SSLP-based subspecies identification in Oryza sativa L.

3. SSLP-based subspecies identification in Oryza sativa L. Y.-Y. FAN, J.-Y. ZHUANG, J.-L. Wu and K.-L. ZHENG National Center for Rice Improvement, China National Rice Research Institute, Hangzhou, 310006 China The classification of Asian cultivated rice (Oryza sativa L.) is important for rice breeding, which was generally based on morphological traits or isozymes (Thou eta!. 1988). The advent of molecular markers has provided new tools to study the genetic diversity of rice. We previously screened a set of RFLP probes for subspecies differentiation in rice (Qian et al. 1995). Simple sequence length polymorphism (SSLP) markers are simply and effectively assayed by PCR and they are as reliable as RFLP markers. In this study, we screened a set of SSLP markers for subspecies differentiation in rice. SSR prime pairs (Research Genetics Inc.) used were all di-nucleotide repeats. The varieties used are listed in Table 1. PCR amplifications were run with a hot start of 94°C for 2 min; 30 cycles of 95°C denaturing for 45 sec; 57°C (or 56°C) annealing for 45 sec and 72°C extension for 1 min, followed by 5 min final extension at 72°C. PCR products were run on 3% MetaPhor® agarose (FMC) gels in 1 x TBE. In all 70 SSLP markers were used to assay the three indica testers and three japonica testers, of which 36 could distinguish the subspecies testers and were then used to assay other 9 indica and 9japonica varieties. Twenty were found to be indica-japonica differentiating markers, which were distributed on all rice chromosomes except chromosome 5. Of the 20 markers which detected different patterns between the subspecies, 7 were identical within the subspecies, 6 were identical within japonica subspecies but different ones within indica subspecies, and 7 were different within both subspecies (Table 2). As no SSLP markers on chromosome 5 were found to be subspecies differentiating, RM 13 detected a 140 bp fragment on 12 indica and 1 japonica varieties, and a 126 bp fragment in the remaining 11 japonica varieties, was also included in the list of indica-japonica differentiating SSLP markers. As compared to the 20 indica-japonica differentiating RFLP markers, we found that SSLP markers shared same regions on chromosomes 2,6, 7, 9 with the indica-japonica differentiating RFLP markers and also appeared on other chromosomes, where no indicajaponica differentiating RFLP markers had been detected. These results indicate that SSLP markers can not only replace but also are complementary to RFLP markers for the subspecies identification. References Chen, X., S. Temnykh, Y. Xu,Y.G. Cho and S.R. McCouch, 1997. Development of a microsatellite framework map providing genome-wide coverage in rice (Oryza sativa L.). Theor. Appi. Genet. 95:553-567. Qian, H.R., J.Y. Zhuang, H.X. Lin, J. Lu and K.L. Theng,1995. Identification of a set of RFLP probes for subspecies differentiation in Oryza sativa L. Theor. AppI. Genet. 90: 878-884. Thou, H., J.C. Glassmann, K.S. Cheng and X.Q. Shi,1988. A comparison of methods in classification of cultivated rice. Chinese J. Rice Sci. 2(1): 1-7.

3. SSLP-based subspecies identification in Oryza sativa L.
Y.-Y. FAN, J.-Y. ZHUANG, J.-L. Wu and K.-L. ZHENG

National Center for Rice Improvement, China National Rice Research Institute, Hangzhou, 310006 China

The classification of Asian cultivated rice (Oryza sativa L.) is important for rice breeding, which was generally based on morphological traits or isozymes (Thou eta!. 1988). The advent of molecular markers has provided new tools to study the genetic diversity of rice. We previously screened a set of RFLP probes for subspecies differentiation in rice (Qian et al. 1995). Simple sequence length polymorphism (SSLP) markers are simply and effectively assayed by PCR and they are as reliable as RFLP markers. In this study, we screened a set of SSLP markers for subspecies differentiation in rice.

SSR prime pairs (Research Genetics Inc.) used were all di-nucleotide repeats. The varieties used are listed in Table 1. PCR amplifications were run with a hot start of 94°C for 2 min; 30 cycles of 95°C denaturing for 45 sec; 57°C (or 56°C) annealing for 45 sec and 72°C extension for 1 min, followed by 5 min final extension at 72°C. PCR products were run on 3% MetaPhor® agarose (FMC) gels in 1 x TBE.

In all 70 SSLP markers were used to assay the three indica testers and three japonica testers, of which 36 could distinguish the subspecies testers and were then used to assay other 9 indica and 9japonica varieties. Twenty were found to be indica-japonica differentiating markers, which were distributed on all rice chromosomes except chromosome 5. Of the 20 markers which detected different patterns between the subspecies, 7 were identical within the subspecies, 6 were identical within japonica subspecies but different ones within indica subspecies, and 7 were different within both subspecies (Table 2). As no SSLP markers on chromosome 5 were found to be subspecies differentiating, RM 13 detected a 140 bp fragment on 12 indica and 1 japonica varieties, and a 126 bp fragment in the remaining 11 japonica varieties, was also included in the list of indica-japonica differentiating SSLP markers.

As compared to the 20 indica-japonica differentiating RFLP markers, we found that SSLP markers shared same regions on chromosomes 2,6, 7, 9 with the indica-japonica differentiating RFLP markers and also appeared on other chromosomes, where no indicajaponica differentiating RFLP markers had been detected. These results indicate that SSLP markers can not only replace but also are complementary to RFLP markers for the subspecies identification.

References
Chen, X., S. Temnykh, Y. Xu,Y.G. Cho and S.R. McCouch, 1997. Development of a microsatellite framework map providing genome-wide coverage in rice (Oryza sativa L.). Theor. Appi. Genet. 95:553-567.

Qian, H.R., J.Y. Zhuang, H.X. Lin, J. Lu and K.L. Theng,1995. Identification of a set of RFLP probes for subspecies differentiation in Oryza sativa L. Theor. AppI. Genet. 90: 878-884.

Thou, H., J.C. Glassmann, K.S. Cheng and X.Q. Shi,1988. A comparison of methods in classification of cultivated rice. Chinese J. Rice Sci. 2(1): 1-7.