1) South China Agricultural University, Guangzhou 510642, China
2) International Rice Research Institute, P. O. Box 933, 1099 Manila, Philippines
It has been reported that the cytoplasmic male sterility in WA-Zhenshan 97 A is caused by the interaction between WA cytoplasm and two recessive genes designated rf-1 and rf-2 and the restorer IR24 has two fertility-restoring genes Rf`1`,Rf`1`,Rf`2`Rf`2` (Zhang and Lu 1987). A near-isogenic restorer line was developed by backcrossing five times to Zhenshan 97 A using IR24 as the donor for the fertility-restoring genes (Lu and Zhang 1986). The backcrossing program was continued in South China Agricultural University, Guangzhou, China. Eight near-isogenic lines (NILS) were selected from a BC`9`F`2` population.
The genotypes for fertility restoration in the eight NILs were determined from the F`1` and F`2` fertility in the testcrosses with Zhenshan 97 A. Among the eight NILs, ZSR1 and ZSR2 had the genotype rf`1`rf`1`Rf`2`Rf`2`, ZSR3, ZSR5 and ZSR7 had Rf`1`Rf`1`rf`2`rf`2`, and ZSR11, ZSR9 and ZSR21 are Rf`1`Rf`1`Rf`2`Rf`2`. In the F`2` populations of testcrosses with Zhenshan 97 A, all NILs with one dominant gene (Rf`1`Rf`1`, or Rf`2`Rf`2`) showed a segregation pattern of 3 fertile (or partial fertile) to 1 sterile plants, while the NILs with two dominant genes (RfRfRf2Rf2) showed a good fit to the ratio of 15 fertile to 1 sterile plants. In the crosses between NILs differing in genetic constitution, F`2` populations segregated a monogenic or digenic mode (Table 1).
To identify introgressed chromosomal segments, Zhenshan 97 A(ZSA), Zhenshan 97 B(ZSB, a maintainer for Zhenshan 97 A), ZSR (a NIL from BC5) and IR24 (a donor for the fertility-restoring genes), as well as the eight NILs were used for RAPD analysis. From the survey of 720 random primers (Operon 10-mer kits, Operon Technologies, Inc.), we identified six that amplified polymorphic bands among the twelve lines. All NILs with Rf`2` showed the same band pattern as IR24 while all NILs with rf`2` showed the same pattern as ZSA. Two of the six RAPD markers, U10-1100 and K5-800, were isolated and used as probes. They were located on chromosome 1 sing a doubled haploid population (Huang et al. 1994). Fifteen RFLP markers flanking the two RAPD markers on chromosome 1 (Causse et al. in press) were then selected to survey the introgressed segment. It was found that the introgressed segment spans about 35 cM from RZ382 to RG458 (Fig. 1). Within the introgressed segment, four RFLP markers, RG532, RG140, RG147 and RG458, showed consisted cosegregation with NILs carrying Rf`2`. Since the region from U10-1100 to RG458 consistently showed association with Rf`2` characteristics in NILs, the gene, Rf`2`, likely resides in this region.
From the survey of the 720 random primers, however, we failed to identify
Table 1. Fertility segregations in the testcrosses of NILs
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No. of plants in F`2` populations
Cross ________________________________________ Ratio Chi2
Fertile Sterile Total
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ZSA x ZSR1 68 21 89 3:1 0.034 (P>0.75)
ZSA x ZSR2 64 22 86 3:1 0
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ZSA x ZSR3 66 22 88 3:1 0
ZSA x ZSR5 71 24 95 3:1 0.004 (P>0.90)
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ZSA x ZSR11 109 7 116 15:1 0.009 (P>0.90)
ZSA x ZSR21 112 8 120 15:1 0
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ZSR2 x ZSR3 151 9 160 15:1 0.027 (P>0.75)
ZSR5 x ZSR1 74 6 80 15:1 0.053 (P>0.75)
ZSR5 x ZSR2 150 10 160 15:1 0
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any RAPD markers cosegregating with Rf, locus. Recent trisomic analysis has shown that Rf`1` is located on chromosome 7 (Virmani, personal communication). To find the introgressed segment carrying Rf`1`, 36 RFLP markers on chromosome 7 from the RFLP map (Causse et al. in press) were surveyed but none of the markers were found to segregate in the NILs. Then, we surveyed ten RFLP markers on chromosome 7 from another RFLP map (Saito et al. 1991) and one marker Npb379 was found to segregate in the NILs (near isogenic lines; Fig. 1). If the Rf`1` gene is located on chromosome 7, this introgressed segment from the donor IR24 is the most likely region for Rf`1` gene.
The analysis of linkage relationship between the putative positive molecular markers and the fertility-restoring genes is being carried out using segregating populations.
References
Causse, M., T. M. Fulton, T. Gucho, S. N. Ahn, J. Chunwongse, K. Wu, J. Xiao, Z. Yu, P. C. Ronald, S. B. Harrigton, G. A. Second, S. R. McCouch and S. D. Tanksley, 1995. Saturated molecular map of the rice genome based on an interspecific backcross population. Genetics. (in press)
Lu, Y. and G. Zhang, 1986. Breeding for an isogenic restorer line of CMS Zhen-Shan 97 A. RGN 3: 90-91.
Saito, A., M. Yano, N. Kishimoto, M. Nakagahra, A. Yoshimura, K. Saito, S. Kuhura, Y. Ukai, M. Kawase, T. Nagamine, S. Yoshimura, O. Ideta, R. Osawa, Y. Hayano, N. Iwata and M. Sugiura, 1991. Linkage map of restriction fragment length polymorphism loci in rice. Japan. J. Breed. 41: 665-670.
Zhang, G. and Y. Lu, 1987. Genetic analysis for the cytoplasmic-nuclear sporophytic male sterility in rice. Acta Agronomica Sinica 13: 23-28.