39. Construction of a contig for a fertility restorer gene, Rf2 in rice using BAG library and its sequence homology with Rf2 gene of maize 

D.C. yang, G.B. magpantay, M. mendoza. N. huang and D.S. brar

International Rice Research Institute, P.O. Box 933. 1099 Manila, Philippines

 Cytoplasmic male sterility (CMS) is the commonly used system in hybrid rice breeding. Four genes have been identified in rice to restore fertility of the various CMS sources, Rf1 and Rf2 were located on chromosomes 1 and 2 respectively by trisomic analysis (Shinjyo and Sato 1994: Bharaj et al. 1995), Rf3 on chromosome 1 based on RAPDs (Zhang et al. 1997) and Rf4 on chromosome 10 through RFLP analysis (Qifa Zhang, pers. comm.). Recently, in maize T-CMS fertility restorer gene, Rf2 was cloned by T-DNA tagging ( Cui et al. 1996). In this communication, we report the construction of a contig for Rf2 gene of rice utsing BAC library and its sequence homology with Rf2 gene of maize,.

We used maize Rf2 cDNA clone, pRf2a (kindly provided by Dr. Cui), as probe to screen the BAG library (Yang et al. 1997). Seven BAG clones, 2019, 28F18, 28H19, 4718, 47J8 and 4612 cross-hybridized with maize Rf2 cDNA. Two contigs or two loci were confirmed by blotting analysis. One consisted of only one BAC clone, 4612, and the other consisted of the rest of 6 BAC clones spanning a total of 210kb. To determine which contig contains rice nuclear restorer gene, a DH mapping population derived from IR64/Azucena was used for mapping. The BAC-end amplified with TAIL-PCR were first used for parental survey. Out of the many PCR probes tested, one BAC-end, 4718R, detected polymorphism with EcoRI digestion. The other locus could not be mapped due to lack of polymorphism. DNA from 60 individual plants of DH population was digested with EcoRI and probed with BAC-end, 4718R. The polymorphic patterns were scored and analyzed with Mapmaker. Rf2 was mapped on chromosome 2 at a distance of 12.1 cM from RZ58 and 2.2cM from CD0686 (Fig. 1). The location of Rf2 is similar to that reported by Shinjyo and Sato (1994). One BAC clone, 4718, was chosen for subcloning. BAC DNA was digested with Hind III, blotted and probed with maize Rf2 DNA. A 10kb fragment with whole coding region was recovered from agarose gel. It was subcloned into Bluescript SK+ vector, and then transformed into E. coli host strain, DH5a . The insert of subclone was digested with DraI and HaeIII, respectively and subcloned by shotgun cloning method. The random clones were picked up for sequencing by ALF express automated sequencer (Pharmacia Biotech). Partial sequences of amino acids from two coding regions of rice Rf2 gene were used for homology analysis. The sequence data was submitted to BLAST to compare the homology. The results indicate that the rice

Research Notes 117

Fig. 1. The physical and genetic map of rice for Rf2 gene on chromosome 2. Total coverage of the contig is 210 kb. 0- indicates position of Rf2. Fig. 2. The homology comparison of partial sequences of Rf2 genes of maize and rice. The numbers on the left refer to the position of amino acids of Rf2 in maize. Rf2 has 86.16% homology to maize Rf2 (Fig. 2). The BAG clone that encompassed Rf2 gene will be used to transform rice and determine its role in fertility restoration of CMS lines. Acknowledgment

We would like to thank Dr. X-Q. Cui for kindly providing maize cDNA clone.

 References 

Bharaj, T.S., S.S. Virmani and G.S. Khush, 1995. Chromosome location of fertility restoring genes for "wild

abortive" cytoplasmic male sterility using primary trisomics in rice. Euphytica. 83: 169-173.

Cui, X-Q., R.P.Wise and P.S. Schuable, 1996. The Rf2 nuclear restorer gene of male-sterile T-cytoplasm 

maize.Science 22:1334-1336.

Shinjyo, C. and S. Sato, 1994. Chromosome location of fertility-restoring gene Rf2. RGN II: 93-95. 

Yang, D.C., A. Parco, S. Nandi, S. Subudhi, Y.G. Zhu, G.L. Wang and N. Huang, 1997. Construction of a

bacterial artificial chromosome (BAC) library and identification of overlapping BAC clones with chro- 118 Rice Genetics Newsletter Vol. 14 mosome 4 specific RFLP markers in rice. Theor Appl Genet 95: 1147-1 154.

Zhang, G., T.S. Bliilraj. S.S. Virmani, and N. Huang, 1997. Mapping of the Rf-3 nuclear fertility restoring gene for WA cytoplasmic male sterility in rice using RAPD and RFLP markers. Theor Appl Genet 94:27-33.