I. NAKAMURA1, H. URAIRONG2, N. KAMEYA3, Y. FUKUTA4, S. CHITRAKON2 and Y.I. SATo5

Japan

Polymorphism of plastid (or chioroplast) DNA has been recognized as a good molecular marker for drawing a framework of plant systematics, especially in an outcrossing or “complex” species, because plastid DNA is inherited primarily from maternal parent (or paternal parent in some species). RFLP analyses of plastid DNAs in Oryza sativa-O. rufipogon complex have been studied by some researchers (Ishii et a!. 1988, Second 1990). Kanno et a!. (1993) found a deletion (69 bp in length) within ORF100 region of plastid DNA in indica cultivars of 0. sativa. Chen et al. (1993) surveyed the distribution of plastid with a deletion in ORF100 over 250 strains of wild rice (0. rufipogon) and cultivars (0. sativa). Although huge data from RFLP analysis of nuclear genes are not in conflict with the monophyletic origin of 0. sativa (Oka and Chang 1962), the data about plastid DNAs seem to support diphyletic origin of indica and japonica cultivars from different populations of 0. rufipogon (Ishii et a!. 1988, Chen eta!. 1993). Therefore, the detailed analysis of plastid DNA is interesting to study the origin and phylogeny of indica and japonica rice cultivars.

Nakamura et a!. (1998) proposed that a linker sequence (ca. 100 bp in length) between rpli6 and rpl14 genes in plastid DNA was considered as a plastid subtype ID (PS-ID) sequence. The PS-ID sequences were thought to have originated from the same ancestral sequence and are shared by plastid DNAs of every higher plant. As PS-ID sequences were found to be different between indica and japonica cultivars, we analyzed variations of PS-ID sequence in 0. sativa-0. rufipogon complex.

Research Notes

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Total genomic DNA was isolated by the procedure of Dellaporta et a!. (1983) from leaf tissues (100 mg) of 92 strains of cultivated and wild rices. Classification of cultivars into indica and japonica was described by Sato (1991). PCR amplification of the plastid DNA fragment containing rpl16 and rpl14 was performed using a pair of primers (A primer:

5’-AAAGATCTAGATTTCGTAAACAACATAGAGGAAG AA-3’ and B primer: 5’- ATCFGCAGCATVFAAAAGGGTCTGAGQTFGAATCAT -3’). The PCR reactions were performed according to Nakamura eta!. (1998). Amplified DNA fragments (ca. 550 bp) were directly sequenced from both strands using the A or B primer with a PRISM dye terminator cycle sequence kit using a 373A sequencer (Applied Biosystems).

PS-ID sequences were the same in 92 strains of 0. sativa-0. rufipogon complex except the numbers of C and A nucleotides in C and A stretches following rpl16 stop codon (Fig. 1). Thus, the differences of PS-ID sequences could be shown in the numbers of C and A nucleotides, for example, as 6C7A. A total of six subtypes (6C7A, 7C6A, 6C8A, 7C7A, 8C8A, 9C7A) of PS-ID sequences were found in 0. sativa-0. rufipogon complex (Table 1). Chen et a!. (1993) found that the deletion type plastid in the ORF100 region

Fig. 1. Schematic representation on the determination of PS-ID sequence. PS-ID sequence is a linker between rpl16 stop codon and rpll4 start codon. Only A and C stretches of PS-ID sequences were variable in 0. sativa- 0. rufipogon complex.

Table 1. Variations of plastid subtypes in 0. sativa-0. rufipogon complex

I); the deletion or non-deletion in

Chen etal. (1993).

ORF100 region was analyzed by PCR according to

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was predominantly found in indica type cultivars and annual strains of 0. rufipogon, whereas the non-deletion type plastid was present in japonica type cultivars and perennial strains of 0. rufipogon. Strains with four plastid subtypes (6C8A, 7C7A, 8C8A, 9C7A) shared the deletion type plastid, but strains containing two subtypes (6C7A, 7C6A) harboured the non-deletion type plastid. Two major subtypes (6C7A and 7C6A) found in japonica cultivars were also found in perennial strains of 0. rufipogon. These data suggest that japonica cultivars containing two different plastid subtypes (6C7A and 7C6A) might have been domesticated from different populations of 0. rufipogon with 6C7A and 7C6A subtypes, respectively. The plastid subtype of 6C8A was predominantly present in annual strains of 0. rufipogon, but not in any cultivars of 0. sativa. On the other hand, 8C8A subtype was found only in indica cultivars of 0. sativa, but not in any wild strain of 0. rufipogon. Interestingly, the origin of indica cultivars is still wrapped up in mystery, because the wild strain having plastid subtype of 8C8A, that is a major plastid subtype of indica cultivars, has not been identified in 0. rufipogon to date.

The plastid subtypes found in this study would be useful as a molecular marker to study the more detailed phylogeny of 0. sativa-0. rufipogon complex.

References

Chen, W.B., I. Nakamura, Y.I. Sato and H. Nakai, 1993. Distribution of deletion type in cpDNA of cultivated and wild rice. Jpn. J. Genet. 68: 597-603.

Dellaporta, S.L., J. Wood and J.B. Hicks, 1983. A plant DNA minipreparation: Version II. Plant Mol. Biol. Rept. 1: 19-21.

Ishii, T., T. Terachi and K. Tsunewaki, 1988. Restriction endonuclease analysis of chioroplast DNA from Agenome diploid species of rice. Jpn. J. Genet. 63: 523-536.

Kanno, A., N. Watanabe, I. Nakamura and A. Hirai, 1993. Variations in chloroplast DNA from rice (Oryza sativa): differences between deletions mediated by short direct-repeat sequences within a single species. Theor. AppI. Genet. 86: 597-584.

Nakamura, I., N. Kameya, Y. Kato, S. Yamanaka, H. Jomori and Y.I. Sato, 1998. A proposal for identifying the short ID sequence which addresses the plastid subtype of higher plants. Breed. Sci. 47: 385-388.

Oka, H.I. and W.T. Chang, 1962. Rice varieties intermediate between wild and cultivated forms and the origin of japonica type. Bot. Bull. Acad. Sinica 3: 109-131.

Sato, Y.I., 1991. Variation in spikelet shape of the indica and japonica rice cultivars of Asian origin. Jpn. J. Breed. 41: 121-134.

Second, G., 1990. Cytoplasmic DNA markers, phylogeny, and systematics in Oryzeae. In: Rice Genetics!! (ed.: IRRI), pp. 475-486. IRRI, Manila, Philippines.