Whole Chloroplast Genome Comparison of Rice, Maize, and Wheat: Implications for Chloroplast Gene Diversification and Phylogeny of Cereals

Yoshihiro Matsuoka*,3, Yukiko Yamazaki{dagger}, Yasunari Ogihara{ddagger} and Koichiro Tsunewaki*

*Fukui Prefectural University, Fukui, Japan;
{dagger}National Institute of Genetics, Mishima, Japan;
{ddagger}Kihara Institute for Biological Research, Yokohama City University, Yokohama, Japan


    Abstract
 TOP
 Abstract
 Introduction
 Data and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The fully sequenced chloroplast genomes of maize (subfamily Panicoideae), rice (subfamily Bambusoideae), and wheat (subfamily Pooideae) provide the unique opportunity to investigate the evolution of chloroplast genes and genomes in the grass family (Poaceae) by whole-genome comparison. Analyses of nucleotide sequence variations in 106 cereal chloroplast genes with tobacco sequences as the outgroup suggested that (1) most of the genic regions of the chloroplast genomes of maize, rice, and wheat have evolved at similar rates; (2) RNA genes have highly conservative evolutionary rates relative to the other genes; (3) photosynthetic genes have been under strong purifying selection; (4) between the three cereals, 14 genes which account for about 28% of the genic region have evolved with heterogeneous nucleotide substitution rates; and (5) rice genes tend to have evolved more slowly than the others at loci where rate heterogeneity exists. Although the mechanism that underlies chloroplast gene diversification is complex, our analyses identified variation in nonsynonymous substitution rates as a genetic force that generates heterogeneity, which is evidence of selection in chloroplast gene diversification at the intrafamilial level. Phylogenetic trees constructed with the variable nucleotide sites of the chloroplast genes place maize basal to the rice-wheat clade, revealing a close relationship between the Bambusoideae and Pooideae.


    Introduction
 TOP
 Abstract
 Introduction
 Data and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The grasses (Poaceae), ca. 10,000 species belonging to 651 genera (Clayton and Renvoize 1986Citation , pp. 29–377), are distributed worldwide. Many species are used by humans, and the importance of grasses as food crops has long been appreciated. Since the very beginnings of agriculture, humans in both the old and new worlds have domesticated cereals. Of these domesticates, maize (Zea mays ssp. mays), rice (Oryza sativa), and wheat (Triticum aestivum), are the major cereals on which large percentages of the world's populations rely for daily sustenance. Taxonomically, maize, rice, and wheat represent different subfamilies of the Poaceae: the Panicoideae (maize), Bambusoideae (rice), and Pooideae (wheat). These cereal species provide important models for evolutionary studies of the grasses because various aspects of their biology have been well documented.

Grass evolution has mainly been studied by analyzing diversity seen in various morphological, cytological, and molecular traits (Kellogg 1998Citation ). Of those traits, variation of chloroplast DNA (cpDNA) has advantages in comparative studies. In general, cpDNA has a low rate of nucleotide substitution, which facilitates comparison of variation in a wide range of plant taxa. Furthermore, uniparental inheritance lowers the impact of intermolecular recombination and helps to simplify theories of chloroplast genome evolution in most plant taxa. Coupled with advances in clarifying entire chloroplast genome sequences (Hiratsuka et al. 1989Citation for rice; Maier et al. 1995Citation for maize), comparative analyses of grass cpDNA variation have complemented morphological studies and given us novel insights into grass evolution.

Although the analysis of cpDNA has contributed significantly to our understanding of grass evolution, most of the conclusions drawn so far are based on nucleotide sequence variation in a single chloroplast gene or gene intron such as rbcL (Doebley et al. 1990Citation ), ndhF (Clark, Zhang, and Wendel 1995Citation ), rpoC2 (Cummings, King, and Kellogg 1994Citation ), rps4 (Nadot, Bajon, and Lejeune 1994Citation ), matK (Hilu and Alice 1999Citation ), and rpl16 intron (Zhang 2000Citation ). In contrast to widespread studies done with the single gene–based approach, there have been only a few studies that addressed grass evolution or grass chloroplast gene evolution based on multiple chloroplast gene sequences (Wolfe, Li, and Sharp 1987Citation ; Wolfe et al. 1989Citation ; Gaut, Muse, and Clegg 1993Citation ). The entire chloroplast genome structure of wheat was reported recently (Ogihara et al. 2002Citation ), and the fully sequenced cpDNAs of three cereals, maize (Maier et al. 1995Citation ), rice (Hiratsuka et al. 1989Citation ), and wheat, now provide a unique opportunity to investigate grass evolution based on whole-genome comparison.

We report results of comparative analyses of nucleotide sequence variations in 106 chloroplast genes of maize, rice, and wheat. The goals were to (1) provide a broad picture of chloroplast gene diversification in cereals; (2) compare relative evolutionary rates of chloroplast genes; and (3) infer the chloroplast genome phylogeny of the three cereals using all the chloroplast gene sequences.


    Data and Methods
 TOP
 Abstract
 Introduction
 Data and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The complete chloroplast genome sequences of maize (Z. mays ssp. mays) (X86563, Maier et al. 1995Citation ), rice (O. sativa) (X15901, Hiratsuka et al. 1989Citation ), tobacco (Nicotiana tabacum) (Z00044, Shinozaki et al. 1986Citation ), and wheat (T. aestivum) (AB042240, Ogihara et al. 2002Citation ) were used. The tobacco sequence was included as the outgroup. All sequences were obtained from DDBJ. The software programs used were CLUSTAL W ver. 1.8 (Thompson, Higgins, and Gibson 1994Citation ) for multiple alignment; MEGA ver. 2.1 (Kumar et al. 2001Citation ) for performing Tajima's relative rate tests (Tajima 1993Citation ), calculating total gene divergence (D) (i.e., the proportion of nucleotide sites at which the two sequences compared differ), estimating synonymous and nonsynonymous substitution rates, and building neighbor-joining trees (Saitou and Nei 1987Citation ); and StatView ver. 5 (SAS Institute, Inc.) for the Kruskal-Wallis, Friedman, and Scheffe's multiple comparison tests. Synonymous and nonsynonymous substitution rates and site degeneracy (see below) were estimated based on nucleotide sequences at the genome level because actual RNA editing sites are unknown for wheat chloroplast genes. Given the small numbers of chloroplast RNA editing sites in maize, rice, and tobacco (21–31 sites per genome) (Maier et al. 1995Citation ; Hirose et al. 1999Citation ; Corneille, Lutz, and Maliga 2000Citation ), the deviation introduced by this approach should be negligible.


    Results
 TOP
 Abstract
 Introduction
 Data and Methods
 Results
 Discussion
 Acknowledgements
 References
 
One hundred six genes chosen from 107 chloroplast genes of known function were analyzed. All the genes are common to maize, rice, wheat, and tobacco. One gene (infA) was excluded because its tobacco homolog is considered to be a pseudogene (Shinozaki et al. 1986Citation ). Of those genes used, rps12 is a divided gene whose 5'- and 3'-regions are encoded at separate loci in the chloroplast genome. In this study, the 5'- and 3'-regions of rps12 are treated as individual genes.

Overall Diversification of Chloroplast Genes
To provide a broad picture of chloroplast gene diversification in the three cereals, total gene divergence (D) from the tobacco homolog was calculated for each of the 106 cereal genes (see Supplementary Material at MBE web site: www.molbiolevol.org). Eight genes, all of them tRNA genes, were invariable in the four species. The 106 chloroplast genes were classified in eight functional gene groups, and the average D was calculated for each group (table 1 ). Average D values of the 106 genes were comparable for maize (0.112), rice (0.112), and wheat (0.113). A Friedman test showed there is no statistical significance for the differences in these average D values (P = 0.35), indicative that most of the genic regions of the three cereal chloroplast genomes evolved at similar rates. Maturase, envelop membrane protein, and proteinase genes seem to have evolved rapidly. In contrast, RNA genes (rRNA and tRNA genes) are highly conservative. Average D values were not statistically uniform between the gene groups of each species (Kruskal-Wallis test, P < 0.0001). Scheffe's multiple comparison test indicated that the average D values of RNA genes always were significantly smaller than those of the other gene groups (P < 0.01 or P < 0.05) and that in each species the average D of photosynthetic genes was significantly smaller than that of ribosomal protein genes (P < 0.01 or P < 0.05).


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Table 1. Average Total Chloroplast Gene Divergence (D) of Three Cereals from Tobacco

 
Synonymous and nonsynonymous substitution rates of the protein-coding chloroplast genes between the three cereals and tobacco also were estimated (table 2 ). Nonsynonymous substitution rate averages vary among the gene groups in each species (Kruskal-Wallis test, P < 0.0001), whereas synonymous substitution rate averages are uniform (Kruskal-Wallis test, P > 0.05). It is noteworthy that photosynthetic genes have low nonsynonymous substitution rates and that the synonymous-nonsynonymous (S-N) ratios are 5–10 times higher for photosynthetic genes relative to the other gene groups. This suggests that photosynthetic genes have been under strong purifying selection during the evolution of the grasses. Of the photosynthetic genes, the pet and psb genes tend to have high S-N ratios (data not shown).


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Table 2. Synonymous and Nonsynonymous Substitution Rate Averages and Synonymous-Nonsynonymous Ratios of Protein-Coding Chloroplast Genes Between Three Cereals and Tobacco

 
Slow Evolution of Some Rice Chloroplast Genes
To examine whether a gene evolves at different rates in the different evolutionary lineages of the grasses, Tajima's relative rate test (Tajima 1993Citation ) was used on each of the 106 chloroplast genes with the tobacco sequence as the outgroup. That test considers two test sequences with one outgroup sequence and compares the numbers of sites at which the test sequences differ from the outgroup sequence under the null hypothesis of homogeneous substitution rates. Of the 318 possible comparisons (106 comparisons each for maize vs. rice, maize vs. wheat, and rice vs. wheat), the relative rate test could not be performed for 79 because there was too little sequence variation (75 for RNA genes and four for protein genes). Totally, significant rate heterogeneity of nucleotide substitution was detected for 14 genes: 12 protein-coding genes and two ribosomal RNA genes (table 3 ). These genes are distributed evenly among the four major gene groups (NADH, photosynthetic, ribosomal protein, and RNA genes) ({chi}2 = 2.09 with Yates' correction, df = 3, P = 0.55). Their physical distribution on the cpDNA molecule does not seem to be clustered, although some are tightly linked (ndhJ-ndhK, psbC-psbD, and psbE-psbF) (data not shown).


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Table 3. Statistically Significant Heterogeneity of Nucleotide Substitution Rates of 14 Chloroplast Genes Common to Three Cereals

 
Relative rate test results showed that rice genes tend to have evolved more slowly than maize or wheat genes (or both) when there was significant substitution rate heterogeneity (table 3 ). In the maize-rice comparison, all five genes that had heterogeneous substitution rates (ndhK, psbD, psbH, rrn16, and rrn23) (P < 0.05 or P < 0.001) had higher total gene divergence in maize. In the rice-wheat comparison, seven (ndhB, psbC, psbE, psbF, psbH, rpl2, and 5'-rps12) of nine genes with a heterogeneous substitution rate (P < 0.05 or P < 0.001) had higher total gene divergence in wheat. There were only two cases of the opposite trend. For ndhJ and rpoA the rice homolog showed higher total gene divergence than the wheat homologs despite the <0.05 probability levels of rate heterogeneity. Of the six genes that showed significant substitution rate heterogeneity in the maize-wheat comparison (P < 0.05, P < 0.01, or P < 0.001), four wheat genes (ndhB, atpE, rpl2, and 5'-rps12) and two maize genes (ndhK and rrn23) had higher total gene divergence than their homologs in the other species.

Nonsynonymous Substitutions as a Driving Force for Gene Divergence
Relative rate tests showed that some cereal chloroplast genes evolved at heterogeneous rates. The rate differences seem to consist of multiple components (table 4 ). One component is the bias regarding transitional and transversional substitutions. For example, the transition-transversion ratio of the wheat psbF gene is 1.7-fold that of rice psbF and 2.1-fold that of maize psbF. This suggests that the high total gene divergence of the wheat psbF gene (table 3 ) may be the result of accelerated transitional substitutions in wheat, decelerated transversions in the others, or both. Similarly, the synonymous and nonsynonymous substitution ratios (S-N ratio) varied among the species (table 4 ). For example, the maize psbD gene had a smaller S-N ratio (15.4) than the rice and wheat genes (38.7 and 22.9) because of the high nonsynonymous substitution rate (0.024 in maize, 0.009 in rice, and 0.015 in wheat). The high nonsynonymous substitution rate may have contributed to the high gene total divergence of maize psbD gene (table 3 ).


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Table 4. Nucleotide Substitution Patterns in 14 Chloroplast Genes with Heterogeneous Substitution Rates

 
The variations in the nonsynonymous substitution rates are particularly interesting because they may reflect different selective constraints on the gene products in the different evolutionary lineages of the chloroplast genome. To further examine nucleotide substitution rate heterogeneity in the cereal lineages, sequences of the 12 protein-coding genes were partitioned into three site classes: nondegenerate (or zerofold degenerate), twofold degenerate, and fourfold degenerate sites. A nondegenerate site is one in which all possible nucleotide substitutions are nonsynonymous, whereas in a fourfold degenerate site all possible nucleotide substitutions are synonymous. A twofold degenerate site is one in which substitution is synonymous or nonsynonymous depending on whether it is transitional or transversional. Relative rate tests were performed on each class (table 5 ).


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Table 5. Heterogeneity of the Nucleotide Substitution Rate at Nondegenerate and Two- and Fourfold Degenerate Sites

 
Of the 50 relative rate tests performed, significant rate heterogeneity was found in 11 genes (table 5 ). The four genes that failed to show significant rate heterogeneity (psbE, psbF, psbH, and 5'-rps12) are small ones, suggesting that the amount of information available may not have been adequate for the tests. Interestingly, except for one (psbC in the rice-wheat comparison), none of the fourfold degenerate sites showed significant rate heterogeneity (table 5 ). In contrast, significant rate heterogeneity was detected at nondegenerate sites in six of 17 genes: psbD in the maize-rice comparison, ndhB and rpl2 in the maize-wheat comparison, and ndhB, rpl2, and rpoA in the rice-wheat comparison. Of these, four were not coupled with rate heterogeneity at the two- or fourfold degenerate sites (ndhB and rpl2 in the maize-wheat comparison; ndhB and rpoA in the maize-wheat comparison). These genes clearly indicate there are nonsynonymous substitution rate differences between the evolutionary lineages of grass chloroplast genomes.

Some genes provided evidence that accelerated nonsynonymous substitution rates enhanced total gene divergence. For the ndhB and rpl2 genes, relative rate tests for the entire sequences indicated that the wheat homologs evolved significantly faster than the maize and rice homologs (P < 0.001) (table 3 ). Although in those genes wheat had larger synonymous and nonsynonymous substitution rates than maize and rice (table 4 ), significant rate heterogeneity (P < 0.001) was found exclusively at nondegenerate sites (table 5 ), indicating that nonsynonymous changes are the major driving force for fast nucleotide substitution in wheat ndhB and rpl2 genes. In the psbD gene, the faster evolution of the maize homolog relative to the rice homolog (P < 0.05, table 3 ) is associated with a marked difference in the nonsynonymous substitution rate (table 4 ) and significant rate heterogeneity at nondegenerate sites (P < 0.001) (table 5 ). Regarding the rpoA gene, the rice homolog evolved significantly faster than the wheat homolog (P < 0.05) (table 3 ). Although compared with wheat, rice had higher synonymous and nonsynonymous substitution rates (table 4 ), and relative rate test results showed significant heterogeneity only at nondegenerate sites (P < 0.05, table 5 ), revealing that accelerated nonsynonymous changes are responsible for the enhanced total gene divergence of the rice rpoA gene.

Chloroplast Genome Phylogeny Based on Genome-Wide Gene Comparisons
Chloroplast genes have been used extensively to reconstruct the phylogeny in the Poaceae. Most of the conclusions drawn have been based on nucleotide sequence variation in a single chloroplast gene. The amount of phylogenetic information that a chloroplast gene provides is not, however, always sufficient to make robust phylogenetic inferences (Doebley et al. 1990Citation ). One way to overcome this is to increase the amount of information available for phylogeny reconstruction by analyzing multiple chloroplast genes. The 106 chloroplast genes provided us the opportunity to infer chloroplast genome phylogeny of cereals on the basis of genome-wide gene comparisons. Because maize (Panicoideae), rice (Bambusoideae), and wheat (Pooideae) represent different subfamilies of the Poaceae, whether phylogeny based on the entire genic region agrees with that based on a single gene is of interest.

To infer a phylogeny based on a genome-wide gene comparison, we focused on nucleotide substitutions in the 106 genes. Nucleotide substitutions are reliable phylogenetic markers for the chloroplast genome (see Golenberg et al. 1993Citation ). We constructed a chloroplast genome type for each species by extracting and concatenating the variable sites from the aligned gene sequences of maize, rice, wheat, and tobacco. Of the 106 genes, 98 had more than one variable site. Because the impact of the rate heterogeneity of the nucleotide substitutions in each gene on phylogeny reconstruction was unknown, we used three gene groups for analysis: (1) all 98 genes; (2) 94 genes, excluding four genes (ndhB, psbD, psbH, and rrn23) that showed highly significant rate heterogeneity of nucleotide substitution (P < 0.001) in relative rate tests on the entire sequences; and (3) 84 genes, excluding all 14 genes that showed significant rate heterogeneity (P < 0.05). The total number of variable sites and average number of variable sites per gene, respectively, ranged from 8324 to 9675 bp and from 98.7 to 99.1 bp (table 6 ).


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Table 6. Phylogenetic Analyses of Three Cereal Chloroplast Genomes Based on Concatenated Variable Sites

 
The use of loci or sites that evolve with homogeneous substitution rates between lineages is ideal for reconstructing the phylogenies. Relative rate tests done on the chloroplast genome types detected significant rate heterogeneity between rice and wheat when all 98 genes were used (table 6 ). On the whole, excluding genes with significant rate heterogeneity is therefore useful for obtaining chloroplast genome types that have homogeneous substitution rates. Variable site–based phylogenetic trees of the three cereal chloroplast genomes were constructed by means of the neighbor-joining algorithm (Saitou and Nei 1987Citation ). All three trees have the same topology (fig. 1 ), which supports a close relationship between the rice and wheat chloroplast genomes (52%–87% for bootstrap, 88%–99% for the interior test) (table 6 ). This suggests that rice and wheat had a common ancestor, which was not involved in the lineage leading to modern maize. The highest statistical supports for the rice-wheat clade (87% for bootstrap, 99% for the interior test) were obtained when the four genes (ndhB, psbD, psbH, and rrn23) that showed highly significant rate heterogeneity of nucleotide substitution (P < 0.001) in the relative rate tests were excluded (table 6 ).



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Fig. 1.—Neighbor-joining tree based on chloroplast genome types constructed from the 94 chloroplast genes of maize, rice, wheat, and tobacco. Chloroplast genome types were constructed by extracting and concatenating variable sites from the aligned gene sequences. The proportion of sites at which two chloroplast genome types differ was used as the genetic distance. Figures on the branch are the bootstrap (numerator) and interior test (denominator) supports for the rice-wheat clade

 

    Discussion
 TOP
 Abstract
 Introduction
 Data and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Mechanism for Chloroplast Gene Diversification
Heterogeneity of the nucleotide substitution rate is well documented for the chloroplast gene, rbcL, at the interfamilial (Bousquet et al. 1992Citation ; Gaut et al. 1992Citation ) and intrafamilial (Doebley et al. 1990Citation ; Soltis et al. 1990Citation ) levels. Those studies showed that rate variation is limited within a family, that synonymous and nonsynonymous substitution rates vary substantially among families, and that rbcL evolved rapidly in the grass family relative to the other families. Furthermore, Gaut, Muse, and Clegg (1993)Citation showed that the same trends hold for several other chloroplast genes. We focused on the chloroplast genomes of three major cereals, which have been fully sequenced and examined the intrafamilial evolution of chloroplast genes by whole-genome comparisons. We performed relative rate tests on 106 chloroplast genes with tobacco sequences as the outgroup and found, in support of previous findings (Gaut, Muse, and Clegg 1993Citation ), that in the cereal species the majority of chloroplast genes (92 out of 106 genes, 86.8%) evolved at homogeneous rates.

Unlike the preponderance of genes that evolve at homogeneous rates, our results showed that 14 genes (13.2%), about 28% of the genic region, evolved at heterogeneous rates in the chloroplast genomes of the three cereals. This raises the question of what generated the heterogeneity of nucleotide substitutions in those genes. The overall nucleotide substitution pattern in the genes suggests that rate heterogeneity was the product of multiple factors, including biased transitional and transversional substitution rates and variation in synonymous and nonsynonymous substitution rates between species (table 4 ). Relative rate tests done on the entire gene sequences showed that the rice homolog tends to have evolved more slowly than the homologs of maize and wheat at loci that showed rate heterogeneity (table 3 ). This may reflect the prolonged generation time of Oryza species, which include many perennials and annual-perennial intermediates, whereas the Zea and Triticum species are primarily annual (exceptions: Zea perennis and Zea diploperennis). If prolonged generation time is the primary factor, one would expect reduced total gene divergence (D) for all rice chloroplast genes because the generation-time effect should affect the entire genome. This expectation is not supported because there are slight differences in the average D values of the 106 genes of the three cereal species (0.112 for maize, 0.112 for rice, and 0.113 for wheat) (Friedman test, P = 0.35). The contribution of the generation-time effect to gene diversification therefore appears to be minor in chloroplast genomes of cereal species.

Unlike the genome-wide effect of generation time, selection may target individual genes, producing rate heterogeneity between evolutionary lineages. We dissected the rate heterogeneities found for 12 protein-coding genes by performing separate relative rate tests on the nondegenerate, twofold degenerate, and fourfold degenerate sites of these genes. Of the 12 genes, four (ndhB, psbD, rpl2, and rpoA) gave results that favor the selection hypothesis. In those genes, nonsynonymous substitutions seem to be accelerated in one of the three species, whereas synonymous substitution rates were homogeneous interspecifically, suggesting that in the different cereal lineages gene products were under different selective constraints. More importantly, the marked association of accelerated rates of nonsynonymous substitution with enhanced total gene divergence indicates that much of the interspecific variation in these genes is attributable to differences in nonsynonymous substitution rates. Selection therefore seems to have had a role in the diversification of these genes.

Our results also provide evidence that other factors contribute to chloroplast gene diversification. The psbC gene, the only one with significant rate heterogeneity at the fourfold degenerate (table 5 ), shows that accelerated synonymous substitution is a driving force for the diversification of this gene in wheat. Relative rate tests failed to detect significant heterogeneity at all site classes in five genes (ndhK, psbE, psbF, psbH, and 5'-rps12) (table 5 ). The variation in these genes may be attributable to factors other than biased rates for synonymous and nonsynonymous substitutions. These findings indicate that the mechanism that underlies gene diversification in cereal chloroplast genomes is complex.

Implications for Phylogeny of Cereals
Morphological and molecular studies of grass phylogeny are not always congruent regarding relationships between the subfamilies that maize (Panicoideae), rice (Bambusoideae), and wheat (Pooideae) represent. A numerical taxonomy study indicated that the Panicoideae is an outgroup to the Bambusoideae and Pooideae (Watson, Clifford, and Dallwitz 1985Citation ), whereas Clayton (1981)Citation proposed that on the basis of geographic distribution patterns rice is an outgroup. Analyses of a chloroplast gene (ndhB, Clark, Zhang, and Wendel 1995Citation ) and a nuclear gene (PHYB, Mathews, Tsai, and Kellogg 2000Citation ) recognized a monophyletic clade that includes the Bambusoideae and Pooideae and placed the Panicoideae as an outgroup. Monophyly of the clade that includes the Bambusoideae and Pooideae, however, is not supported by findings for other chloroplast genes (e.g., matK gene, Hilu and Alice 1999Citation ). Recently, Ogihara et al. (2002)Citation suggested that rice and wheat are more closely related to each other than to maize based on the structural similarity of the chloroplast genomes.

One problem regarding such molecular studies is the limited amount of phylogenetic information a single gene provides. In this study, we constructed chloroplast genome trees for the three major cereals based on variable nucleotide sites in the genes. They all place maize basal to the rice-wheat clade; moreover, statistical support for the rice-wheat clade is a high 87% (bootstrap) and 99% (interior test) (fig. 1 and table 6 ). These findings support a close relationship between the Bambusoideae and Pooideae in the grass phylogeny and suggest that the Panicoideae split from the rice-wheat lineage during the early stage of grass evolution. Given the increasing number of fully sequenced chloroplast genomes in the databases, a variable site–based approach should provide a simple method to reconstruct a reliable phylogeny based on the entire genome comparison, but the performance and application of this approach require further evaluation.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Data and Methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Yves Vigouroux for his helpful comments. This work was supported in part by a Grant-in-Aid (No. 10309008) from the Ministry of Education, Science, Sports, and Culture of Japan.


    Footnotes
 
Geoffrey McFadden, Reviewing Editor

Abbreviations: cpDNA, chloroplast DNA. Back

Keywords: chloroplast genome gene evolution relative rates substitution rate Poaceae Back

Address for correspondence and reprints: Yoshihiro Matsuoka, Fukui Prefectural University, Matsuoka-cho, Yoshida-gun, Fukui 910-1195, Japan. E-mail: matsuoka{at}fpu.ac.jp Back


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 TOP
 Abstract
 Introduction
 Data and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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Accepted for publication July 15, 2002.





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