17. Mapping of the Temperature sensitivE gene, TS

Y. HATTORI¹, M. Kaneko¹, M. Ashikari², M. Matsuoka² and K. Hattori¹

1) Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601 Japan

2) Bioscience and Biotechnology Center, Nagoya University, Nagoya, 464-8601 Japan

A chloroplast is an important organelle in the plant cell, which fixes solar energy, supplies a photosynthesis product, and releases oxygen into the atmosphere by photosynthesis including photosynthetic pigments, such as chlorophyll and carotenoid. Chlorophyll-deficient mutant develops chlorotic leaf including immature chloroplasts because of poor accumulation of chlorophyll, and many of them cannot be grown. In order to avoid it, relatively mild chlorophyll mutants, such as virescent-2 and zebra (Sugimoto et al. 2004, Kusumi et al. 2000), have been analysed for chloroplast differentiation mechanism, but the cause genes are still unknown. To investigate the genetic mechanism for chloroplast differentiation, we are isolating the rice Temperature sensitivE gene, TS, by positional cloning. ts mutant was a temperature sensitive chlorophyll mutant and generated by gamma-ray irradiation to japonica rice cv. Nipponbare. Under low-temperature condition, ts mutant fails to form normal thylakoid membrane systems (Fig. 1) and develops chlorotic leaves independent of its growth stage (Fig. 2). The F₁ population crossed ts mutant and Nipponbare showed normal phenotype (WT) and F₂ population segregated 3:1 (WT: ts) following to the Mendelian low; indicating that ts mutant is controlled by a single recessive nuclear gene. For mapping, we crossed the ts mutant with indica rice variety, Kasarath, homozygous plants for the wild-allele (TS/TS). Then, F₁ plants were cultivated and self-pollinated to obtain F₂ seeds. In the F₂ seedlings, we screened homozygous plants for ts by low-temperature treatment and isolated the genomic DNAs for PCR analysis. We used 50 F₂ ts homozygous plants for mapping. The TS locus was roughly mapped on the long arm of chromosome 1. For further analysis, we screened about 300 F₂ ts homozygous plants, and investigated the linkage between TS locus and two markers, S13528 and S10581, located near the TS locus. Linkage analysis revealed that TS was located between S13528 and S10581, with the genetic distance between them is 14.7cM (Fig. 3). We are determining more precise location of TS for cloning.

Fig. 1. Electron micrograph of ts mutant chlorophyll. A: high-temperature　(30°C) condition. B: low-temperature (16°C) condition.

Fig. 2. Phenotype of ts mutant under low-temperature condition. A: seedling stage. B: vegetative stage. C: heading stage.

Fig. 3. Map position of the TS locus.

References

Kusumi, K., H. Komori, H. Satoh and K. Iba, 2000. Characterization of a zebra mutant of rice with increased susceptibility to light stress. Plant Cell Physiol. 41: 158-164.

Sugimoto, H., K. Kusumi, Y. Tozawa, J. Yazaki, N. Kishimoto, S. Kikuchi and K. Iba, 2004. The virescent-2 mutation inhibits translation of plastid transcripts for the plastid genetic system at an early stage of chloroplast differentiation. Plant Cell Physiol. 45: 985-996.