6

Plant Breeding Laboratory, Faculty of Agriculture, Yamagata University, Tsuruoka, 997, Japan

The classification and evolutionary characterization of rice subspecies, indica and japonica, has been studied mainly based on morphological and biochemical traits including isozymes (Morishima and Oka 1981). Recently, RFLP analysis has been shown to be useful for classification of rice cultivars. Chloroplast and mitochondrial DNAs differed between indica and japonica subspeciese. (Dally and Second 1990; Ishii and Tsunewaki 1989; Second and Wang 1992). The present paper aims to identify critical mtDNA makers for characterization of rice subspecies.

RFLPs of mtDNA were investigated using 44 rice varieties including indica, japonica and javanica subspecies, and one strain of 0. glaberrima. Total DNA was digested Hind III or Xba 1. RFLPs were visualized by Southern hybridization with non-radioactive mtDNA probes labeled with digoxigenin-dUTP. After agarose gel electrophoresis and blotting the separated DNAs onto nylon membrane, they were hybridized with eight mtDNA probes. Mitochondrial DNA probes used were atpA, atp6, atp9, coxl, coxIl, cob, rrnl8 and rrn26 from a rice cultivar, Nipponbare (lwahashi et al. 1992).

When the probes of atp6 and rrnl8 were used, RFLPs could be detected (Fig. 1 and 2). Atp6 patterns showed that rice cultivars were classified into four types (I, II, III and IV), and rrnl8 patterns showed that they were classsified into two types (type I and II). Table I shows the mtDNA patterns of 44 cultivars of 0. sativa and one strain of 0. glaberrima detected with two mtDNA probes. Differences of the mtDNA patterns between japonica and typical indica were recognized. Almost all the japonica and javanica cultivars and some indica cultivars showed type I in both the atp6 and rrnl8 patterns, whereas many indica cultivars were type II in both probes. It is noteworthy that some cultivars (Ambar, BlueBelle, British Honduras Creole and Zenith) which were classified as indica type because of their grain shape and plant statues showed type I in the both probes, the same as the type of japonica. In type I of atp6 patterns, one Hind III fragment (8.6 kb) was found, whereas two fragments (8.6 and 6.9 kb) were observed in type II. Silewah included in javanica cultivar group showed a different pattern and was designated as type III which has the two fragments (8,6 and 7.4 kb). However, two indica cultivars (Chinsura Boro II and Gaiya Dhan Tosar) showed another pattern and

Table 1. Classification of rice subspecies, indica, japonica and javanica based on the RFLP of

The authors thank Prof. A. Hirai, and Dr. M. Nakazono, Laboratory of Radiation Genetics, Faculty of Agriculture, The University of Tokyo, for providing the mtDNA clones. The authors also thank Prof. H. Morishima, National Institute of Genetics, for providing seeds of 0. glaberrima strain. W0440 and for her valuable advice.

Cultivar	Probe		Cultivar	Probe
	atp6	rrn18		Atp6	rrn18
japonica			indica	II	II
Aikoku	I	I	Boushitou	II	II
Kiyonishiki	I	I	Josaeng Tongil	II	II
Koshihikari	I	I	Kinandang	II	II
Kamenoo	I	I	Panbira	II	II
Kinmaze	I	I	Te-tep	II	II
Nipponbare	I	I	Tadukan	II	II
Norin 1	I	I	Chinsura Boro II	IV	I
Reirnei	I	I	Ambar	I	I
Sasanishiki	I	I	BlueBell	I	I
Sakaikaneko	I	I	British Honduras Creole	I	I
Somewake	I	I	Zenith	I	I
Senshou	I	I	Gaiya Dhan Tosar	IV	I
Taichung 65	I	I
Toyonishiki	I	I	javanica
Yoneshiro	I	I	Arditane	I	I
Calrose	I	I	Lady Wright	I	I
Choukoutou	I	I	Lomelto	I	I
			Rinnatto	I	I
indica			Rizzotto	I	I
Akamai	II	II	Secia	I	I
Dee-geo-woo-gen	II	II	Senatore	I	I
Taichung Native 1	II	II	Stripe 136	I	I
Dojinkyo	II	II	Stripe 136 anthocyane	I	I
Konansou	II	II	Silewah	III	I
Konansen	II	II
			0. glaberrima
			W0440	II	II