Genes Genet. Syst. (2007) 82, p. 217–229 Phylogenetic analysis of Oryza rufipogon strains and their relations to Oryza sativa strains by insertion polymorphism of rice SINEs Jian-Hong Xu, Chaoyang Cheng, Suguru Tsuchimoto, Hisako Ohtsubo and Eiichi Ohtsubo* Institute of Molecular and Cellular Biosciences, the University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan (Received 22 January 2007, accepted 6 March 2007) Oryza rufipogon, the progenitor of the cultivated rice species Oryza sativa, is known by its wide intraspecific variation. In this study, we performed phyloge- netic analyses of O. rufipogon strains and their relationships to O. sativa strains by using 26 newly identified p-SINE1 members from O. rufipogon strains, in addi- tion to 23 members previously identified from O. sativa strains. A total of 103 strains of O. rufipogon and O. sativa were examined for the presence and absence of each of the p-SINE1 members at respective loci by PCR with a pair of primers that hybridize to the regions flanking each p-SINE1 member. A phylogenetic tree constructed on the basis of the insertion polymorphism of p-SINE1 members showed that O. rufipogon and O. sativa strains are classified into three groups. The first group consisted of O. rufipogon perennial strains mostly from China and O. sativa ssp. japonica strains, which included javanica strains forming a distinct subgroup. The second group consisted of almost all the O. rufipogon annual strains, a few O. rufipogon perennial strains and O. sativa ssp. indica strains. These groupings, in addition to other results, support the previous notion that annual O. rufipogon originated in the O. rufipogon perennial population, and that O. sativa originated polyphyletically in the O. rufipogon populations. The third group consisted of the other perennial strains and intermediate-type strains of O. rufipogon, in which the intermediate-type strains are most closely related to a hypothetical ancestor with no p-SINE1 members at the respective loci and to those belonging to the other rice species with the AA genome. This suggests that O. rufipogon perennial strains are likely to have originated from the O. rufipogon intermediate-ecotype population. Key words: O. sativa, O. rufipogon, phylogeny of rice strains, SINE, insertion polymorphism Previous studies of the cultivated and wild rice strains of INTRODUCTION the AA-genome species, on the basis of morphology (Oka The rice genus, Oryza, consists of 22 species, including and Chang, 1962; Oka, 1974), isozymes (Second, 1982), two cultivated rice species Oryza sativa and Oryza glab- RFLPs (Ishii et al., 1988; Wang et al., 1992), AFLPs errima (Khush, 1997; Ge et al., 1999; Vaughan et al., (Aggarwal et al., 1999), inter-simple sequence repeat 2003). O. sativa is now cultivated worldwide, whereas O. (ISSR) polymorphisms (Joshi et al., 2000), RAPDs glaberrima is grown in a limited area in Africa. Both (Bautista et al., 2001), and microsatellite polymorphisms cultivated rice species are diploid (2n = 24) and have the (Bautista et al., 2001; Ishii et al., 2001), have indicated AA genome. There are five wild rice species with the AA with little controversy that the progenitors of the two cul- genome, Oryza rufipogon, Oryza barthii, Oryza glumae- tivated rice species O. sativa and O. glaberrima are O. patula, Oryza longistaminata and Oryza meridionalis. rufipogon and O. barthii, respectively. O. sativa has been classified into two subspecies, Edited by Yoshio Sano japonica and indica (Kato et al., 1928). A third subspe- * Corresponding author. E-mail: [email protected] Note: Supplementary materials in this article are at cies, called javanica, has also been reported for certain http://wwwsoc.nii.ac.jp/gsj3/sup/82(3)Xu/ strains (Matsuo, 1952; Morinaga, 1954). Javanica strains 218 J.-H. XU et al. are, however, thought to be tropical components of a sin- netic relationships of strains of the species with the AA gle japonica group (Oka, 1958). O. rufipogon has been genome. Recently, Cheng et al. (2003) identified 23 p- classified into two ecotypes; perennial and annual (Oka SINE1 members from O. sativa, which show insertion and Morishima, 1967; Oka, 1988; Morishima et al., 1984; polymorphism in strains of O. sativa and O. rufipogon. 1992). An intermediate type has been noted for some O. Most of the members contain three common substitution rufipogon strains (Morishima et al., 1961; Sano et al., mutations (T at nucleotide position 7, A at 63, and A at 1980). The particular ecotype of O. rufipogon that is the 114), and form a subfamily, named RA (recently- ampli- actual progenitor of O. sativa remains controversial. fied). Phylogenetic analysis based on the insertion poly- Based on the results of analyses of morphological and eco- morphism of the members revealed that the O. sativa ssp. logical characteristics, many researchers proposed that O. japonica and javanica strains are closely related to a sativa originated monophyletically from O. rufipogon group of O. rufipogon perennial strains, and that the (Oka, 1964; Chang, 1976; Sano et al., 1980; Oka and indica strains are related to the O. rufipogon annual Sano, 1981). Biochemical and molecular studies, how- strains (Cheng et al., 2003). This result indicates that O. ever, suggest that the japonica and indica strains are sativa has been derived polyphyletically from O. rufi- much closer to different O. rufipogon strains than they pogon. Note, however, that no p-SINE1 members iden- are to each other (Second, 1982; Wang et al., 1992; tified from O. rufipogon were used for the phylogenetic Mochizuki et al., 1993; Hirano et al., 1994; Bautista et al., analysis in the previous study. It was therefore difficult 2001; Ishii et al., 2001), suggesting that the two subspe- to determine detailed relationships between some O. rufi- cies japonica and indica of O. sativa originated diphylet- pogon strains that are distantly related to O. sativa ically or polyphyletically from O. rufipogon. strains. Short interspersed elements (SINEs) are 70–500 bp In this study, to obtain more detailed information about repetitive DNA sequences that have proliferated via tran- the origin of O. sativa and O. rufipogon, we identified 26 scription, followed by reverse transcription. SINEs are new p-SINE1 members from several O. rufipogon strains found in a wide variety of eukaryotes, including animals, representing three ecotypes that show insertion polymor- fungi and plants (Umeda et al., 1991; Okada, 1991; phism among O. rufipogon strains. A total of 51 p- Kachroo et al., 1995). SINEs have served as useful SINE1 members, including the new members and those markers for phylogenetic studies owing to their specific previously identified, were then examined for their pres- characters: once inserted, SINE remains in that genomic ence or absence at the respective loci in 103 strains of O. locus; the probability of insertion occurring more than rufipogon and O. sativa, and a phylogenetic tree was con- once at any single site has been presumed to be extraor- structed on the basis of the p-SINE1 insertion polymor- dinarily low (Batzer and Deininger, 1991; Batzer et al., phism. Based on the results of phylogenetic analysis, we 1994; Takahashi et al., 1998; Nikaido et al., 1999; for a discuss the possible origins of the strains of O. rufipogon review, see Shedlock and Okada, 2000). In particular, and O. sativa, each of which shows ecotype or subspecies the presence or absence of a SINE inserted at a locus is differentiation. easy to assay by PCR, and thus SINEs have found increasing use as phylogenetic markers to study relation- MATERIALS AND METHODS ships among species of primates (Bailey and Shen, 1993; 1997; Hamdi et al., 1999; Salem et al., 2003), whales and Rice strains A total of 103 rice strains (68 O. sativa even-toed ungulates (Shimamura et al. 1997; Nikaido et and 35 O. rufipogon strains) were chosen for the phyloge- al. 1999), salmonid fish (Murata et al., 1993; Hamada et netic analysis (Table 1). These include strains of two al., 1998) and plants (Mochizuki et al., 1993; Tatout et al., subspecies (japonica and indica) of O. sativa and strains 1999; Cheng et al., 2002). of three ecotypes (perennial, annual and intermediate) of The first plant SINE, named p-SINE1, was identified in O. rufipogon, all of which were originally collected in Asia the genomes of O. sativa and O. glaberrima (Umeda et al., or New Guinea. In addition to the 103 strains, another 1991; Mochizuki et al., 1992). A large number of p- five strains representing each of the other rice species SINE1 members that are present at particular loci were with the AA genome were chosen for the present phyloge- further identified in the strains of O. sativa [Nipponbare netic analysis (see Table 1). Total genomic DNA was (ssp. japonica), IR36 and C5924 (ssp. indica)], as well as isolated from some of these rice strains as described pre- in those of wild rice species with the AA genome, such as viously (Ohtsubo et al., 1991). Total genomic DNA sam- O. barthii, O. glumaepatula, O. longistaminata and O. ples of the rest of the strains have been obtained from meridionalis (Mochizuki et al., 1993; Hirano et al., 1994; elsewhere, as described previously (Cheng et al., 2003). Motohashi et al., 1997; Cheng et al., 2002; 2003). Some members were found to show insertion polymorphism in Polymerase chain reaction (PCR) Adaptor-ligation strains of one or more species. Such polymorphic p- based-PCR (ADL-PCR) (Cheng et al., 2003) was per- SINE1 members were used for the study of the phyloge- formed to identify new p-SINE1 members present at dif- Phylogenetic analysis of O. rufipogon and O. sativa strains 219 ferent loci from those previously identified, as follows. generated two PCR amplified fragments with or without The total DNA of an O.