Genes Genet. Syst. (2007) 82, p. 217–229 Phylogenetic analysis of rufipogon strains and their relations to strains by insertion polymorphism of 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 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 (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 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. rufipogon strain (W1681, W2007, a p-SINE1 member, indicative of both the presence and W1943, W0120, or W0593) was digested with BamHI, absence of the member. Such cases were coded 1 to indi- EcoRI, HindIII, or XbaI (New England Biolabs), none of cate the presence of a p-SINE1 member (Cheng et al., which cut the p-SINE1 member. T4 DNA ligase (New 2003). The neighbor-joining (N-J) method or the UPGMA England Biolabs) was used to ligate the digested DNA method were used for tree construction with the computer with an oligonucleotide adaptor. First, PCR was per- program PAUP* 4.0b10 (Swofford, 2002). Bootstrap val- formed with ExTaq DNA polymerase (Takara) using a ues of the N-J tree were calculated with the same pro- ligated sample as the template and using primers that gram. The structure program (Pritchard et al., 2000) was hybridize to the adaptor and to the p-SINE1 sequence in used to infer population structure with burn-in 10,000, order to obtain fragments with the proximal portion of p- run length of 100,000, and a model with admixture (Gar- SINE1 and its flanking sequence. Second, PCR was per- ris et al., 2005). formed with primers that hybridize to the adaptor and to a different portion of the p-SINE1 sequence. Fragments Accessions Nucleotide sequence data with information that included the entire p-SINE1 sequence were obtained for p-SINE1 members (r401–r406, r411–r414, r421–r423, by an ADL-PCR with primers that hybridize to the flank- r431–r433, and r441–r452) from O. rufipogon appear in ing sequence of each of the identified members and to the the DDBJ/EMBL/GenBank International Nucleotide Sequ- adaptor. Inverse PCR (IPCR) was also performed to ence Databases under the accession numbers AB201718– identify new p-SINE1 members, as described elsewhere AB201745. (Tenzen et al., 1994), with the total DNA from an O. rufi- pogon strain as the template. Primers used for PCR are RESULTS AND DISCUSSION listed in Table 2. The presence or absence of each p-SINE1 member was Identification and characterization of new p-SINE1 determined by identifying one unique PCR fragment with members from O. rufipogon strains A total of 26 or without a p-SINE1 member after electrophoresis in a new p-SINE1 members were isolated from five strains of 1.8% agarose gel, as described previously (Motohashi et O. rufipogon (W1681, W2007, W1943, W0120 and al., 1997). When the fragments differed in size or when W0593), which represent perennial, annual, or intermedi- two or more bands were present, the presence or absence ate ecotypes (see Table 1). Their nucleotide sequences of p-SINE1 in the fragments was confirmed by Southern were aligned together with 25 previously identified p- hybridization or by direct sequencing of the PCR products SINE1 members from O. sativa (see Fig. 1). With the (Cheng et al., 2003). Failure of the amplification (no exception of seven members (r1, r69, r441, r444, r445, bands) was treated as no data. Primers used for PCR to r447 and r452), each of the p-SINE1 members contains identify the presence or absence of each of the represen- three common substitution mutations (T at nucleotide tative p-SINE1 members are listed in Table 2. Primer position 7, A at 63, and A at 114) (Fig. 1), which have also sequences useful for the presence or the absence of each been identified in members forming the RA (recently- of the other p-SINE1 members will be provided upon amplified) subfamily. Of 44 RA-subfamily members that request. have been identified to date, 27 members contained three additional common substitution mutations (C at 77, T at Cloning and sequencing PCR products were cloned 113, and T at 117), and 11 members contained two addi- into the pGEM-T Easy Vector System I (Promega), tional common substitution mutations (T at 10 and T at according to the supplier’s instructions. Sequencing was 117) (Fig. 1). These suggest that RA-subfamily members performed on an ABI 377 automated DNA sequencer with consist of three groups, of which two newly identified a BigDye Terminator Cycle Sequencing Reaction kit (PE groups are referred to as RAα and RAβ (Fig. 1). Applied Biosystems). Phylogenetic analysis of strains of O. rufipogon Computer analysis Primary nucleotide sequences were and O. sativa A total of 108 rice strains (68 of O. analyzed with the GENETYX-Mac 11.2 system program. sativa, 35 of O. rufipogon, and 5 of other rice species with Multiple sequences were aligned using the programs, the AA genome; see Table 1) were examined for the pres- GENETYX-Mac 11.2 and CLUSTAL W (Version 1.7). ence or absence of the 26 new p-SINE1 members, as well The phylogenetic tree for various rice strains was con- as the 25 previously isolated p-SINE1 members, by PCR structed by organizing the presence or absence of the p- with primer pairs that hybridize to the regions flanking SINE1 members at particular loci in the strains into a each p-SINE1 member. Most of the RA-subfamily mem- data matrix (Cheng et al., 2003), such that the presence bers were found to show insertion polymorphisms among of a p-SINE1 member at a given locus was coded 1, and the O. sativa and O. rufipogon strains (Table 1). All the its absence at the same locus was coded 0. Some strains RA-subfamily members, except two (r34 and r443), were 220 J.-H. XU et al.

Table 1. The presence or absence of p-SINE1 members at respective loci in various rice strains

p-SINE1c Straina species Ecob Origin r1 r2 r501 r502 r503 r505 r506 r507 r510 r30 r34 r51 r53 r54 r55 r56 r57 r58 r59 r62 r69 Km5 sativa In Cambodia + + – + + – – – – + – + + + + + – + + – + OKA710 sativa In China Cen + + – + + – – – – + – – + + + + – + + – + OKA715 sativa In China Cen + + – + + – – – – + – – + – + + – + + – + Zheda8044 sativa In China Eastern + + + + + – – – – + – + + + + + – + / – + Minghui86 sativa In China South + + + + + – – – – + + + / + + + – + + – + OKA724 sativa In China South + + – + + – – – – + + + + + + + – + + – + Nanjing11 sativa In China? + + – + + – + – – + – + + + + + – + / – + OKA421 sativa In + + – + + – – – – + – + + + + + – – / – + M4 sativa In Malaysia + + – + + – – – – + – + – + + + – + / – + Bs19 sativa In Myanmar + + – + + – – – – + – + + + + + – + + – + N7 sativa In Nepal + + – + + – – – – + – / + + + + – + / – + Sm13 sativa In Sikkim + + – + + – – – – + – – + + + + – + / – – OKA111 sativa In Taiwan + + – + + – – – – + + + + + + + – + / – – C9329 sativa In Thailand + + – + + – – – – + – – + + + + – + / – + C9374 sativa In Thailand + + – + + – – – – + – / + + + + – + – – + C9392 sativa In Thailand + + – + + – – – – + – – – + + + – + / – + Th17 sativa In Thailand + + – + + – – – – + – – + + + + – – + – + Th27 sativa In Thailand + + – + + – – – – + – – + – + + – + + – – Th61 sativa In Thailand + + – + + – – – – + – – + + + + – – + – +/– Bs3 sativa In* Myanmar + + – + + – – – – + – / + + + + – – + – + C9356 sativa In* Thailand + + – + + – – – – + – – + + + + – + / – + Ac130 sativa In Taiwan + + – + + – – – – + – + + – + + – – / – – Ba13 sativa In* Bangladesh + + + + + – – – – + – + + – + + – + + – – C5924 sativa In Nepal + + + + + – – – – + + – – + + + – + – – – A39 sativa In Assam + + + + + – – – – + + – – – + + – + – – – Ac419 sativa In India + + – + + – – – – + + – – – + – – + / – – OKA451 sativa In India + + + + + – – – – + + – – + + – – + – – – C9439 sativa In* Thailand + + – + + – – – – + + – – – + – – + – – – N16 sativa In Nepal + + + + + – – – – + + – – – + – – + – – – IR36 sativa In IRRI + + + + + – – – – + + + + + + + + + + + + Milyang23 sativa In Korea + + + + + – + – – + + + – + + + – + + + – C9463 sativa In Thailand + + – + + – – – – + + / – + + + – + + + – CR94.13 sativa In IRRI + + + + + – – – – + + + + + + + + + + + – Is24 sativa In Indonesia + + + + + – – – – + – + – + + + – + + + + IR24 sativa In Philippines + + + + + – – – – + + / – + + + – + + + – OKA318 sativa Jv Indonesia + + + + + – + – + + + + – – – + – + + + + Ketang Nangka sativa Jv Indonesia + + + + + – + – + + + + – – – + – + – + + OKA642 sativa Jv Indonesia + + + + + – + – + + + + – – – + – + / + + Is108 sativa Jv Indonesia + + + + + – + – + + + + – – – + – + – + + Is6 sativa Jv Indonesia + + + + + – + – + + + + – – – + – + – + + Is83 sativa Jv Indonesia + + + + + – + – + + + + – – – + – + + + + Is67 sativa Ja* Indonesia + + + + + + + – + + + + – – – + – + / + – Ac221 sativa Jv Philippines + + + + + + + – + + + + – – – + – + + + – OKA259 sativa Jv Philippines + + + + + + + – + + + + – – – +/– – + + + – P30 sativa Jv Philippines + + + + + + + – + + + + – – – + – + + + + P9 sativa Jv Philippines + + + + + + + – + + + + – – – + – + + + – OKA272 sativa Jv Philippines + + + + + + + – + + + + – + – + – + / + + M24 sativa Jv Malaysia + + + + + + + – + + + + – – – + – + + + – OKA701 sativa Jv China North + + + + + + + + + + + + + – – – – – / + – OKA718 sativa Jv China Cen + + + + + + – + + + + + – – – – – – + + + OKA446 sativa Jv Thailand + + + + + + + + + + + + – – – + – + – + – Bs48 sativa Jv Myanmar + + + + + + + + + + + + – – – + – +/– + + + Akihikari sativa Ja Japan + + + + + + + + + + + + – – – – – – – + – J362 sativa Ja Japan + + + + + + + + + + + + – – – – – – – + – J67 sativa Ja Japan + + + + + + + + + + + + – – – – – – / + – Koshihikari sativa Ja Japan + + + + + + + + + + + + – – – – – – – + – T65 sativa Ja Taiwan + + + + + + + + + + + + – – – – – – – + – Sasanishiki sativa Ja Japan + + + + + + + + + + + + – – – – – – – + – Ba22 sativa Ja Banguladesh + + + + + + + – + + + – – – – – – – / + – Phylogenetic analysis of O. rufipogon and O. sativa strains 221

r60 r61 r210 r215 r401 r402 r403 r404 r406 r411 r412 r413 r414 r421 r422 r431 r432 r433 r441 r442 r443 r444 r445 r446 r447 r448 r449 r450 r451 r452 + + + – + + + + + + – + – – / + + + + + + + + – / – – + – + + + – – + + + + + + – – – – + + + – + + + + + – + – – + – + + + – – + + + + + + – – – – + + + – + + + + + – + – – + – + + + + – / + / + + + – + – / + + + – + + + + + – + – – + – + + + + – + + + + + + – + – / + + + – + + + + + – + – – / – + + + – – + + + + + + – + – / + + + – + + + + + – + – – + – + + + + – + + + + + + – + – – – + + – + + + + + – + – – + – + + + + – + + + + + + – – – – + + + – + + + + + – + – – + – + + + – – + + + + + + – + – – + + + – + + + / + – + – – + – + + + + – / + + + + + – / – – / + + + + + + + + – + – – / – + + + + – + + + + + + – + – – + + + – + + + + + – + – – + – + + + + – + + + + / + – – – – + + + – + + + + + – + – – + – + + + – – / + + + + + – + – – + + + + + + + + + – + – – + – + + + + – + + + + + + – + – – / + + – + + + + + – + – – + – + + + + – + + + + + + – + – – / + + + + + + + + – + – – + – + + + + – + + + + + + – + – – / + + – + + + + + – + – – + – + + + + – + + + + + + – + – – + + + + + + + + + – + – – + – + + + + – + – + + + + – + – – + + + – + + + + + – + – – + – + + + + – + + + + + + – + – – + + + + + + + + + – + – – + – + + + + – + + + + + + – – – – + + + – + + + / + – + – – + – + + + + – + + + + + + – + – – + + + + + + + + + – + – – + – + + + + + + + + + + + – + – – + + + – + + + + + – + – – + – + + + + + + + + + + + – – – – + + + + + + + + + – + – – + – + + + + + + – + + / + – – – – + + + – + + + + + – + – – / – + – + + + + – + + + + – – – – / + + – + + + / + – + – – + – + – + – + + + + + + + – – – – / + + – + + + / + – + – – + – + + + – – + + + + + + – – – – + + + – + + + + + – + – – + – + + + – + + + + + + + – – – – / + + – + + + / + – + – + + – + – + – + + + + + + + – – – – / + + – + + + / + – + – – / – + + + + – + + + + + + – – + – – + + – + + + + + – + – – + – + + + + – + + + + + + – + + – + + + + + + + + + – + – – + – + + + + – + + + + + + – + + – + + + + + + + + + – + – – + – + + + + – + + + + + + – – + – – + + – + + + + + – + – – + – + + + + – / – + + + + – + + / + + + – + + + + + – + – – + – + + + + – + + + + + + – + + – + + + + + + + + + – + – – + – + – + – – – – + + – + – + + – – + + – + + + + + – + – – – – + – + – – – – + – – + – + + – – + + – + + + + + – + – – – – + – + – / – – + – – + / + + – – + + – + + + + + – + – – – – + – + – – – – – – – + – + + – – + + – + + + + + – + – – – – + – + – – – – – – – + – + + – – + + – + + + + + – + – – – – + – + – – – – – – – + – + + – – + + – + + + + + – + – – – – + – + – – / – – + – + – + + – – + + – + + + + + – + – – – – + + + – – – – + – – + – + + – – + + – + + + + + – + – – – – + – + – – – – + – – + – + + – – + + – + + + + + – + – – – – + + + – – – – + – – + – + + – + + + – + + + + + – + – – – – + + + – – – – + – – + – + + – – + + – + + + + + – + – – – – + + – – – – – + – – + – + + – – + + – + + + + + – + – – – – + – – – – – – + – – + – + + – – + + – + + + + + – + – – / – + – – – – – – – – – + – + + – – + + + + + + + + – + – – – – + – – – – – – – – – + – + + – – + + – + + + + + – + – – – – + – – – – – – + – / + – + + – – + + + + + + + + – + – – – – + – + – – – – – – – + – – + – – + + – + + + + + – + – – – – + – – – – – – – – – + – + + – – + + + + + + + + – + – – – – + – – – – – – – – – + – + + – – + + + + + + + + – + – – – – + – – – – – – – – – + / + + – – + + + + + + + + – + – – – – + – – – – – – – – – + – + + – – + + + + + + + + – + – – – – + – – – – – – – – – + – + + – – + + + + + + + + – + – – – – + – – – – – – – – – + – + + – – + + + + + + + + – + – – – – + + – + – – – – – – + – + + – + + + – + + + + + – + – – – – + 222 J.-H. XU et al.

p-SINE1c Straina species Ecob Origin r1 r2 r501 r502 r503 r505 r506 r507 r510 r30 r34 r51 r53 r54 r55 r56 r57 r58 r59 r62 r69 OKA712 sativa Ja China Cen + + + + + + – + + + + + – – – – – – – + + OKA709 sativa Ja China Cen + + + + + + – + + + + + – – – – – – – + + Zheda22 sativa Ja China Eastern + + + + + + + + +/– + + + – – – – – – – + + OKA703 sativa Ja China North + + + + + + + + + + + + – – – – – – – + – OKA563 sativa Ja Indonesia + + + + + + + + + + + + – – – – – – – + – Nippobare sativa Ja Japan + + + + + + + + + + + + – – – – – – – + – K8 sativa Ja Korea + + + + + + + + + + + + – – – – – – + + – Bs63 sativa Ja Myamar + + + + + + + + + + + + – – – + – – + + + OKA869 sativa Ja Taiwan + + + + + + + + + + + + – – – – – – + + –

W1822 rufipogon Int Bangladesh + + – + + – – – – + + – – – + – – / – – – W0610 rufipogon Ann Myamar + + – + + – – – – + + + + + + + – + – – – W0107 rufipogon Ann India + + / + + – – – – + + – + + + + – + – – – W0106 rufipogon Ann India + – – + + – – – – + + – – + + – – + – – – W1681 rufipogon Ann India + + / / + – – – – / + – + + / – – + – – + W1983 rufipogon Ann India + + +/– / + – – – – + + – – – + – – – – + – W1987 rufipogon Ann India + + + + + – – / +/– + + + – + + – – + – + – W0630 rufipogon Ann Myanmar + + – + + – – – – + + – – + + – – + / – – W1551 rufipogon Ann Thailand + + – + + – – – – + + – – + + – – + + – – W1690 rufipogon Ann Thailand + + – + + – – – – + – – – + + + – + + – – W0168 rufipogon Ann Thailand + + – + + – – – – + + – – + + + – + + – – W1680 rufipogon Ann Thailand + + + + + – – – – + + – + – + – – + – – – W1865 rufipogon Ann Thailand + + – + + – – – – + + – + – + – – + – – – W0558 rufipogon Int Cambodia + + – + + – – – – + + – – + + – – + + – – W1654 rufipogon Per China + – + + + – – – – + + + – – + +/– – – / – – W0120 rufipogon Per India + – + +/– – – – – – + + + – +/– + + +/– – +/– + – W1943 rufipogon Per China + – + + + – + – – + – + – – – – – – – + – W1945 rufipogon Per China + – + + – – + – + + – + – – – – – – / + – W1954 rufipogon Per China + – – + + – + – + + – + + – +/– – – – – + + W1976 rufipogon Per Indonesia + – – + + – – – – + – + – – – – – – – + – W1956 rufipogon Per China + + – + + +/– – – + + – + +/– – – – – – – +/– – W1958 rufipogon Per China + + + + +/– – +/– – – + – +/– + – + – – + – +/– – W1965 rufipogon Per China + +/– – + – – – + – + – +/– +/– + + – – + / – + CP25 rufipogon Per Nepal + – – + + – – – – + – – – / – +/– – – – – – W0108 rufipogon Per India + – +/– + + – – – – +/– + + – – + +/– – + – – + W1981 rufipogon Per Indonesia + – – + + – – – – – – / – + – – – – + – +/– W1939 rufipogon Per Thailand + – – + + – – – – + – – – / – + – – – – + W1238 rufipogon Per New Guinea + + – + – – – – – – + / – – + + – – / – – W2005 rufipogon Per India + +/– – + + – – – – – + – + – + – + / / – + W2004 rufipogon Ann India + + – + – – – – +/– – + – + – – – – – – – – W0593 rufipogon Per Malaysia + + – + – – – – – – + – + – – – + – / – – W0596 rufipogon Int Malaysia + + – +/– – – – – – – + – + – – – – – / – – W1244 rufipogon Int Nepal + + – + – – – – – – + – + – – – +/– – – – – W0137 rufipogon Int India + + – +/– – – – – – – + + + – – – +/– – – – +/– W2007 rufipogon Int India + + – + – – – – – – + – + – – – – / – – +/–

W0025 glaberrima Ann Sierra Leone + – – – – – – / – – + – – – – – / / – – +/– W1581 barthii Ann Chad + – – – – – – / – – + – – – – – – – – – + W1192 glumpaetula Per Brazil + – – – – – – / – – + – / – – – – – / – + W1452 longistaminata Per Ivory Coast + – – – – / / / / – – / / – – / – – – – + W1625 meridionalis Ann Australia + – – – – – – – / – – / / – – / – – – – + Phylogenetic analysis of O. rufipogon and O. sativa strains 223

r60 r61 r210 r215 r401 r402 r403 r404 r406 r411 r412 r413 r414 r421 r422 r431 r432 r433 r441 r442 r443 r444 r445 r446 r447 r448 r449 r450 r451 r452 – – – – – – – – – + – + + – – + + – + + + + + – + – – – – + – – – – – – – – – + / + – – – + + + + + + + + – + – – – – + – – – – – – +/– +/– +/– + – + + / – + + + + + + + + – + – – +/– – + – – – – – – – – – + – + + – – + + – + + + + + – + – – – – + – – – + – – – – – + / + + – – + + + + + + + + – + – – – – + – – – – – – – – – + – + + – – + + – + + + + + – + – – – – + – – – – – – – – – + – + + – – + + – + + + + + – + – – – – + – + – – – – – – – + – + + / – + + – + + + + + – + – – – – + – + – – – – – – – + – + + – – + + – + + + + + – + – – – – +

– + / + + + – + + + – – – – + + + – + – + + + – / – – + – + + + + + + + + + + + – – – – + + + – + + + + + – + – – + – + + + – + + + + + + + – – – – + + + – + + + / + – + – – + – + + + – + + + + + + + – – – – + + + – + + + + + – + / – + – + + + – + + + + + + + – – – / / + + – + + + + + – + – – + – + +/– + – + + + + +/– + + – – + – + + + + + + + + + – + / + + – + + + + + + – + – + + – + + – / + + – + + + + + – + / – + – + – + + + + + + + + + – – – – + + + – + + + / + – + – – + – + + + + + + + – + + + – – – / + + + – + + + + + – + – – + – + + + + + + + + + + + – – – – + + + – + + + + + – + – – + – + + + – + + + + + + + – – – – / + + – + + + + + – + – – + – + + + – + + + + + + + – – – – + + + – + + + / + – + – – + – + + + + + + + + + + + – – – – + + + – + + + + + – + – – + – + + + – + + + + + + + – + – – + + + – + + + + + – + – – + – + – + – – + – – + + + – + – – – + + – + + + / + – + – – + – + – + + / +/– + – – +/– + – – + + + + + – + – + + + – + – – +/– – + – – – – – – – – – + + + + – – + + – + + + + + – + / – – – + – – – – – – – – – + + – + – – + + + + – + + + – + – – – – + – – – – – – – + / + + – + – – + + – + – + + + – + – / + – + +/– – – – – – + + / + + + + – + + + – + + + / + – + / – – – + +/– – – – – – – – + + + + – – + + + – + – + + + – + – – + – + + – – – – + – – +/– + – +/– – – +/– + + – + – + + + – + – – +/– – + + – – – / + – + – + – +/– – – – + + + + – + + + – + – – / – + – – – – – + + – – + – / – – + + + + + – + + + – + / / – – + – – – – – + / – – + – / – / + + + + + – + + + – + / – – – + – – – +/– – / – + + + + / – – + + + + + – + + + – + – – + – + +/– – – – – + +/– +/– – + – +/– – / +/– + + + + – + + + – + / – – – + – – – – – + – – + + – / – + + + + + + – + + + – + – – + – + – – – – – + – – + + – / – – – + + – + + + / + – + / + + – + – – – – – + – – + + – – – – – + + + + – + + + +/– + +/– +/– + – + – + – – – – – – + + – – – – – + + – + + + + + + + + + + + + – + – – – – – – + + – – – – – + + – + + + + + + + + + + + + – – – – – – / – + + – – – – – + + – + + + / + + + + + + + + – – – – – + – + + + – – – / – + + + + +/– + + + +/– + +/– +/– + + + – – – – – + – – – + – / – – – + + + + + + + + + + + + – – +

– – – – – – – – – – – / – – – – / – + – + + + – + – – – – + – – – – – – – – – – – – – – – – – – + – + + + – + – – – – + – – – – – – – / – / – – – – – / – – + – + + + – + – – – – + / – – – / / / – / / – / – – – / / / + – – / + / / / / / – + – – / – / / / / / / – – – – – / / / + – – + + – + – – / – + aRice strains chosen for the phylogenetic analysis have been used in previous studies (Cheng et al., 2003; Xu et al., 2005). bSubspecies of the Oryza sativa strains japonica (Ja) and indica (In) are shown. Strains shown by Ja* and In* are classified by p-SINE1-insertion polymorphism in the previous study (Cheng et al., 2003). Ecotypes of the Oryza rufipogon strains annual (Ann), perennial (Per) and intermediate (Int) are also shown (H. Morishima, N. Kurata, and Y. Sano, personal communication; see Cheng et al., 2003). cp-SINE1 members identified from O. sativa cv. Nipponbare (Ja), IR36 (In), C5924 (In) (Cheng et al., 2003), and those from O. rufipogon W1681 (Ann; r401–r404, r406), W1943 (Per; r411–414), W0120 (Per; r421–422), W2007 (Per; r431–433) and W0593 (Per; r441–452). The presence (+) or absence (–) of p-SINE1 members at particular loci was determined by analysis of PCR-amplified fragments (see Materials and Methods). ± indicates a strain that generates fragments both with and without p-SINE1. A slash indicates that no PCR fragments were amplified by PCR. The boxed plus corresponds to the p-SINE1 member identified originally from the respective strains. 224 J.-H. XU et al.

Fig. 1. An alignment of nucleotide sequences of p-SINE1 members. Twenty-six p-SINE1 members (named r401–404, r406, r431–433, r411–414, r421–422, and r441–452) were newly isolated from O. rufipogon strains, and the other 25 members had been previously iso- lated from O. sativa strains. p-SINE1 members marked with an x are present in all the rice strains examined; those with solid circles indicate insertion polymorphism among strains with the AA genome and with the non-AA genome; those with solid triangles show poly- morphisms among strains of all species with the AA genome; and those with open circles are specifically present in strains of O. sativa and/or O. rufipogon. In each p-SINE1 member, nucleotide sequences identical to those in the consensus sequence are indicated by dashes; deleted nucleotides are indicated by slashes; and duplicated sequences are indicated by asterisks. Nucleotide sequences underlined are target-site duplication. Note that members of the RA subfamily (RA, RAα or RAβ) have characteristic mutations in common. not present in the rice strains of species other than O. the three groups (I, II and III) were relatively high when sativa and O. rufipogon (Table 1). several perennial-type strains were excluded from the To determine the relationships of the rice strains exam- analysis (see Supplementary Fig. 1D by way of example). ined, they were bar-coded on the basis of the presence or These facts suggest that the O. rufipogon perennial-type absence of p-SINE1 members at the respective loci, and strains have more or less mixed features of the three a phylogenetic tree of the strains was constructed using groups, which might be partly the results of natural the bar codes assigned to each of the strains with the hybridizations between different groups. neighbor-joining (N-J) method (Fig. 2; see Materials and Because the bootstrap values of the N-J tree were low, Methods). According to the phylogenetic tree obtained, we also constructed a phylogenetic tree with the UPGMA the strains examined were classified into three groups, method using the same dataset as the N-J tree, and found named I, II and III (Fig. 2). that the strains were also classified into three groups, During the course of the phylogenetic analysis, we which contain the same members as were observed in the noticed that bootstrap values of the three groups were Groups I, II and III deduced by N-J method, except that relatively low (Supplementary Fig. 1A). We deduced several O. rufipogon perennial strains of Group III tree of strains without all the O. rufipogon strains or all (W1965, CP25, etc.) were clustered together with those of the O. sativa strains, to know how the bootstrap values Group II (Supplementary Fig. 2). Furthermore, we infe- fluctuate. The tree without O. rufipogon strains showed rred population structure of the strains with the Struc- high bootstrap values, whereas the values of the tree ture program (Pritchard et al., 2000). When the number without O. sativa strains stayed low (Supplementary Fig. of populations (K) was set at three, the three clusters 1B, C). These suggest that the low bootstrap values were almost corresponded with Groups I, II and III, except that caused by the presence of O. rufipogon strains. We then several O. rufipogon perennial strains of Groups I and III deduced trees by excluding various combination of O. (W1954, W1965, etc.) showed somewhat admixed popula- rufipogon strains, and found that the bootstrap values of tions (Supplementary Fig. 3). These results suggest that Phylogenetic analysis of O. rufipogon and O. sativa strains 225

Table 2. Primers used for Adaptor-ligation PCR and IPCR nial strains are from China. This suggests that japonica Primera Sequences (5’ –> 3’) rice strains originated in the O. rufipogon perennial pop- ulation in China, as has been suggested previously AC1-1 (RA) GGCTAGACGACCTGGGTTCA (Cheng et al., 2003), if there were no introgression from AC1-2 (RA) AGCCGGAAGACCCCTGGGCA cultivated rice into these O. rufipogon strains. AC1-3 (RA) ATTAGAAGGGGTGAGGCTTT Japonica strains include those from the temperate area AC2-1 (RA) GGTCCTTCCCTAATATTCGA of East Asia and those from the tropical area of Southeast AC2-2 (RA) AAGACCCCTGGGCATTTCTC Asia (Table 1). Interestingly in the tree, the strains from AC1-1 GGCTAGACGACCTGGGTTCG the tropical area of insular Southeast Asia (such as Indonesia, Philippines, and Malaysia) (OKA318, Ketang AC1-2 AGCCGGAAGACCCCTGGGCG Nangka, etc.) were clustered together, forming a branch AC1-3 ATTAGAAGGGGTGAGGCTTC that is distinct from those of the strains from the temper- AC2-1 GGTCCTTCCCTAATATTCGC ate area of East Asia and mainland Southeast Asia (such AC2-2 AAGACCCCTGGGCGTTTCTC as Myanmar and Thailand) (Fig. 2). This was not previ- AP1 GGATCCTAATACGACTCACTATAGGG ously noted by Cheng et al. (2003). Most of the strains AP2 CACTATAGGGCTCGAGCGG from the tropical area of insular Southeast Asia have been classified as the strains of the third subspecies, PFr55 CATCATCTCAATGGCACATC javanica (Morishima, H. and Kurata, N., personal com- PRr55 TCTCAGTTGGCGAACGCCTG munication). Consequently, these findings suggest that javanica strains distributed in insular Southeast Asia can PFr215 CCATCCATAAATTATTAAGG be distinguished from the other japonica strains by our PRr215 TGGTAAGAGTTCTAACCTCT method. Note that these javanica strains formed a PFr502 GATCTGGCATGGGAACGAAC branch also in the UPGMA tree (see Supplementary Fig. PRr502 ACCATGTCTGCTCATAGTCA 2), which supports the findings above. Interestingly, PFr503 ACTGTACACTGCATACCTTG javanica strains forming a subgroup were much closer PRr503 ATGGCATAGATCGATGAAGT than the other japonica strains to the O. rufipogon peren- nial strains in Group I. This observation suggests that PFr507 GACTCCAGCCAACATGGAGA japonica and javanica rice strains have originated in the PRr507 AACCGTCCTCTACCTGATTC common ancestral population, which may have consisted aPrimers with (RA) were used to isolate p-SINE1 members of javanica-type strains. Note, however, that several with homology to a consensus sequence derived from the other javanica strains from mainland Southeast Asia RA subfamily members. Primers without (RA) are used to (Myanmar and Thailand) (Bs48 and OKA446) and China isolate other p-SINE1 members with homology to a consensus sequence derived from all the p-SINE1 members. AP1 (OKA701 and OKA718) are not clustered with those from and AP2 are oligonucleotide adapters. Forward and insular Southeast Asia (Fig. 2 and Table 1). It is possi- reverse primers (designated with PF and PR, respectively) ble that these strains are cultivars, which have been are the primers used to amplify the fragment containing improved through hybridization by breeders or farmers in each p-SINE1 member, such as r55, r215, etc. these areas from the typical javanica strains. It is also possible that these strains have been misclassified as the classification by the N-J method is valid in spite of the javanica strains. low bootstrap values, which were probably caused by sev- eral O. rufipogon perennial strains with mixed features of Polyphyletic origin of indica strains from O. different groups. Based on these results, we will rufipogon Group II of the N-J tree consisted of almost describe features of the three groups more in detail in the all the O. rufipogon annual strains and two perennial following three sections. strains (W0120 and W1654) (Fig. 2). Note that these two O. rufipogon perennial strains have been previously Origin of japonica strains from the Chinese O. rufi- shown to cluster with the other perennial strains (Cheng pogon perennial population Group I of the N-J tree et al., 2003), probably because no p-SINE1 members from consisted of six O. rufipogon perennial strains (W1943, O. rufipogon have been included in the previous phyloge- W1945, etc.) and O. sativa ssp. japonica strains. The six netic study. This provides further support to the previ- O. rufipogon strains were grouped with japonica strains ous idea that the annual O. rufipogon was derived from also in the UPGMA tree (see Supplementary Fig. 2). primitive perennial O. rufipogon (Cheng et al., 2003). These results support the previous idea that the O. Several additional O. rufipogon perennial strains (W1965, rufipogon perennial strains and O. sativa ssp. japonica CP25, etc.) were grouped together with the Group II strains originated from a common ancestor (Cheng et al., strains in the UPGMA tree (see Supplementary Fig. 2). 2003). Interestingly, five of the six O. rufipogon peren- This also supports the above idea. 226 J.-H. XU et al.

Fig. 2. A phylogenetic tree showing relationships among the O. sativa and O. rufipogon strains. The tree was constructed on the basis of p-SINE1-insertion polymorphism in all the strains shown in Table 1 by the N-J method. I, II, and III indi- cate the three major groups. Japonica strains of O. sativa are indicated by letters in blue; and indica strains are in red; tropical japonica (javanica) strains are underlined and those in insular Southeast Asia are circled. Annual, perennial and intermediate strains of O. rufipogon are respectively shown by letters in green, cyan (greenish-blue) and pink. The other five AA genome species strains are shown in black; a hypothetical ancestor is shown with a dashed line. Strains with an asterisk are O. rufipogon from China.

Group II included O. sativa ssp. indica strains, which was also discussed previously (Cheng et al., 2003). are clustered with O. rufipogon annual strains (Fig. 2). Interestingly in the phylogenetic tree, six indica This shows that O. rufipogon annual strains and O. sativa strains, such as IR36, IR24, Milyang 23, etc., are clus- ssp. indica strains are closely related to each other. Of tered to form one subgroup. These strains formed a indica strains, however, most (Km5, OKA710, etc.) subgroup also in the UPGMA tree (see Supplementary formed a branch clearly distinct from that of the annual Fig. 2). Such a subgroup was not observed previously by strains of O. rufipogon, indicating both types originated Cheng et al. (2003), probably because no p-SINE1 mem- from a common ancestor, and this ancestor is most likely bers identified from O. rufipogon were included in the O. rufipogon of the perennial type as described above. previous phylogenetic analysis. Note that these indica Several O. sativa ssp. indica strains (C5924, A39, etc.) are strains are the progenies, which have been made by clustered together with annual strains (Fig. 2), as also breeders in different institutes, such as IRRI, in different noted previously (Cheng et al., 2003). This cluster was countries through hybridization using various indica also formed in the UPGMA tree (see Supplementary Fig. strains, as well as a few japonica strains. These indica 2). These results indicate that these indica strains, strains have some p-SINE1 members, r62 for example, which include those from mountainous regions of South which are not present in the other indica strains at the Asia, such as Assam (India) and Nepal (see Table 1 and corresponding loci, but are present in japonica strains. Fig. 2), may have originated in the annual population, as This suggests that these strains have more sequence Phylogenetic analysis of O. rufipogon and O. sativa strains 227 homology in such loci, to japonica strains than other members have been identified so far from various species indica strains. with the AA genome (340 from O. sativa, 26 from O. The SINE insertion analyses suggest that japonica and rufipogon, 4 from O. barthii, 6 from O. glumaepatula, 8 indica strains are closely related to different groups of O. from O. longistaminata, and 8 from O. meridionalis) rufipogon strains, respectively. This supports the previ- (Umeda et al., 1991; Mochizuki et al., 1992; 1993; Moto- ous idea that O. sativa originated polyphyletically from hashi et al., 1997; Cheng et al., 2002; 2003; Ohtsubo et al., perennial O. rufipogon (Cheng et al., 2003). 2004). There are 112 RA-subfamily members, including 64 RAα and 18 RAβ members. Interestingly, p-SINE1 Possible origin of O. rufipogon strains of the peren- RA-subfamily members have not been identified from nial ecotype from those of the intermediate ecotype strains of species with non-AA genomes until now, sug- Group III consisted of O. rufipogon strains of perennial gesting that RA-subfamily members have been inserted and intermediate ecotypes (see Fig. 2). Note that it is into the loci after divergence of species with the AA reasonable that the ancestral state of the species with the genome. Furthermore, 27 RAα and 11 RAβ members AA genome would have had no insertion of any p-SINE1 investigated were specifically present in strains of O. member, because the insertion of SINE is thought to be sativa and/or O. rufipogon, but not in strains of the other irreversible. Such a hypothetical ancestor with no p- species with the AA genome (Table 1; Fig. 1). This obser- SINE1 members at the respective loci was placed with the vation suggests that RAα and RAβ group members have strains of Group III in the phylogenetic tree (Fig. 2). The retroposed after the divergence of O. sativa and O. representative strains of the other rice species with the rufipogon from the other rice species with the AA genome. AA genome have only a few p-SINE1 members at their From seven non-RA subfamily p-SINE1 members, respective loci (see Table 1) and thus they appeared to three (r441, r447 and r452) were present at particular loci also be close to the hypothetical ancestor in the phyloge- in the strains of species with the AA genome (Table 1), netic tree (Fig. 2). Interestingly, O. rufipogon strains of which include O. sativa, O. rufipogon, O. glaberrima, O. the intermediate type (W0596, W1266, etc.) were clus- barthii, O. glumaepatula, O. longistaminata and O. tered together, forming a subgroup that is the closest to meridionalis, but were absent in a strain of species with the hypothetical ancestor among O. rufipogon strains. non-AA genome (Xu, 2004). This result indicates that These strains were also clustered together in the UPGMA these three members have been inserted in a common tree (see Supplementary Fig. 2). ancestor of strains of the AA-genome species. This also Morishima et al. (1961) and Morishima et al (1984) suggests that rice species with the AA genome are mono- have discussed that if the habitat is always submerged in phyletically derived. water, plants once established may propagate asexually, while propagation by may to some extent be hin- The presence of p-SINE1 members specific to sub- dered. On the other hand, if the habitat is dry once a species or ecotypes It is worthwhile to note that there year, propagation may be obligatory unless the are several p-SINE1 members that are very useful for plants can survive the drought. Furthermore, differ- readily distinguishing different subspecies strains of O. ences between populations of the perennial and annual sativa and different ecotypes strains of O. rufipogon. The types seem to be regarded as the difference between spe- p-SINE1 member r502, which is present only in the cies of “stable” and “unstable” habitats. In the paper of strains of O. sativa and O. rufipogon, but not in those of Morishima et al. (1984), it was suggested that the inter- the other species with the AA genome, can be used to dis- mediate plants are ecologically unstable and when tinguish strains of O. sativa and O. rufipogon from those disruptive selection is strong enough, they can be differ- of the other species with the AA genome (Table 3; see also entiated into perennial and annual types. Therefore, it Table 1). As shown in Table 3, the p-SINE1 member r55 is likely that the ancestral population of O. rufipogon had is present in the indica strains, but not in the japonica the intermediate characters between perennial and and javanica strains, whereas the p-SINE1 member r507 annual types. The p-SINE1 insertion analysis above is present in the japonica strains, but not in the javanica showed that O. rufipogon intermediate strains forming a and indica strains (see also Table 2). These two p- subgroup are closer to the hypothetical ancestor. This SINE1 members are therefore very useful to distinguish leads us to suggest that the perennial and intermediate three different subspecies strains of O. sativa. As also O. rufipogon strains originated from the common ances- shown in Table 3, the p-SINE1 member r215 is present tral O. rufipogon population, which may have consisted of in the annual strains, but not in the perennial and inter- intermediate-type strains. mediate strains, whereas the p-SINE1 member r503 is present in the perennial and annual strains, but not Notes on p-SINE1 members present in strains of present in the intermediate strains (see also Table the AA-genome species Including the p-SINE1 mem- 1). These two p-SINE1 members are therefore very use- bers identified in this study, about four hundred p-SINE1 ful for distinguishing between three different ecotype 228 J.-H. XU et al.

Table 3. Useful p-SINE1 members to distinguish different RAPD, RFLP and SSLP analysis of phylogenetic relation- subspecies strains of O. sativa and different ecotypes ships between cultivated and wild species of rice. Genes strains of O. rufipogon Genet. Syst. 76, 71–79.

b Chang, T. T. (1976) The origin, evolution, cultivation, dissemi- p-SINE1 nation and diversification of Asian and African rice. Species Subspeciesa r502 r55 r507 r215 r503 Euphytica 25, 425–441. or ecotype Cheng, C., Motohashi, R., Tsuchimoto, S., Ohtsubo, H., and Ohtsubo, E. (2003) Polyphyletic origin of cultivated rice: O. sativa Japonica + – + based on the interspersion pattern of SINEs. Mol. Biol. Javanica + – – Evol. 20, 67–75. Indica + + – Cheng, C., Tsuchimoto, S., Ohtsubo, H., and Ohtsubo, E. (2002) Evolutionary relationships among rice species with AA O. rufipogon Perennial + – + genome based on SINE insertion analysis. Genes Genet. Annual + + + Syst. 77, 323–334. Intermedi- + – – Garris A. J., Tai, T. H., Coburn, J., Kresovich, S., and McCouch, ate S. (2005) Genetic structure and diversity in Oryza sativa L. Genetics 169, 1631–1638. a Japonica indicates the strains from the temperate area, Ge, S., Sang, T., Lu, B. R., and Hong, D. Y. (1999) Phylogeny of whereas Javanica indicates the strains from the tropical rice genome with emphasis on origins of allotetraploid spe- area of insular Southeast Asia. cies. Proc. Natl. Acad. Sci. USA 96, 14400–14405. b The p-SINE1 member r502 is present in the strains of O. Hamada, M., Takasaki, N., Reist, J. D., DeCicco, A. L., Goto, A., sativa and O. rufipogon, but not in those of the other and Okada, N. (1998) Detection of the ongoing sorting of species with the AA genome. ancestrally polymorphic SINEs toward fixation or loss in populations of two species of charr during speciation. Genetics 150, 301–311. strains of O. rufipogon. Further characterization and Hamdi, H., Nishio, H., Zielinski, R., and Dugaiczyk, A. (1999) classification of more rice strains based on the p-SINE1 Origin and phylogenetic distribution of Alu DNA repeats: irreversible events in the evolution of primates. J. 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