35. Identification of RAPD markers linked to rice thermosensitive genetic male
        sterility gene by bulk segregant analysis

P. K. Subudhi, Reena P. Borkakati, S. S. Virmani and N. Hung
Genome Mapping Laboratory, Plant Breeding, Genetics and Biochemistry Division, IRRI. Philippines

     Exploitation of heterosis through production of hybrids is a major approach to increase productivity in rice. Usually the hybrid seed production is carried out by the 3-line breeding system utilising cytoplasmic genetic male sterility, which is the major limiting factor for large scale production of hybrid seeds in tropics and subtropics. The discovery and application of thermosensitive genetic male sterility (TGMS) is likely to have great impact in those areas because of many advantages in its use compared to CMS system such as easier multiplication of TGMS seeds, no need of maintainers for establishing male sterile line, no need of restorer parents etc. The cytoplasmic base can also be diversified, since TGMS genes can be incorporated into any rice cultivar to use it as female parent of hybrids. The incorporation of TGMS gene through conventional breeding procedure is quite cumbersome, it involves identification of TGMS plants in segregating generations and induction of seed fertility by ratooning under appropriate fertility inducing temperature regime. Mapping of this gene using molecular markers should help in exercising marker-aided selection for transferring the TGMS gene to different genetic background.
    A TGMS mutant line IR32364TGMS developed through mutagenesis at IRRI was used, which showed complete male sterility at day/night temperature of 32°/24°C, but partial fertility at 27°/21°C and 24°/18°C in IRRI phytotron (IRRI 1992; Virmani and Voc 1991). A mapping population was developed from the cross IR32364TGMS X IR68. The genetic analysis indicated this trait to be under control of a single recessive gene. The individuals in F2 generation were phenotyped and progenies of fertile F2 individuals were grown to distinguish homozygous and heterozygous genotypes. Single plants of these population were evaluated for pollen fertility (determined by using 1 % IKI solution) and spikelet fertility (determined by using two bagged panicles per plant). Individuals with less than 20% pollen fertility were grouped under sterile class and partial fertile individuals were excluded from analysis.
    DNA of two parents and 64 F2 individuals was isolated following the procedure of Dellaporta et a/.(1983) and the bulk segregant analysis (Michelmore et al. 1991) was performed in conjuction with RAPD (Random Amplified Polymorphic DNA) technique. Equal amounts of DNA from 10 completely sterile and 10 fully fertile F2 individuals were pooled to constitute the sterile and fertile bulks respectively. The two bulked samples along with parents were then subjected to RAPD analysis using 10-mer random primers obtained from Operon Technologies Inc. Alameda, California. The amplification reaction condition of Williams et al. (1990) was followed with minor modification in Techne thermocycler. Amplification profile was 94°C for 2 min followed by 45 cycles of 1 min at 94°C, 1 min at 36°C, 2 min at 72°C with a final extension time of 7 min at 72°C. Amplification products were analysed in 1.5% ethidium bromide containing agarose gel.
    After surveying 389 random primers, five RAPD markers generated by five primers; OPF18, OPB19, OPAC1, OPAC3 and OPAA7 were found to be putatively linked to TGMS gene. Further analysis with all 34 homozygous sterile and homozygous fertile F2 individuals showed cosegregation of these markers with the trait. Fig. 1 shows a typical result of the bulk segregant analysis with one of the primers. Out of 5 markers, four were sterility specific (OPE 18-2600, OPB 19-750, OPAC1-300, OPAA7-550) and one fertility specific (OPAC3-640). All the polymophic fragments were of multicopy sequences except OPF18-2600. The OPF1 8-2600 fragment when used as probe also showed cosegregation with TGMS in Southern blot analysis of progenies. Preliminary analysis revealed linkage of all these markers to the TGMS gene flanking on both the sides (Fig. 2). These fragments are now being cloned. Mapping and sequencing of these clones are underway, which can be helpful for marker assisted selection, while transferring to different genetic background. Recently a TGMS gene has been reported to be mapped on chromosome 8 (Wang et al. 1995), but we could not detect linkage with any of the marker from this chromosome thus indicating that the mutant gene of IRRI line may be different.
    We gratefully acknowledge the technical help rendered by Arnold Parco for this work. PKS was supported with a post-doctoral fellowship by the Rockefeller Foundation.   Fig. 1. DNA amplification pattern obtained with random primer OPF18 in bulked segregant analysis. The polymorphic fragment of 2600 bp is indicated by an arrow. M. Marker, SP. Sterile parent, IR32364TGMS, FP. Fertile parent IR68, FB. Fertile bulk, SB. Sterile bulk. Fig. 2. RAPD map of the region surrounding the gene for thermosensitive genetic male sterility (TGMS), based on segregation data from the cross IR32364TGMS X IR68. Distances(cM) are given on the left side and the markers are on the right side of the vertical line.

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