The complete nucleotide sequence of wild rice (Oryza nivara) chloroplast genome: first genome wide comparative sequence analysis of wild and cultivated rice

Gene 340 (2004) 133 – 139 www.elsevier.com/locate/gene

The complete nucleotide sequence of wild rice (Oryza nivara) chloroplast genome: first genome wide comparative sequence analysis of wild and cultivated rice M. Shahid Masood a,1, Tomotaro Nishikawa a, Shu-ichi Fukuoka a, Peter K. Njenga a, Takahiko Tsudzuki b, Koh-ichi Kadowaki a,* a

Molecular Biodiversity Laboratory, Genetic Diversity Department, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Ibaraki, Tsukuba 305-8602, Japan b Department of Information and Policy Studies, Aichi-Gakuin University, Araike 12, Nisshin 470-0195, Japan Received 20 April 2004; received in revised form 15 May 2004; accepted 1 June 2004 Available online 27 July 2004 Received by W. Martin

Abstract We determined the complete nucleotide sequence of the chloroplast genome of wild rice, Oryza nivara and compared it with the corresponding published sequence of relative cultivated rice, Oryza sativa. The genome was 134,494 bp long with a large single-copy region of 80,544 bp, a small single-copy region of 12,346 bp and two inverted repeats of 20,802 bp each. The overall A + T content was 61.0%. The O. nivara chloroplast genome encoded identical functional genes to O. sativa in the same order along the genome. On the other hand, detailed analysis revealed 57 insertion, 61 deletion and 159 base substitution events in the entire chloroplast genome of O. nivara. Among substitutions, transversions were much higher than transitions with the former even more frequent than the latter in the coding region. Most of the insertions/deletions were single-base but a few large length mutations were also detected. The frequency of insertion/deletion events was more in the coding region within inverted repeats. In contrast, a very few substitution events were identified in the coding region. Polymorphism was observed among rice cultivars at loci of large insertion/deletion events. This is the first report describing comparative and genome wide chloroplast analysis between a wild and cultivated crop. D 2004 Elsevier B.V. All rights reserved. Keywords: Wild rice; Oryza nivara; Chloroplast; Comparative genome

1. Introduction Chloroplasts are intracellular organelles present in plants where photosynthesis is processed. Similar to mitochondria, they have their own genome distinct from the nucleus. The first complete chloroplast DNA sequences are reported in

Abbreviations: CP, chloroplast; CH, chromosome; DEL, deletion; indel, insertion/deletion; ins, insertion; IR, inverted repeat; LSC, large single copy; PCR, polymerase chain reaction; rRNA, ribosomal RNA; SSC, small single copy; Ts, transition; Tv, transversion. * Corresponding author. Tel.: +81-29-838-7449; fax: +81-29-8387408. E-mail address: [email protected] (K. Kadowaki). 1 Present address: Plant Genetic Resources Institute, National Agricultural Research Center, Park Road, Islamabad, Pakistan. 0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.06.008

tobacco (Nicotiana tabacum, Shinozaki et al., 1986) and liverwort (Marchantia polymorpha, Ohyama et al., 1986). Since then the entire sequence of chloroplast DNA has been determined from various plant species such as rice (Oryza sativa, Hiratsuka et al., 1989), beechdrops (Epifagus virginiana, Wolfe et al., 1992), black pine (Pinus thunbergii, Wakasugi et al., 1994), maize (Zea mays, Maier et al., 1995), whisk fern (Psilotum nudum, Wakasugi et al., 1998), mouse-ear cress (Arabidopsis thaliana, Sato et al., 1999), wheat (Triticum aestivum, Ogihara et al., 2000), evening prim-rose (Oenothera elata, Hupfer et al., 2000), lotus (Lotus japonicus, Kato et al., 2000), spinach (Spinacia oleracea, Schmitz-Linneweber et al., 2001), deadly nightshade (Atropa belladonna, Schmitz-Linneweber et al., 2002) and maiden-hair fern (Adiantum capillus-veneris, Wolf et al., 2003).

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The chloroplast genome of photosynthetic plants consists of multiple copies of homogeneous circular double-stranded DNA molecules ranging in size from 120 to 217 kb. Most of the genomes examined contain a pair of large inverted repeats (IR) harboring the ribosomal RNA (rRNA), six tRNAs, and seven protein genes (Palmer, 1991; Sugiura, 1992; Gaut, 1998) The two repeat sequences are identical and are separated by a large (LSC) and a small single-copy (SSC) region. Although the overall structure of the chloroplast genome is generally well conserved, a number of mutations have been observed. These are small structural changes such as inversions (Hiratsuka et al., 1989), translocations (Ogihara et al., 1988), and insertions/deletions (Ogihara et al., 1991; Kanno et al., 1993; Maier et al., 1995) as well as base substitutions (Morton and Clegg, 1995). Three inversions characterize all examined members of grass family (Doyle et al., 1992). These inversions caused extensive rearrangements within the rice chloroplast LSC region were elucidated by comparison with the tobacco chloroplast genome (Hiratsuka et al., 1989). Studies of wheat (Howe et al., 1988) and maize (Palmer and Thompson, 1982) are also consistent with conservation of this gene arrangement among cereals. It is reported that insertions/deletions (indels) do not occur at random locations within organelle genomes, but they are often associated with specific features of DNA sequences. Regions containing repeats that lead to slippedstrand mispairing and intramolecular recombination are thought to cause the majority of indel mutations (reviewed by Kelchner, 2000). Furthermore, comparison of the complete DNA sequence among three cereals (rice, maize and wheat) indicates the presence of some hot-spot regions for length mutations (Ogihara et al., 2002). To date, sequences of chloroplast genome have been reported from a wide variety of crops, however, there are no reports of comparison with their wild relatives. Rice is one of the most important food crops and its production must increase to meet the demand of the growing population. In addition to the chloroplast genome, sequencing information of the rice nuclear (Goff et al., 2002) and mitochondrial (Notsu et al., 2002) genomes are also available. Here, we report the entire chloroplast genome nucleotide sequence of the wild rice, Oryza nivara which is an important reservoir of useful genes for rice breeding. Both annual (O. nivara) and perennial (Oryza rufipogon) wild species are believed to be the immediate progenitors of rice and share the same AA type nuclear genome (Vaughan et al., 2003). In this study we also compare the chloroplast genome of O. nivara with rice and wheat.

2. Materials and methods Chloroplast DNA from wild rice, O. nivara (SL10, NIAS) from a Sri Lanka was used. The chloroplast DNA was isolated from young green leaves using CTAB-based

protocol according to Murray and Thompson (1980). Primers were designed using available information from the complete chloroplast sequence analysis of O. sativa (Hiratsuka et al., 1989). Template DNA was prepared with 50 ng of genomic DNA in a PCR system 100 (MJ Research, USA) following the enzyme manufacturer’s instructions (KOD-plus, Toyobo, Japan). The thermal cycling program was as follows: 94 jC for 2.5 min, then 30 cycles of 94 jC for 15 s, 50– 55 jC for 30 s and 68 jC for 4 – 6 min (depending upon the size of the target region). PCR products were analyzed by electrophoresis on 0.9% agarose gel. To eliminate unused dNTPs and primers, the PCR products were purified using SUPRECk-02 (TaKaRa, Japan) according to the manufacturer’s instruction. The purified DNA was subsequently used for cycle sequencing using Dye Terminator Cycle Sequencing (DTCS) Quick Start Kit (Beckman Coulter, USA). The PCR was performed using final volume of 10 Al with a first step at 96 jC for 1 min followed by 30 cycles of 96 jC for 20 s, 55 jC for 20 s and 60 jC for 4 min. PCR products were precipitated and directly subjected to sequence analysis using CEQ 2000 XL DNA Analysis System (Beckman Coulter). Sequences were assembled and evaluated using Sequencher 3.0 (Gene Codes, USA). The complete chloroplast genome sequence of rice (X15901, Hiratsuka et al., 1989) was obtained from DDBJ as a reference sequence. The DNA sequences were aligned using GENETYX program (Software Development, Tokyo). Substitutions were scored assuming a single substitution at a site. Comparative analysis of O. nivara and O. sativa was performed according to (Tsudzuki et al., in press). The O. nivara chloroplast DNA indels were compared with rice cultivars namely Nipponbare, Chuumoushi (Japonica), Junsouhaku (Indica) and Shoutan Zairai (Japonica) using PCR. The phylogenetic tree was constructed using Unweighted Pair Group Method with Arithmatic Means (Sokal and Michener, 1958) based on Kimura two-parameter distance matrix (Kimura, 1980).

3. Results and discussion 3.1. Overall organization of O. nivara chloroplast genome The entire chloroplast genome of O. nivara was determined (DDBJ/EMBL/GenBank accession no. AP006728). It was a circular double-stranded DNA molecule of 134,494 bp, shorter than rice, O. sativa, by 31 bp (134, 525 bp, Hiratsuka et al., 1989). It exhibited a quadripartite structure, typical of other angiosperm chloroplast genomes, with two single copy regions [SSC of 12,346 bp, and LSC of 80,544 bp] separating two identical copies of IR each 20,802 bp long containing the rRNA operon. The A + T content (61.0%) was similar to those of O. sativa (61.1%), T.

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Fig. 1. Comparative sequence analysis of the O. nivara and O. sativa chloroplast genome. Arrows to right direction indicate deletion event in O. nivara (insertion in O. sativa) and arrows to opposite direction indicate deletion event in O. sativa (insertion event in O. nivara). Length of arrow indicates size of the indel event.

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aestivum (61.7%), and Z. mays (61.5%). The A + T contents of the LSC and SSC region were 62.9% and 66.7%, respectively. The O. nivara chloroplast genome encoded a similar number of functional genes like that of O. sativa (Hiratsuka et al., 1989) and they are arranged in an identical order along the genome.

Table 2 Deletion events in the IR, LSC, and SSC region of O. nivara chloroplast genome

3.2. Comparative chloroplast genomics (O. nivara vs O. sativa)

The values in parentheses represent proportion (%). IR = inverted repeat, LSC = large single copy, SSC = small single copy.

One of the purposes of this study was to evaluate the genetic diversity between wild (O. nivara) and cultivated rice (O. sativa) using entire chloroplast DNA sequence. Comparative sequence analysis revealed a large number of mutations throughout the genome that includes indels and base substitutions. In total, 57 insertion, 61 deletion, and 159 base substitution events were observed in the entire chloroplast genome of O. nivara. Overall genome wide indel events in O. nivara relative to O. sativa are shown in Fig. 1. These mutations are described below. 3.2.1. Insertion/deletion events Of the 57 insertion events, 11 (19.3%) were identified in the coding region, 14 (24.6%) in intron and 32 (56.1%) in the intergenic region (Table 1). Most of the insertions (42) were single-base, however multi-nucleotide insertions were also identified in the coding, intron and intergenic regions. A 6-bp insertion was located in the coding region of ORF178, whereas another insertion of 4-bp was found within ORF63. These results suggest that ORF63 is not a functional gene since a 4-bp insertion may disrupt the coding sequence. In addition to these events in the SSC, a 2-bp insertion was also observed in the coding region of the 23S rRNA gene which is present in the IR-region. Although most insertions in the intron and intergenic region were short, six bases or less, a 21-base duplication was present in the intergenic spacer region between infA and rps8. The second large insertion of 16-bp was found in the intergenic region of atpB and rbcL. The two events happened in the LSC region. The deletion events in three regions (coding, intergenic and intron) are given in Table 2. Of 61 deletion events, only 6 (9.8%) were in the coding region, 16 in the intron (26.3%) and 39 (63.9%) in the intergenic region. Overall, the indels were observed more frequently in the intergenic

Region

IR

LSC

SSC

Total

Coding region Intron Intergenic region

4 1 6

1 15 32

1 0 1

6 (9.8) 16 (26.3) 39 (63.9)

region compared to that of intron and coding region. Hiratsuka et al. (1989) also identified many indels in the intergenic region when comparing rice and tobacco. Similar to insertions, numerous one base pair deletions (51) exist in the chloroplast genome of O. nivara. A single large deletion of 69-bp was located in the coding region of ORF100 within the LSC region. Since a 13-bp direct repeat sequence was found at the boarder of the deleted portion, this deletion is assumed to have been caused by a recombination via the 13 bp direct repeat. The same size of deletion within this locus has previously been reported by Kanno et al. (1993) in indica cultivar (O. sativa, ssp indica). It is known that direct repeats contribute significantly to variability of chloroplast genomes (Wolfe et al., 1992; Ogihara et al., 2002). The four single-base deletions were located in the coding region of genes in the IR whereas one base pair deletion was found within ndhF which is present in the SSC region. As for the intergenic region and intron, most of the multi-nucleotide deletions were 2- to 6-bp in length. The only exception was a 16-bp deletion that occurred in the intergenic region of ORF106 and ORF36. 3.2.2. Substitution events Comparison of the entire sequence of the O. nivara chloroplast genome to that of O. sativa revealed 159 base substitutions. Of these, 81 (50.9%) events were in the coding region, 14 (8.8%) in intron and 64 (40.3%) in the intergenic region (Table 3). The frequency of substitution events per 10 kb in the coding, intron and intergenic region was 10, 8, and 18, respectively. A large proportion of point mutations was found in the coding region within the LSC (83.9%) and SSC (12.4%) regions. Conversely, mutation rate (3.7%) was very low in the IR. This is a

Table 1 Insertion events in the IR, LSC, and SSC region of O. nivara chloroplast genome

Table 3 Substitution events in the IR, LSC, and SSC region of O. nivara chloroplast genome

Region

IR

LSC

SSC

Total

Region

IR

LSC

SSC

Total

Coding region Intron Intergenic region

6 0 7

2 13 24

3 1 1

11 (19.3) 14 (24.6) 32 (56.1)

Coding region Intron Intergenic region

3 2 7

68 11 49

10 1 8

81 (50.9) 14 (8.8) 64 (40.3)

The values in parentheses represent proportion (%). IR = inverted repeat, LSC = large single copy, SSC = small single copy.

The values in parentheses represents proportion (%). IR = inverted repeat, LSC = large single copy, SSC = small single copy.

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contrast to indels that were mainly the IR region. Sequences located in the IR diverge at a slower rate compared to the sequences in the SSC region (Wolfe et al., 1987). The difference in the substitution rate between IR and LSC/ SSC may be attributed to a reduced mutation rate in the IR or to a bias against mutant sequences during DNA repair. Substitutions often occur with unequal probability resulting in substitution bias. The 159 base substitutions that are transversions (Tv) or transitions (Ts) for the coding, intron and intergenic region are shown in Table 4. The proportion of Tv are much higher than Ts at all the regions indicating a bias in favor of transversions. It was even more pronounced in the coding region where transversions (50) were 60% more than transitions (31). Morton and Clegg (1993) noted that the ratio of Tv to Ts is much lower in sequences with a lower A + T content in the noncoding regions of chloroplast DNA. Morton (1995) analyzed the frequency of base substitutions for the two sequences with different A + T contents and found that ratio of Tv to Ts increased with an increasing A + T content. These results were further supported by Xu et al. (2000) when nine of the eleven substitutions detected in the non-coding region of subgenus Soja were either A/C or T/ G transversions. The high ratio of Tv to Ts in the present study may also be due to high A + T content of the sequences. The chloroplast genome has high A + T contents that might affect the substitution dynamics in the DNA of the chloroplast. 3.3. Evaluation of O. nivara chloroplast DNA indels compared with several rice cultivars A few large length mutations were detected in the chloroplast genome of O. nivara (SL10) relative to O.

Table 4 Transversion (Tv) and transition (Ts) events in the coding region, intergenic region, and intron of the chloroplast genome of O. nivara vs. O. sativa Region

On

Coding region

A G C T Total A G C T Total A G C T Total

Intergenic region

Intron

Os A

G

C

T

Tv/Ts

– 10 4 6

6 – 12 7

6 10 – 5

2 3 10 –

– 6 6 6

10 – 2 7

8 0 – 4

2 7 6 –

– 0 0 2

2 – 1 1

1 2 – 0

1 1 3 –

8/6 13/10 16/10 13/5 50/31 10/10 7/6 8/6 13/4 38/26 2/2 3/0 1/3 3/0 9/5

Os and On stand for O. sativa and O. nivara, respectively.

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Fig. 2. Amplification patterns of specific chloroplast DNA fragment (indels) of O. nivara in comparison with O. sativa cv. Nipponbare and several other rice cultivars. M, Molecular marker (100 bp DNA ladder); Lane 1, Nipponbare; Lane 2, SL10; Lane 3, Chuumoushi (Japonica); Lane 4, Junsouhaku (Indica); Lane 5, Shoutan Zairai (Japonica). A, B, and C indicate PCR amplification with primer 8k-1, 8k-2; 57k-1, 57k-2, and 76k1, 76k-2 respectively.

sativa (Nipponbare). These include: a 69-bp deletion in the coding region of ORF100, a 21-bp and a 16-bp insertion in the intergenic spacer region of infA and rps8, and atpB and rbcL, repectively (Fig. 2). All these indels are accumulated in the LSC region. We surveyed several rice (O. sativa) cultivars to determine the distribution of these indel events. With regard to the 69-bp deletion, some of the cultivars had non-deletion type whereas the others were found to have deletion type chloroplast DNA (Fig. 2). These results indicate polymorphism within the cultivated rice in terms of presence/absence of this 69-bp deletion. Chen et al. (1993) has already surveyed 137 rice cultivars for distribution of this deletion, our results agreed with their report. However, their evaluation was restricted only to one locus at ORF 100. In this study another two novel loci (21 and 16 bp) were identified after genome wide analyses, we examined their polymorphism in the cultivated rice. Insertion of the 16-bp chloroplast DNA fragment was observed in some cultivars but not in others. On the other hand, it is of interest that insertion of the 21-bp sequence has not yet been detected in cultivated rice. Taken together, the present data suggest that the extent of polymorphism is different within O. sativa as regard to these indel events. These data will be useful to understand the process of domestication in cultivated rice.

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3.4. Comparative analysis of current chloroplast genomes and its ancestral DNA sequence migration into the rice nuclear genome Gene transfer from the chloroplast to the nucleus has occurred during a course of symbiosis. Some of the gene transfer events are functional whereas the others are nonfunctional. A large chloroplast DNA segment of 130,882 bp was identified by computer search in the rice (O. sativa cv. Nipponbare) nuclear genome at chromosome 10. We were interested to see whether this event occurred before or after the divergence of Pooideae (wheat) and Bambusoideae (rice), and to see which kind of chloroplast genome integrated into the nucleus. The entire chloroplast genomes of O. sativa, O. nivara, T. aestivum, and the DNA sequence of rice nuclear genome at chromosome 10 are now available and were compared. We constructed a phylogenetic tree that clearly showed that chloroplast DNA migration into the nuclear genome occurred after the split of Bambusoideae and Pooideae (Fig. 3). It is believed that O. nivara is an immediate progenitor of cultivated rice, and a chloroplast sequence has been detected in the nuclear genome of O. sativa. We analysed whether this migration occurred from the O. sativa or O. nivara chloroplast genome. Investigation of homologous sequence using PCR amplification showed presence of corresponding chloroplast DNA sequence in O. nivara (data not shown). Our analyses revealed 6 indels (Loci 1, 3, 4, 5, 6, and 9) in the nuclear DNA sequence at chromosome 10 of O. sativa but not in the chloroplast genomes of O. nivara and O. sativa (Table 5; Fig. 3). The identification of such indel events in the nuclear genome was not surprising as in plant cells the mutation rate has been shown to be much

Table 5 Indel events revealed in chloroplast genome of O. nivara, O. sativa, and T. aestivum with homologous sequence in nuclear genome (ch. 10) of O. sativaa Locus Position (length in bp) 1 2 3 4 5 6 7 8 9

Os

Os (ch.10) On Ta

Between 117,563 and 117,564 (122) – Ins Between 8,552 and 8,553 (69) Ins – Between 17,746 and 17,747 (32) – Ins 19,610 to 19,643 (34) – Del Between 32,102 and 32,103 (45) – Ins 45,169 to 45,205 (37) – Del Between 56,964 and 56,965 (16) Ins – 76,639 to 76,659 (21) – – 92,505 to 92,522 (18) – Del

– – – Ins – – – – – – – – – – Ins – – –

Position is based on O. nivara chloroplast genome in this study. Os, On and Ta stand for O. sativa, O. nivara and T. aestivum, respectively. Ins = Insertion, Del = Deletion. a The detailed results in Table 5 are available at http://www.nias.affrc. go.jp/gene_suppl/ as supporting information.

lower in chloroplast genome than in the nucleus (Wolfe et al., 1987). Further, the chloroplast sequence in the nuclear genome is non-functional and no bias for keeping integrity of sequence. In O. nivara the homologous sequence shows one insertion event (Locus 8) not seen in chromosome 10 of rice and two insertion events (loci 2, 7) in the homologous sequence of O. sativa chloroplast DNA not seen in chromosome 10 (Table 5; Fig. 3). The insertion (locus 2) in O. sativa chloroplast DNA sequence was also found in wheat chloroplast sequence. This conserved sequence requires further study. In conclusion, the O. nivara chloroplast genome is very similar to O. sativa. However, a number of insertion/ deletion and base substitution events were detected. A substantial amount of polymorphism was observed within cultivated rice (O. sativa) based on large indels detected in the O. nivara chloroplast genome relative to O. sativa. Our data shows that the 130,882 bp inserted chloroplast sequence in the nuclear genome (chromosome 10) of rice has specific similarity to both the homologous sequences in O. nivara chloroplast genome and O. sativa chloroplast genome. The fully sequenced chloroplast genome of O. nivara in comparison with its counterpart O. sativa will be extremely helpful for understanding diversity among AA genome taxa and understanding the process of domestication in cultivated rice.

Acknowledgements Fig. 3. Identification and evolution of a 131 kb chloroplast DNA sequence identified in rice chromosome 10. Solid filled triangles represent deletions and solid open triangles indicate insertions identified only in one genome but not in others. A dotted open triangle shows insertion but further analysis is required. A black circle represents timing of chloroplast DNA insertion into nuclear genome and an arrow shows its presence in chromosome 10. The order and size of the triangles does not mean anything. The numbers represents loci of indel events that are given in Table 5.

The authors are grateful to D.A. Vaughan for critically reading the manuscript. The skillful technical assistance of Ms. N. Nohara and K. Miyashita is gratefully acknowledged. We are thankful to S. Kawakami for his generous help. This research was supported by fellowship from the Japan Society for the Promotion of Science (JSPS, L 03564) to M.S. Masood.

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