Polyphyletic Origin of Cultivated Rice: Based on the Interspersion Pattern of SINEs

Chaoyang Cheng*, Reiko Motohashi*,1, Suguru Tsuchimoto*, Yoshimichi Fukuta{dagger}, Hisako Ohtsubo* and Eiichi Ohtsubo*

* Institute of Molecular and Cellular Biosciences, the University of Tokyo, Bunkyo-ku, Tokyo, Japan
{dagger} Hokuriku National Agricultural Experimental Station, Joetsu, Japan


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
The wild rice species Oryza rufipogon with wide intraspecific variation is thought to be the progenitor of the cultivated rice species Oryza sativa with two ecotypes, japonica and indica. To determine the origin of cultivated rice, subfamily members of the rice retroposon p-SINE1, which show insertion polymorphism in the O. sativaO. rufipogon population, were identified and used to "bar code" each of 101 cultivated and wild rice strains based on the presence or absence of the p-SINE1 members at the respective loci. A phylogenetic tree constructed based on the bar codes given to the rice strains showed that O. sativa strains were classified into two groups corresponding to japonica and indica, whereas O. rufipogon strains were in four groups, in which annual O. rufipogon strains formed a single group, differing from the perennial O. rufipogon strains of the other three groups. Japonica strains were closely related to the O. rufipogon perennial strains of one group, and the indica strains were closely related to the O. rufipogon annual strains, indicating that O. sativa has been derived polyphyletically from O. rufipogon. The subfamily members of p-SINE1 constitute a powerful tool for studying the classification and relationship of rice strains, even when one has limited knowledge of morphology, taxonomy, physiology, and biochemistry of rice strains.

Key Words: Oryza sativa • indica and japonica rice • Oryza rufipogon • SINE • insertion polymorphism • polyphyletic origin


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
Oryza sativa is the species of rice cultivated worldwide. It has five wild relatives which belong to the Oryza genus with the AA genome. Of these, Oryza rufipogon is the species closest to O. sativa and is generally thought to be its progenitor (Oka 1988; Morishima, Sano, and Oka 1992; Khush 1997). O. sativa and O. rufipogon, however, show high intraspecific variation (see Morishima, Sano, and Oka 1992). Morphologically, O. sativa strains are classified into two ecotypes, indica and japonica (Kato, Kosaka, and Hara 1928). A third ecotype, javanica, has also been reported for certain strains (Matsuo 1952; Morinaga 1954), but these are thought to be tropical components of a single japonica group (Oka 1958). Even the ecotypes indica and japonica show overlapping morphological variations, and there is no single criterion by which to distinguish them with certainty (Morishima, Sano, and Oka 1992). O. rufipogon strains are classified as two ecotypes, perennial and annual, which differ markedly in life-history traits and habitat preference (Oka and Morishima 1967; Oka 1988; Morishima, Sano, and Oka 1984, 1992). An intermediate type has been noted for some O. rufipogon strains (Morishima, Oka, and Chang 1961; Sano, Morishima, and Oka 1980). The intraspecific variation of O. sativa has been investigated on a large scale by restriction fragment length polymorphism (RFLP) analysis (Wang and Tanksley 1989) and by isozyme analysis (Glaszmann 1987), whereas few extensive molecular investigations have been conducted on variation in O. rufipogon.

The origin of O. sativa has been questioned. Perennial type O. rufipogon is considered to be a possible progenitor, because the high genetic variability in the perennial population would provide higher evolutionary potential than that in the annual population, and because some wild-cultivated hybrid plants have been shown to form a bridge that connects the perennial type of O. rufipogon with O. sativa in various traits (Oka 1964, 1974). Annual type O. rufipogon is also considered to be a possible progenitor of O. sativa (Chang 1976), because annual strains have high seed productivity and certain other characters similar to O. sativa. Some O. rufipogon strains have been shown to be capable of evolving into both the indica and japonica types of O. sativa (Oka 1974; Oka and Morishima 1982), which supports the idea that the two ecotype strains have been derived monophyletically by domestication. Biochemical and molecular studies, however, show that the indica and japonica strains are closer in some characters or loci to different O. rufipogon strains than they are to each other (Second 1982; Morishima 1986; Dally and Second 1990; Wang, Second, and Tanksley 1992; Mochizuki et al. 1993; Hirano et al. 1994), which may support the idea that the two ecotype strains originated diphyletically. Note, however, that the number of genetic markers or O. sativa and O. rufipogon strains used in these studies are not sufficient to discuss the origin of the two ecotype strains.

Short interspersed elements (SINEs) are retroelements found in eukaryotic genomes (see Maraia 1995). SINEs range from 70 to 500 bp, and each has an internal promoter for RNA polymerase III (pol III). Many SINEs appear to be related to tRNAs, whereas a few, such as the primate Alu and rodent B1 family elements, are related to 7SL RNA. SINEs have been used as markers for phylogenetic studies owing to their character: The probability of independent retroposition at the same chromosome site is virtually nil, and a SINE insertion is irreversible (for a review, see Shedlock and Okada 2000). The insertion polymorphism of SINEs has been used to infer the phylogenetic relationship among species of primates (Bailey and Shen 1993, 1997; Hamdi et al. 1999), other animals (Murata et al. 1993; Shimamura et al. 1997; Nikaido, Rooney, and Okada 1999), and plants (Mochizuki et al. 1993; Tatout et al. 1999). Although SINEs are useful for phylogenetic studies, most published studies have been limited to an analysis of interspecies relationships. This mainly reflects the fact that most of the identified SINEs are fixed at particular loci in the population of a given species. Some Alu members, however, show insertion polymorphism in humans (Batzer et al. 1994; Roy et al. 1999), and several members of the salmon SmaI family show intraspecific polymorphism in chum salmon (Takasaki et al. 1997). These polymorphic members must have retroposed recently in the evolutionary time scale, and therefore they are not fixed in the human and salmon populations.

Here, we report the identification of members of a subfamily of p-SINE1, the first plant SINE identified in O. sativa and its related rice species (Umeda, Ohtsubo, and Ohtsubo 1991; Mochizuki et al. 1992, 1993; Motohashi et al. 1997). We show that these p-SINE1 members have been recently amplified in the O. sativa and O. rufipogon populations. A phylogenetic tree of the various O. sativa and O. rufipogon strains was constructed from "bar codes" based on the presence or absence of p-SINE1 members at particular loci in each strain. In this tree, the ecotypes of O. sativa or O. rufipogon strains are clearly distinguished. The O. sativa japonica strains and some perennial O. rufipogon strains appear to constitute one group. Most O. sativa indica strains and the annual O. rufipogon strains are separated but appear to constitute another group. This indicates that the japonica and indica strains are descended from different ancestors. The phylogenetic tree also suggests that temperate japonica strains originated from a common source and that a few indica strains originated by recent domestication in the annual O. rufipogon population. We also show that two types of chloroplast DNA are distributed in the rice strains tested and discuss the origin of the maternal source of O. sativa.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
Rice Strains
The rice strains used are listed below. O. sativa cv. Nipponbare, IR36, and C5924 have been described previously (Motohashi et al. 1997). Some of the wild rice strains were obtained from Dr. N. Kurata, the Genetic Stock Research Center of the National Genetic Institute (Japan). Total genomic DNAs were isolated from these strains, as described previously (Ohtsubo, Umeda, and Ohtsubo 1991). DNA samples of O. sativa cv. Zheda 22, Zheda 8044, and Minghui 86 were supplied by Dr. Q. Xue, Zhejiang University (China). DNA samples of the other rice strains were obtained from the Hokuriku Agricultural Experiment Station (Japan).

Polymerase Chain Reaction
The polymerase chain reaction (PCR) analysis was done with Ex Taq DNA polymerase (Takara), as described elsewhere (Motohashi et al. 1997). The presence or absence of each p-SINE1 member was determined by identifying one unique PCR fragment with or without a p-SINE1 member after electrophoresis in the agarose gel. When the fragments differed in size or when two or more bands were present, the presence or absence of p-SINE1 in the fragments was confirmed by Southern hybridization or by direct sequencing of the PCR products (Mochizuki et al. 1993; Motohashi et al. 1997). Primer sequences will be provided upon request.

Adaptor-ligation based–PCR (ADL-PCR) (Spertini, Beliveau, and Bellemare 1999) was performed with Ex Taq DNA polymerase (Takara) as follows: The total DNA of O. sativa cv. IR36 or C5924 was digested with EcoRI, HindIII, BamHI, XbaI, NheI or SpeI (New England Biolabs), none of which cut the p-SINE1 sequence. T4 DNA ligase (New England Biolabs) was used to ligate the digested DNA with the oligonucleotide adaptor. PCR first was performed with a ligated sample as the template and with primers that hybridize to the adaptor and the p-SINE1 sequence to obtain fragments with the proximal portion of p-SINE1 and its flanking sequence. PCR then was accomplished with primers that hybridize to the adaptor and a different portion of the p-SINE1 sequence (see fig. 1 for primers used). Fragments that included the entire p-SINE1 sequence were obtained by an ADL-PCR with primers that hybridize to the flanking sequence of each identified member and the adaptor. Two pairs of primers [P1 and P2; P1(RA) and P2(RA) (see fig. 1)] were used to isolate general p-SINE1 members and RA subfamily members, respectively.



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FIG. 1. Nucleotide sequences of the p-SINE1 members, most showing insertion polymorphism among the rice species. A, Seventeen p-SINE1 members described in the first section of Results (see table 1). A nonpolymorphic p-SINE1 member, r1, is shown for comparison. A consensus sequence (CON) of p-SINE1 (Motohashi et al. 1997) is shown at the top. Sequences corresponding to the A-box and the B-box of the pol III promoter are shown in boldface letters. Note that the p-SINE1 members include six RA subfamily members with three diagnostic mutations, as indicated by the square brackets. B, All RA-subfamily p-SINE1 members, except for r64 and r65. p-SINE1 members with an x are those present in all the rice strains examined; those with a circle are specifically present in O. sativa, O. rufipogon, or both. The other members showing insertion polymorphism are marked with an asterisk. In each p-SINE1 sequence, nucleotides identical to those in the consensus sequence are shown by dashes; deleted nucleotides are indicated by slashes. RA-subfamily diagnostic mutations are boxed. RA-subfamily members may be further divided into four groups, as indicated by the square brackets. Group-specific mutations are boxed. Member r63 exceptionally has a solo long terminal repeat of a retrotransposon whose target site sequence is boxed. Positions of two pairs of primers [P1 and P2; P1(RA) and P2(RA)] used to isolate the p-SINE1 members by ADL-PCR are shown at the bottom by horizontal arrows. Note that the 3' ends of the primers P1(RA) and P2(RA) match the mutated nucleotides in the p-SINE1 sequence of the RA subfamily

 
Inverse PCR (IPCR) was performed as described elsewhere (Tenzen et al. 1994) with the total DNA from O. sativa cv. C5924 as the template and with primers that hybridize to the additional sequence of each of the three p-SINE1 transcripts that were identified as cDNA by 5' RACE-PCR (unpublished results).

Cloning and DNA Sequencing
In this study, PCR products were cloned into the pGEM-T Easy Vector plasmid (Promega), according to the supplier's instructions. DNA sequencing was done with a BigDye Terminator kit (PE Biosystem) and an ABI PRISM DNA sequencing system. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB086069AB086091.

Computer Analysis
Primary nucleotide sequences were analyzed with the GENETYX-Mac 10.1 system program. Nucleotide sequence searches of the DDBJ/Genbank/EMBL databases were done with the program BLAST (Altschul et al. 1990) and a Smith-Waterman search (Smith and Waterman 1981). Multiple sequences were aligned by use of the program CLUSTAL W (version 1.7).

To construct the phylogenetic tree for the various rice strains, the presence or absence of the p-SINE1 members at particular loci in the strains was organized into a data matrix, such that the presence of a p-SINE1 member at a given locus was coded 1, and its absence at the same locus was coded 0. Some strains generated two PCR-amplified fragments with or without a p-SINE1 member, indicative of both the presence and the absence of the member. Such a case was coded 1, the presence state. The Neighbor-Joining method was used with the computer program PAUP* 4.0b8 (Swofford 1998).


    Results and Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
Identification of p-SINE1 Members Recently Amplified in O. sativa and O. rufipogon
Forty-seven members of p-SINE1, present at different loci in the genome of O. sativa cv. Nipponbare (japonica), IR36, or C5924 (indica), were collected. Of these, 34 had been identified previously (Motohashi et al. 1997) and 13 were newly found by ADL-PCR or IPCR (see Materials and Methods) or by a computer-based homology search of the databases. The presence or absence of these members at particular loci in strains of six rice species with the AA genome, O. sativa, O. rufipogon, O. glumaepatula, O. meridionalis, O. longistaminata, and O. barthii, was determined by PCR analysis with a pair of primers that hybridize to the flanking regions of each p-SINE1 member. Seventeen p-SINE1 members were found to be absent at corresponding loci in one or more of the rice strains examined (table 1), suggesting that they are polymorphic p-SINE1 members which may have been amplified during the divergence of the rice species.


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Table 1 The Presence or Absence of p-SINE1 Members in Oryza Strains with the AA Genome

 
Of the polymorphic members, five (r2, r30, r210, r215, and r216) were absent in rice species other than O. sativa and O. rufipogon and showed insertion polymorphism in O. sativa and O. rufipogon (table 1). This means that these p-SINE1 members were amplified recently during speciation of O. sativa and O. rufipogon and/or after the divergence of the two species. A comparison of nucleotide sequences of the above members with a p-SINE1 consensus sequence derived previously (Motohashi et al. 1997) showed that these members share three common mutations; two in the A-box and B-box sequences in the pol III promoter and one in the distal end region (fig. 1A). None of the other polymorphic members, except for r34, nor any of the nonpolymorphic members had three mutations (fig. 1A). Member r34 showed insertion polymorphism among the O. sativa and O. rufipogon strains, but it was also present in the strains of several species other than O. sativa and O. rufipogon, unlike the above five members (table 1). The insertion polymorphism of r34 may represent genetic introgression during speciation. Alternatively, it may reflect differential sorting of an ancestral polymorphism, as previously proposed (Wu 1991; Hamada et al. 1998). We have recently identified other polymorphic members like r34 and analyzed their distribution among many wild rice strains of all the species with the AA genome, and we discussed two possibilities based on the results obtained (Cheng et al. 2002).

Note that three members (r210, r215, and r216) were isolated by IPCR based on the sequences of transcripts in suspension-cultured cells of O. sativa cv. C5924 (see Materials and Methods). This indicates that some members with the common mutations are transcribed and supports the speculation that the six p-SINE1 members have recently been amplified.

Recently Amplified p-SINE1 Members Forming a Subfamily
To ascertain whether members other than the six with the three diagnostic mutations are present in O. sativa, we used ADL-PCR to isolate p-SINE1 members with mutations in the pol III promoter and obtained 16 members (r51–r66; table 1). Except for two members, r64 and r65, these members carried the same three mutations as those in the six p-SINE1 members identified above (fig. 1B), suggesting that these and the six members form a subfamily. Two members (r64 and r65) did not have the third diagnostic mutation in the distal end region of p-SINE1 (fig. 1B), suggesting that they are not subfamily members. A computer-aided homology search of the databases further identified seven members (named r501–r507) that have the three diagnostic mutations (table 1 and fig. 1B). Alignment of all members with three diagnostic mutations showed that they are highly homologous but divided into several groups, the members of which carry an additional diagnostic mutation(s) (fig. 1B).

To determine whether the newly identified 23 members have been recently amplified or not, their presence or absence at their respective loci in the rice strains was investigated. Twenty members were absent from the four rice species other than O. sativa and O. rufipogon and showed insertion polymorphism in O. sativa and O. rufipogon (table 1). One member, r63, which had a retrotransposon insertion within the p-SINE1 sequence, however, was present not only in O. sativa and O. rufipogon but also in two of the other four species (table 1), as was one (r34) of the six members identified earlier. Two members, r64 and r65, which were considered not to be subfamily members because of lack of a third diagnostic mutation in the distal end region of p-SINE1, were present in all the rice species (table 1), as expected. These findings indicate that p-SINE1 members that carry all three diagnostic mutations have been amplified in recent evolutionary time. We therefore named them RA (recently amplified) subfamily members.

As stated above, RA subfamily members have two mutations in the A-box and B-box sequences of the internal pol III promoter. These members have another mutation in the distal end region, which is thought to be important for priming reverse transcription and integration into the target site. All mutations that occurred in p-SINE1 may have had a positive influence on the transcription and retroposition of p-SINE1, leading to the beneficial amplification of RA subfamily members in the O. sativa and O. rufipogon populations.

Phylogenetic Analysis of O. sativa Strains Based on p-SINE1 Insertion Polymorphism
We collected 101 strains of O. sativa and O. rufipogon. O. sativa strains have been classified morphologically into indica and japonica (fig. 2). Except for seven strains, the O. rufipogon strains have been classified into two ecotypes, annual and perennial (fig. 2). Of the exceptional strains, five have been classified as an intermediate type and two as the annual type based on one classifier or as the perennial type based on another (fig. 2).



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FIG. 2. The rice strains used. Ecotypes of the O. sativa strains, japonica (JA) and indica (IN), are shown. Ecotypes of the O. rufipogon strains, annual (A), perennial (P), and intermediate (I) (Morishima, Oka, and Chang 1961; Morishima, Kurata, and Sano, personal communication) are also shown. O. rufipogon strains, whose ecotypes are reported as perennial based on one classifier or annual based on another, are indicated by (P, A). Twenty-four columns indicate the loci where p-SINE1 members are present. Each rice strain shows a characteristic pattern of the presence (+) or absence (-) of p-SINE1 members. The presence of p-SINE1 members identified originally in Nipponbare, IR36, or C5924 is shown by a boxed +. The plus-or-minus signs indicate that PCR-amplified fragments both with and without a p-SINE1 member were generated. A slash indicates that no PCR fragments were amplified. Relationships among the rice strains are shown at left by the cladogram based on the results in figure 3. The four groups (I–IV) defined in figure 3 are shown

 
The presence or absence of the 22 RA polymorphic members, one non-RA polymorphic member (r69), and one non-RA, nonpolymorphic member (r1) of p-SINE1 at particular loci in each rice strain was investigated by PCR. Note here that of the p-SINE1 members examined, eight are those identified in one japonica strain, O. sativa cv. Nipponbare, whereas 16 are those identified in two indica strains, O. sativa cv. IR36 and C5924 (see fig. 2, in which the presence of p-SINE1 members identified originally in each of the three rice strains is shown by a boxed +). As expected, the p-SINE1 member r1 was present in all rice strains examined, but the other p-SINE1 members showed insertion polymorphism (fig. 2). None of the polymorphic p-SINE1 members, however, exclusively distinguished the strains of one ecotype from those of another (fig. 2), as would be expected from the gene flow that may have occurred spontaneously among the strains of closely related species O. sativa and O. rufipogon (see Morishima et al. 1992). To show the relationships among the rice strains, each was bar-coded based on the presence or absence of p-SINE1 members at the respective loci, and a phylogenetic tree of the rice strains was constructed by the Neighbor-Joining method based on the bar codes given to all the strains (fig. 3). In the phylogenetic tree, the rice strains are divided into four major groups, I–IV. O. sativa strains are clearly separated into two groups, I and II. Group I comprises 32 japonica strains and one indica strain, whereas group II comprises 32 indica and 4 japonica strains (fig. 3). This means that the two ecotype strains of O. sativa can be distinguished almost exclusively by the presence or absence of p-SINE1 members and that the present method provides a means of studying the relationships of these rice strains. The exceptional one indica strain in group I and four japonica strains in group II may be the result of a mishap in handling the rice strains or of wrong classification, because indica and japonica show overlapping morphological variations.



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FIG. 3. A phylogenetic tree showing the relationships among the O. sativa and O. rufipogon strains. The four groups (I–IV) identified are shown by four deep internal branches indicated by thick lines. The large star indicates a hypothetical ancestor, whose position is shown by the dashed arrow. All the rice strains are listed in figure 2. The japonica strains of O. sativa are indicated by letters in blue, and the indica strains are in pink. Tropical japonica strains are underscored. The annual, perennial and intermediate strains of O. rufipogon are respectively shown by letters in green, red, and orange. The O. rufipogon strains, whose ecotypes are reported as annual based on one classifier or perennial based on another, are shown by black letters. The wild rice strains of the four species other than O. rufipogon are circled. Rice strains with an asterisk are those carrying the chloroplast DNA with a deletion. The scale bar equals a distance of 0.1

 
The japonica strains in group I include those from the tropical area of South Asia and those from the temperate area of East Asia (fig. 2). The tropical and temperate japonica strains form a branch of the tree on which the temperate strains are clustered together (fig. 3). This suggests that the temperate japonica strains originated from a common source. Note that eight japonica strains, including Nipponbare and J67, cannot be distinguished from one another (fig. 3). This suggests that these japonica strains are derived from a narrow source.

Phylogenetic Analysis of O. rufipogon Strains
O. rufipogon strains are divided among all four groups on the tree (fig. 3). The five O. rufipogon intermediate-type strains examined are in group II or IV, whereas the two O. rufipogon strains, whose ecotype identification is inconsistent among different classifiers, also are in groups II and IV (fig. 3). This is significant because O. rufipogon shows remarkably high intraspecific variation (Morishima, Sano, and Oka 1992). Interestingly, all the annual O. rufipogon strains, except for one strain (W2004 in group IV), are present in group II, whereas the perennial strains are in each of the other three groups, I, III, and IV (fig. 3). This finding supports the speculation that the annual strains are derived from the primitive perennial strain (Morishima, Sano, and Oka 1992). The annual O. rufipogon strains have been referred to as an independent species, Oryza nivara (Sharma and Shastry 1965), whose classification is still accepted by some rice geneticists. Because annual type strains appear to form a distinct group in the O. rufipogon population (fig. 3), differentiation into the annual type should be considered an intraspecific variation, as reported previously (Oka 1988).

The insertion of SINE is thought to be irreversible. Therefore, it is reasonable that the ancestral state of the species with the AA genome would have had no insertion of any p-SINE1 member. The perennial strains of group IV have only a few p-SINE1 members at particular loci (fig. 2), which suggests that these strains may represent the progenitor of the other perennial strains as well as of the annual strains. In the phylogenetic tree, the hypothetical ancestor with no p-SINE1 members at the respective loci has been placed with the strains of group IV (fig. 3).

Polyphyletic Origin of O. sativa
The phylogenetic tree shows the japonica strains of O. sativa and six perennial strains of O. rufipogon are in group I (fig. 3), indicating that the japonica and perennial strains of this group originated from a common source, probably O. rufipogon of the perennial type. Note that five of the six perennial O. rufipogon strains in this group are from China (fig. 2).

The indica strains of O. sativa and annual strains of O. rufipogon are both in group II (fig. 3). Most indica strains are clearly distinct from the annual strains, indicating that both types of strains are originated from another common source. When we consider the fact that the O. sativa cultivars are essentially perennial plants (Morishima, Sano, and Oka 1992), the source is most likely O. rufipogon of the perennial type. However, a few indica strains, including those from Assam (India) and Nepal, are clustered with the annual strains (fig. 3). This suggests that these indica strains may have originated in the annual O. rufipogon population by recent domestication. These findings show that the O. sativa strains originated polyphyletically.

Relationship Between O. rufipogon and Other Wild Rice Species with the AA Genome
The presence or absence of all the p-SINE1 members at particular loci in the four strains belonging to the other four wild rice species with the AA genome (O. glumaepatula, O. meridionalis, O. longistaminata, and O. barthii ) was also investigated by PCR. These strains carry only some of the p-SINE1 members at the corresponding loci (fig. 2), and are clustered together with the perennial strains of O. rufipogon of group IV and with the hypothetical ancestor (fig. 3).

The four strains in the tree appear to be closely related (see fig. 3). This is due to the use of RA-subfamily p-SINE1 members, which are specifically present in strains of the O. rufipogonO. sativa population, but not in those of the other species. Therefore, to analyze the relationships among the strains of the other species in more detail, p-SINE1 members showing insertion polymorphism in one or more of the other species have to be used. In fact, the non-RA polymorphic p-SINE1 members identified in this study (see table 1) proved to be useful for the phylogenetic study of all the rice species with the AA genome, which showed that the O. sativa and O. rufipogon strains appear as one of several major branches of the phylogenetic tree (Cheng et al. 2002).

Analysis of the Maternal Source of the Rice Strains Investigated
Previous RFLP analysis indicated that there are overlaps in variation in the chloroplast DNA of O. sativa and O. rufipogon (Murata et al. 1993). It is reported that indica strains carry chloroplasts whose genome has a 69-bp deletion at the ORF100 locus, whereas japonica strains do not (Kanno et al. 1993; Kaneda et al. 1996). To clarify the variation in chloroplast DNA in the rice strains used in this study, the presence or absence of a deletion in chloroplast DNA was investigated by PCR with a pair of primers hybridizing to the sequences flanking ORF100. O. rufipogon strains of each of the four groups have chloroplast DNA with or without the deletion (see fig. 3 for strains [asterisk] with deleted chloroplast DNA). All four strains of the other wild rice species with the AA genome had chloroplast DNA without deletion (fig. 3). This suggests that chloroplast DNA deletion occurred after O. rufipogon diverged from its ancestral rice species.

As for O. sativa, 63% of the indica strains had chloroplast DNA with deletion, but the rest had the chloroplast DNA without deletion (fig. 3), suggesting that the indica strains have a polyphyletic maternal source. All the japonica strains, however, had chloroplast DNA without deletion (fig. 3), suggesting that japonica strains have a monophyletic maternal source. This finding is interesting but inconsistent, considering that some japonica strains have been derived by hybridization with indica strains in the course of variety improvement. The combination of cytoplasm carrying the deleted chloroplast DNA with the japonica-type nucleus may be restricted during natural selection by a mechanism as yet unknown. Moreover, nuclear substitution in japonica strains by indica strains may be frequent during variety improvement, whereas nuclear substitution in indica strains by japonica strains is rare. This rare form of nuclear substitution would lead to the production of japonica strains carrying chroloplast DNA without deletion. This theory may be supported by distorted segregation in indica–japonica hybrids, in which the segregation ratios usually deviate from the mendelian ratio owing to an increase in the number of genes derived from the indica parent (Oka 1988).


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
We thank Drs. N. Kurata and Q. Xue for providing the rice strains. We are grateful to Drs. H. Morishima and Y. Sano for the information on the ecotypes of the O. rufipogon strains and for their critical reading of the manuscript. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan and from the Ministry of Agriculture, Forestry and Fisheries of Japan.


    Footnotes
 
1 Present address: R. Motohashi, RIKEN Tsukuba Institute, Tsukuba, Ibaraki, 305-0074, Japan. Back

E-mail: eohtsubo{at}ims.u-tokyo.ac.jp. Back


    Literature Cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 

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Accepted for publication September 8, 2002.