Fig. 1. Autoradiogram of clone RG345 probed onto EcoRV-digested DNAs from 7 indica (lanes 1-7) and 7 japonica varieties used as controls, showing that the two subspecies can be differentiated with a single probe-enzyme combination.
Biotechnology Department, China National Rice Research Institute (CNRRI), Hangzhou, 310006 China
Assessment of genetic differences between rice varieties is carried out usually for classification, phylogenetic analysis and selection of varieties for breeding. The development of DNA restriction fragment length polymorphism (RFLP) technology provides a new tool to assess genetic differences between rice varieties and explore their evolutionary relationships. However, RFLP analysis is relatively expensive, and labor and time consuming. We have tried to select potentially powerful RFLP probes to reduce the number of probes required in subspecies classification.
One hundred and sixty probes (2 G # from Dr. Uchimiya and all others from Dr. Tanksley's lab.) were used to survey RFLPs among 3 indica varieties, Nan-te-hao, Nanjing 11 and IR36, and 3 japonica varieties, Ballila, Akihikari and Zao-sha-keng. These were adopted as standard subspecies-testers for screening 44 wide-compatibility" varieties in China (Gu et al. 1991).
The results showed that the proportion of shared fragments within subspecies was about 42% and that between subspecies was about 18%. Sixty-eight probes produced identical hybridization patterns within indica and japonica testers, and different patterns between indica and japonica testers.
The 68 probes were screened further in 7 typical indica and 7 typical japonica varieties combined with 4 enzymes (EcoRI, EcoRV, HindIII and DraI). The rice varieties were obtained from different regions of China and other countries. Twenty-one probes were isolated as subspecies-specific, as they showed different hybridization patterns between indica and japonica with at least one enzyme (Fig. 1). Nineteen of them revealed polymorphisms between indica and japonica varieties by two or more enzymes (Table 1). RG358, RG375 and G318 were indica-specific probes, and hardly hybridized to DNAs from japonica varieties. These probes may play an important role in differentiating the subspecies of O. sativa.
Fig. 1. Autoradiogram of clone RG345 probed onto EcoRV-digested DNAs from 7
indica (lanes 1-7) and 7 japonica varieties used as controls, showing that
the two subspecies can be differentiated with a single probe-enzyme
combination.
M: Molecular weight marker lambda/HindIII 1: Ai-zi-zhan 2: Di-jiao-wu-jian 3: Xian-feng 1 4: Guang-lu-ai 4 5: Gul-chao 2 6: Zhe-fu 802 7: Nanjing 14 8: Daikoku dwarf 9: Lao-lai-ging 10: Xue-he-ai-zao 11: Tai-zhong 12: Reimei 13: Koshihikari 14: Nong-ken 58A set of 13 probes for subspecies differentiation were selected in view of their performance in Southern blotting and distribution on chromosomes (Table 1, shown by *). They were designated as core probes for subspecies differentiation. Based on data from these core probes, a dendrogram of 12 varieties recognized as "wide-compatible" was constructed by the unweighted paired group method with arithmetic mean (UPGMA method, Sokal and Michener 1958; Fig. 2). Seven typical indica and 7 typical japonica varieties taken as controls were also included in this dendrogram. The varieties tested were divided into two groups. The genetic distance, estimated according to Nei (1987, p.122-124, formula 5.53- 5.55), was much greater between the groups than within each group.
The selection of potentially powerful RFLP probes is useful in genetic research and breeding, especially in hybrid rice breeding. It is believed that stronger heterosis is obtained from crosses between distantly related varieties (Yuan 1987). Using the present set of probes, enough information can be obtained for selection of appropriate varieties by a small number of hybridizations for RFLP survey, and will help establish a low-cost RFLP-based analysis in rice breeding.
Table 1. Probes differentiating between rice subspecies
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Probe Chr1 Enzyme2 No. of
enzymes
RG64 6 EV 1
RG81 12 EI,EV,Hd,DI 4
RG96 3 EI,EV,Hd,DI 4
RGIOI* 3 EI,EV,HD 3
RG256* 2 EI,EV,DI 3
RG345* 1 EI,EV,HD 3
RG35 1 * 7 EI,EV,HD 3
RG358* 9 EI,EV,Hd,DI 4
RG375* 4 EI,EV,Hd,DI 4
RG437 2 EI,EV,HD 3
RG462* 1 EI,EV,Hd,DI 4
RG482* 3 EI,EV,Hd,DI 4
RG553 9 EI,EV,Hd,DI 4
RG570 9 EI,EV 2
RG620* 4 EI,EV,Hd,DI 4
RG667* 9 EI,HD,DI 3
RG684* ? EV,HD,DI 3
RG869* 12 EI,HD,DI 3
RG944 3 El 1
G318* 12 EI,EV,Hd,DI 4
CDO281 1 EI,DL 2
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1 Chr=Chromosome, ? not mapped yet;
2 Enzymes with which a probe detected different hybridization patterns
between 7 indica and 7 japonica varieties. EI=EcoRI, EV=EcoRV, Hd=HindIII,
DI=Dral;
* Core probes for subspecies differentiation.
ReferencesGu, M., Z. Pan, Z. Chen and W. Yang, 1991. A study on compatibility of standard wide compatibility testers of rice in China. Sci. Agric. Sinica 24: 27-32.
Nei, M., 1987. Molecular Evolutionary Genetics. Columbia Univ. Press, New York, 512 pp.
Sokal, R. R. and C. D. Michener, 1958. A statistical method for evaluating systematic relationships. Univ. Kansas Sci. Bull. 28: 1409-1438.
Yuan, L., 1987. Breeding strategies for hybrid rice. Hybrid Rice 1: 1-3.
Fig. 2. Dendrogram of 12 varieties taken as "wide-compatible", and indica
and japonica controls based on 13 core probes for subspecies differentiation.
The numbers below and above are scales showing genetic distance estimated
according to Nei (1987).