35. Development of PCR-based markers for thermosensitive genetic male sterility gene, tms3 (t) in rice

N.T. LANG, P.K. SUBUDHI, S.S. VIRMANI, N. HUANG and D.S. BRAR
International Rice Research Institute, P. O. Box 933. 1099 Manila, Philippines

Thermosensitive genetic male sterility (TGMS) refers to sterility regulated by temperature. It is becoming an important male sterility system in hybrid rice breeding (2-line system). Three genes for TGMS are known in rice, of which two have been mapped with molecular markers; tms1 on chromosome 8 (Wang et al. 1995), and tms3 (t) on chromosome 6 (Subudhi et al. 1997). One TGMS line (IR32364) with tms3 (t) was crossed with a high yielding cultivar IR68 to develop mapping population for fine mapping of this gene.

IR32364 TGMS shows complete male sterility at 32/24°C (day/night) and partial fertility at 27/2°C. The F2 and recombinant inbred lines (RIL) grown at 32/24°C were evaluated for pollen and spikelet fertility. DNA from both fertile and sterile bulks and of the parents was used as template for PCR analysis. The nucleotide sequence of 4 RAPD markers OPF18, OPAC3, OPB19 and OPAA7 linked to tms3 (t) was used to design and synthesize oligonucleotide primers.

Amplicon length polymorphism was observed. The combination of primer pairs F18/ F18RM produced 2300bp DNA band for IR32364 TGMS and 1050bp band for IR68. Another set of primers F18FM/F18RM produced 1900bp band for IR32364 TGMS and 1000bp band for IR68.

In case of primers B19FR/B19RK only IR32364 TGMS allele was amplified. In contrast when combination pair of primers AC3FR/AC3RK was used, only IR68 allele was amplified. Sequence tagged sites (STSs) used in F2 population from the cross IR32364 TGMS/IR68 could classify individual genotypes into fertile and sterile groups. The sterile F2 individuals could be classified on the basis of banding pattern as homozygote for IR32364 TGMS (2300bp band), homozygote for IR68 fertile type (1050bp band) and heterozygotes showed bands from both parents using primer combination F18F/F18RM (Fig. 1). Similarly the combination of primers F18FM/F18RM, thermosensitive and insensitive individuals could also be classified based on the banding pattern for IR32364 TGMS (1900bp band) and 1000bp band for the IR68.

To estimate linkage between tms3 (t) and the STS markers data from 62 F2 plants was analyzed using Mapmaker. The results showed that primers F18F/F18RM and F18FM/ F18RM were linked to tms3 (t) at a distance of 2.7cM.

Selection for TGMS which is controlled by a single recessive gene is difficult under field condition by conventional breeding methods. The development of PCR based markers for tms3 (t) has made it possible to use them in marker assisted selection for thermosensitive genetic male sterility and to precisely identify genotypes in a segregating population in the early generations, especially when selected plants are to be used in further crosses. Moreover selection can be carried out independent of temperature conditions based on markers closely linked to tms3 (t).

References 

Subudhi, P.K.. R.P. Borkakati, S.S. Virmani and N. Huang, 1997. Molecular mapping of a thermosensitive genetic male sterility gene in rice using bulked segregant analysis. Genome 40: 188-194.

Wang, B., W. Xu. J.V. Wang, V. Zheng, H. Yang, J.D. Rang and H.T. Nguyen, 1995. Tagging and mapping the thermosensitive genetic male sterile gene in rice (Oryza sativa L.) with molecular markers. Theor Appl Genet 91:1111-1114.


Fig. 1. PCR products of F2 generated with primers F18F/F18RM.
Lane M = molecular weight marker, lane I = P, (IR32364 TGMS), lane 2 = P; (IR68), lane 3-
8 and 22 = homozygote (sterile),
lane 9-15 = heterozygote, lane 16-21 = homozygote (fertile).