Ensuring the genetic purity of parental lines and hybrids is a prerequisite to realize their full potential (Yashitola et al. 2004). The cytoplasmic male sterile (CMS) lines that are used for developing the popular “three-line” hybrids often get contaminated with their isonuclear maintainer lines and genotypes with restorer genes during CMS line multiplication. Use of such CMS lines in hybrid seed production results in the production of genetically impure hybrid seed. The present study combines cpDNA and SSR (simple sequence repeat) diversities to identify genetic purity of rice sporophytic CMS lines during multiplication.

Fourteen sporophytic CMS genotypes and six restorer genotypes, which were all extensively used for developing the popular “three-line” hybrids in China, were analyzed by SSR marker RM258 reported to be linked with restorer gene (He et al. 2002). Four polymorphisms were discovered among them and these suggested that SSR marker was prone to distinguish between CMS lines and restorer lines (Fig. 1).

CMS lines contained one more AGAAAA than their isonuclear maintainer lines on cpDNA (Hou et al. 2000, He et al. 2003), and this diversity could be used to remove maintainer off-types from sporophytic CMS lines during multiplication. Regardless of restorer genes’ contamination, it was simple and veracious to identify genetic purity of CMS lines by using three primers PCR due to the 6bp diversity (Sang et al. 2006). New available primers (Forward: 5´-TTCCATTGAGTCTCTGCACC-3´; Reverse: 5´-GCACAAGGAATCCTGGTCTC-3´) were then redesigned to combine with SSR markers to more authentically identify the genetic purity of CMS lines. The SSR diversity rejects off-types contaminated by restorer genes and the cpDNA diversity rejects off-types contaminated by fertile cytoplasm, and their combinations should be ideal in identifying the genetic purity of sporophytic CMS lines during multiplication.

Zhenshan97A has been most widely used as female parent in three-line hybrid rice seed production since 1975. Up to 2003, Shanyou63, crossed by Zhenshan97A and Minghui63, had been cultivated over 61.3 millions hectare since its application on production. The CMS line Zhenshan97A was then regarded as a sample to assess the feasibility of this idea in genetic purity identification. Select 490 seedlings from Zhenshan97A field and 5 seedlings from Zhenshan97B and Shanyou63 field respectively, mix randomly and transplant them into a new field. Each plant was picked out a piece of leave and numbered for PCR identification, and field test was conducted at heading stage by observing anthers and pollens. Genome DNA was extracted according to Sang et al. (2003), and DNA amplification was carried out in 12.5μL reaction mixture containing 1×PCR buffer, 1.5mM MgCl₂, 100μM dNTPs, 5ng each of primers, 0.5U Taq DNA polymerase, 1μL template. The PCR reactions were run 5 min at 94°C, followed by 35 cycles for 30 s at 94°C , 30s at 56°C 1 min at 72°C, and 5 min at 72°C for the final extension. The PCR products were separated on a 10% polyacrylamide gel and the bands were revealed using silver nitrate staining as described by Wang et al. (2004) (Fig. 2). PCR amplification detected fourteen off-types including all purposively contaminated maintainers and F₁ hybrids, and this was consistent with field test.

This result leads to a new idea about using molecular marker to identify genetic purity of sporophytic CMS lines during multiplication, and this must generalize the application of molecular markers on the seed genetic purity identification.

Acknowledgements

This study was partially funded by National Natural Science foundation of China (30200173), Excellent Seed Development Project of Chongqing and Key Project of Chongqing Natural Science foundation.

References

He G. H., L. Hou, X. Y. Luo, Y. H. Xiao, G. Q. Niu, G. W. Yang and Y. Pei, 2003. A common sequence difference between cytoplasmic male sterile lines and their maintainer lines existing in rice (Oryza sativa L.) chloroplast tRNA-Leu gene region. Euphytica. 131(3): 269.274

He G. H., W. M. Wang, G. Q. Liu, L. Hou, Y. H. Xiao, M. Tang, Zh. L. Yang and Y. Pei, 2002. Mapping of two fertility-restoring genes for WA cytoplasmic male sterility in minghui63 using SSR markers. Acta. Genetica. Sinica. 29(9): 798-802.

Hou L., G. W. Yang, G. H. He, B. Tang, Y. H. Xiao, and Y. Pei, 2000. AFLP markers and sequence analysis in rice cytoplasmic male sterility line, Zhenshan97A, and its maintainer line. Acta. Botanica. Sinica. 42(6): 591-594.

Sang X. C., Z. L. Yang, B. Q. Zhong, Y. F. Li, H. Lou and P. Yan, 2006. Assessment of purity of rice CMS lines using cpDNA marker. Euphytica.

Sang X. C., G. H. He, Y. Zhang, Z. L. Yang and Y. Pei, 2003. The simple gain of the templates of rice genomes DNA for PCR. HEREDITAS(Beijing). 25(6): 705-707.

Wang F. G., J. R. Zhao, J. L. Guo, H. D. She and L. Z. Liu, 2004. An improved PAGE/rapid silver staining method used in maize SSR marker. J. Agric. Biotechnol. 12(5): 605-607.

Yashitola J., R. M. Sundaram, S. K. Biradar, T. Thirumurugan, M. R. Vishnupriya, R. Rajeshwari, B. C. Viraktamath, N. P. Sarma and R. V. Sonti, 2004. A sequence specific PCR marker for distinguishing rice lines on the basis of Wild Abortive cytoplasm from their cognate maintainer lines. Crop Sci. 44(3): 920-924.