Fig. 1. Fine-mapping of blast resistance genes.
International Rice Research Institute, P. 0. Box 933, Manila, Philippines
Major genes for rice blast resistance Pi-1(t), Pi-2(t) and Pi-4(t) have been mapped previously on chromosome 11, 6, and 12, respectively (Yu et al. 1991). Pi-1(t) is reported to be linked to RFLP marker RZ536 with a distance of 14.0+/- 4.5 cM, Pi-2(t) is closely linked to RG64 with a distance of 2.8+/- 1.4 cM and Pi-4(t) is 15.4+/- 4.7 cM away from RG869 and 18.1+/-5.5 cM from RZ397. The distances between the markers and Pi-1(t) and Pi-4(t) are relatively large as compared with Pi-2(t). On the other hand, flanking marker for Pi-2(t) is not yet determined. Tightly linked markers are needed for marker-aided breeding to ensure higher selection accuracy and efficiency. Recently, more molecular markers were added to the rice RFLP map (Causse et al. 1994). With the saturation of the rice map, the probability of identifying markers more closely linked to the resistance genes has increased. In this study we fine-mapped the three major genes using RFLP markers (RG, RZ and CDO) from Cornell University, U.S.A. and (Npb) National Institute of Agricultural Resources, Japan.
Table 1. Plant material, restriction enzymes and RFLP markers used in
Southern analysis
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Blast resistance Isoline Donor Population Restriction RFLP marker Polymorphic
gene parents size enzymes tested tested markers
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pi-1(t) C101LAC Lac23 160 30 10 5
Pi-2(t) C101A51 5173 120 12 9 5
Pi-4(t) C101PKT Pai-kan-tao 80* 30 14 6
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F`3` lines were used to represent the F`2` genotypes.
We selected at least 9 RFLP markers close to the regions of each of the blast
resistance genes in the respecitve chromosomes (Table 1). To identify more
polymorphism in the parental survey we included more restriction enzymes in
addition to the five enzymes previously tested. Three mapping populations
(F`2` and F`3`) were derived from crosses between the susceptible recurrent
parent CO39 and each of the 3 isolines. DNA samples were extracted from the
fresh leaf tissues collected from the F`2` or F`3` mapping populations, CO39,
isolines and the donor parents (Table 1). The DNA extracted from the parents
and isolines was used for the parental survey filters and the extracted DNA
from the F`2` plants and F`3` lines (15-20 bulked plants/line) for
verification filters for linkage analysis.The F`3` mapping populations were test-inoculated with IRRI blast isolates 101 and V89013 in two separate trials. At least 15 plants per family were grown and inoculated 21 days after sowing. Disease was evaluated 7 days after inoculation and individual plants of an F`3` family were scored as resistant and susceptible based on disease reaction. A family with both resistant and susceptible plants was scored as segregating. Similarly, each F`2` plant or F`3` family was given a score for each RFLP marker, according to whether the F`2` was homozygous for the allele of the resistant parent (3), homozygous for the allele of the susceptible parent (1), or heterozygous (2). In cases wherein the RFLP pattern of heterozygous was not easily distinguishable (eg. null allele) from the homozygous resistant, a score of 4 was assigned. Linkage of donor alleles and resistance phenotype was confirmed by Chi-square analysis. The genetic distances between markers and resistance genes were estimated using the computer program MAPMAKER (Fig. 1).
As reported by the previous workers, we also found RG64 to be closely linked with Pi-2(t) on chromosome 6 and RG869 and RZ397 to be linked with Pi-4(t) on chromosome 12. However, we obtained a closer distances from the target resistance genes. We estimated RG64 to be 2.1 cM from Pi-2(t), RG869 to be 5.4 cM and RZ397 to be 3.3 cM away from Pi-4(t). A new flanking marker, RG241 was identified to be 5.2cM from Pi-4(t), marker Npbl8l was found to be more closely linked (3.5 cM) than RZ536 (11.4 cM). The closer distances from the resistance genes that we obtained may be attributed to the larger mapping population sizes we used as compared with the previous study (Table 1). With the identification and confirmation of these closely linked flank-
Fig. 1. Fine-mapping of blast resistance genes.
ing markers to blast resistance genes, it is now possible to use these markers for marker-aided breeding. Marker-assisted plant selection with these flanking markers is being used for pyramiding the three major blast resistance genes.
References
Causse, M., T. M. Fulton, Y. G. Cho, S. N. Ahn, K. Wu, J. Xiao, J. Chuwongse, Z. Yu, P. C. Ronald, S. B. Harington, G. A. Second, S. R. McCouch, and S. D. Tanksley, 1994. Saturated molecular map of the rice genome based on an interspecific backcross population. Genetics. (in press)
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.