr (44)         1    43                      97.7%
           rr (5)                       5
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           Rr (1)                 1
rr(25)     rr (24)                      24               100%
_______________________________________________________________________________
^a Number of plants predicted to carry the indicated genotype.

References

Tanksley, S. D., 1983. Molecular markers in plant breeding. Plant. Mol. Bio. Rep. 1: 3-8.

Williams, M. N. V., N. Pande, S. Nair, M. Mohan and J. Bennett, 1991. Restriction fragment length polymorphism analysis of polymerase chain reaction products amplified from mapped loci of rice (Oryza sativa L.) genomic DNA. Theor. Appl. Genet. 82: 489-498.

Yu, Z. H., D. J. Mackill, J. M. Bonman and S. D. Tanksley, 1991. Tagging genes for blast resistance in rice via linkage to RFLP markers. Theor. Appl. Genet. 81: 471-476.