Rice Genetics Newsletter, Vol. 18

Rgn18

40.	Subspecies specific RAPD markers in rice
	Y.-Z. JIANG^1,2, S.-X. TANG², D.S. BRAR¹, Z.-K. LI¹ 1)Plant Breeding, Genetics and Biochemistry Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines, 2)China National Rice Research Institute, Hangzhou, China

The cultivated rice (Oryza sativa L.) is known to contain two major subspecies, indica (O. sativa L. ssp indica) and japonica (O. sativa L. ssp japonica). The indica and japonica differentiation is most pronounced in Chinese rice germplasm and has resulted in significant levels of hybrid sterility and hybrid breakdown (Oka 1991, Li et al. 1997, unpublished data), which are barriers of gene flow between the two major subgene pools within O. sativa Traditional classification of indica and japonica germplasm based on isozymes and DNA markers typically utilizes a relatively large number of markers and thus is more expensive. With the advent of PCR based markers, such as RAPDs classification of rice cultivars have become much easier. Here, we report identification of several random amplified polymorphic DNA (RAPD) markers that have alleles specific in indica or japonica varieties and thus provide a quick and accurate tool to distinguish japonica lines from indica ones.

A total of 94 accessions were used in this study, including 46 indica and 46 japonica varieties collected from different parts of China (according to the Catalog of China Rice Germplasm Resources 1985) and two check varieties, IR36 (indica) and Azucena (japonica). A total of 40 decamer primers from Operon Technologies were used genotype 94 accessions. Of these, two

primers, OPAA07 and OPAG20 produced unique bands of 600bp and 370bp in size, respectively, specific only to the indica lines (Fig. 1). OPAG20 also amplified a unique band of 1300bp in size specific in all 47 japonicas plus an indica variety "Wushanziangzhan". A third primer, OPQ12 produced a band of 150bp specific in japonica varieties. Four additional primers, OPM02, OPAH13, OPP01, and OPP07, each amplified a unique band predominant in japonica or indica varieties (Table 1). Although the probability of misclassification by each of these four markers ranged from 4.1 - 9.4%, data combining any three of the four markers could reach the accuracy over 99%. Our data suggested that the overall degree of introgression between the two subgene pools was approximately 3.9%. For instance, five japonica lines (Azucena, Shuangfeng 1, Sujing 2, Dongfanghong 1 and Hongqi 5) were found to have the indica band of 340 bp amplified by OPM02, and 3 indica varieties (Shuanggui 1, Yunanhaixiannuo, and IR36) shared a japonica band of 1900bp produced by OPP07. Finally, we conclude that OPAA07, OPAG20, and OPA12 primers can clearly differentiate indica varieties from japonica ones and thus can be used as reliable markers for the identification of subspecies of O. sativa

References

Li, Zhikang, S.R.M. Pinson, A.H. Paterson, W.D. Park, and J.W. Stansel. 1997 Genetics of hybrid sterility and hybrid breakdown in an inter-subspecific rice (Oryza sativa L.) population. Genetics, 145: 1139-1148.

Oka, H.I. (1988) Origin of Cultivated Rice. Japan Sci. Soc. Press, Tokyo