30. Identification of RAPD markers linked to fertility restoring gene of rice cytoplasmic male sterile line Y. shen, Z. guam, H. qiang J. Lu, K. zheng and X. wanG

1) Institute of Genetics, Fudan University, Shanghai 200433, P.R. China

2) National Rice Research Institute, Hangzhou 310006, P.R. China

 Rice cytoplasmic male sterility (CMS) is a complicated phenomenon yet to be elucidated. Numerous studies have focused on the difference in mitochondrial genomes between CMS line and its maintainer counterpart without breakthrough. Induction of fertility restoration of CMS line may open a new way in CMS study. We have obtained a fertile revertant induced from CMS line II-32A with 60^CO gamma-rays treatment. The revertant is able to restore not only the fertility of its parental CMS line, but also that of other indica CMS lines such as Zhenshan 97A, Xieqingzao A. The restoration is conditioned by one nuclear gene, which is different from other restorer genes so far in practical use, such as those of IR24 and Minghui 63 (Shen et al. 1996).

In the present study, we attempted to tag and map the fertility restoring gene. At first, the DNA polymorphism between II-32A and the revertant line was analyzed by using RAPD-PCR with 70 10-mer primers (Operon Technologies) in comparison with that among II-32A(B), Minghui 63 and IR24. It was shown that there exist about 6% RAPD polymorphism between II-32A and its revertant, but much less than that between II-32A and Minghui 63 as well as IR24 (21.1%-23.6%), and even smaller than that between II-32A and II-32B (10.2%). These results revealed that irradiation caused changes in numerous loci throughout the genome besides the mutation for the fertility restoring gene.

To pinpoint the fertility restoring gene, an effort was made to search the RAPD between bulked sterile segregants and homogeneous fertile segregants of an F2 population of a cross between II-32A and the revertant E15. Unfortunately, no polymorphism was found between the two gene pools after RAPD analysis with 512 single primers and 860 primer pairs. This indicates that the mutation might be very minute and beyond the sensitivity of RAPD analysis, or the scale of the search was still not large enough.

Alternatively, the genomic background around the reversion locus between II-32A and the revertant was not severely disturbed by irradiation, so that it is difficult to Find a RAPD marker closely linked to the restoring locus.

A direct screening of RAPD between II-32A and the revertant was then made by using 516 primers in an attempt to find RAPD markers loosely linked to the target gene. A few primers were found to produce RAPD between II-32A and the revertant, which were scaled down to OPB07, OPB18, OPJ04 and OPJ10 by analyzing a proportion of the segregants. Among these primers, OPB07 and OPB 18 generated polymorphic DNA fragments OPB07-640 and OPB 18-1000, which are most closely linked to the fertility restoring gene. The whole F2 population of 94 individuals was then analyzed with primers OPB07 and OPB18 (Fig. 1). It was found that OPB07-640 and OPB 18-1000 co-segregated and were 5.3cM away from the restoring gene, Un-

116 Rice Genetics Newsletter Vol. 13

Fig. 1. RAPD analysis of the sterile individuals of the F2, population of II-32A and the
revertant line E15. Arrow shows the RAPD fragment. fortunately, both OPB07-640 and OPB18-1000 are multicopy sequences, which are not suitable for mapping with RFLP.

When OPB07 and OPB18 were used to analyze the parents of a mapping population Tesanai/CB, polymorphic DNA fragments OPB07-640 and OPAB18-1000 were present in CB. These RAPDs were proved by southern hybridization with OPB-640 and OPB18-1000 as probes to be homologus to those found between II-32A and E15. Therefore, one of the RAPDs, OPB076-640, was used to analyze 91 F2 individuals of

Fig. 2. Partial linkage map of chromosome 1 showing location (shaded part) of fertility
reversion gene.
Research Notes 117
Tesanai/CB. Among them, 69 plants showed OPB07-640 fragment, indicating normal segregation. Linkage analysis with Mapmaker showed that OPB07-640 is on chromosome 1, 10.8cM from RG374 and 8.8cM from RG394.

Fine mapping was made by using RFLP analysis with probes around the OPB07-640 marker on the F2 population of Tesanai/CB. Unfortunately, no polymorphism was found between II-32A and E15 even with DNAs digested with 16 restriction enzymes. At present, the fertility restoring gene was, therefore, tentatively localized to chromosome 1 between RG374 and RG394 (Fig. 2). It is plausible that the fertility restoring gene induced from CMS line may not be allelic to other restoring genes of WA type CMS lines possessed by IR24 (Zhang et al. 1994), IR36 (Bharaj et al. 1995) and the restoring gene of BT type CMS line (Yu et al. 1995)

References

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