Complete Chloroplast Genome Sequence of Omani Lime (Citrus aurantiifolia) and Comparative Analysis within the Rosids
Identifieur interne : 000219 ( Pmc/Corpus ); précédent : 000218; suivant : 000220Complete Chloroplast Genome Sequence of Omani Lime (Citrus aurantiifolia) and Comparative Analysis within the Rosids
Auteurs : Huei-Jiun Su ; Saskia A. Hogenhout ; Abdullah M. Al-Sadi ; Chih-Horng KuoSource :
- PLoS ONE [ 1932-6203 ] ; 2014.
Abstract
The genus
Url:
DOI: 10.1371/journal.pone.0113049
PubMed: 25398081
PubMed Central: 4232571
Links to Exploration step
PMC:4232571Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Complete Chloroplast Genome Sequence of Omani Lime (<italic>Citrus aurantiifolia</italic>) and Comparative Analysis within the Rosids</title><author><name sortKey="Su, Huei Jiun" sort="Su, Huei Jiun" uniqKey="Su H" first="Huei-Jiun" last="Su">Huei-Jiun Su</name><affiliation><nlm:aff id="aff1"><addr-line>Institute of Ecology and Evolutionary Biology, National Taiwan University, Taipei, Taiwan</addr-line></nlm:aff></affiliation></author><author><name sortKey="Hogenhout, Saskia A" sort="Hogenhout, Saskia A" uniqKey="Hogenhout S" first="Saskia A." last="Hogenhout">Saskia A. Hogenhout</name><affiliation><nlm:aff id="aff2"><addr-line>Department of Cell and Developmental Biology, John Innes Centre, Norwich, United Kingdom</addr-line></nlm:aff></affiliation></author><author><name sortKey="Al Sadi, Abdullah M" sort="Al Sadi, Abdullah M" uniqKey="Al Sadi A" first="Abdullah M." last="Al-Sadi">Abdullah M. Al-Sadi</name><affiliation><nlm:aff id="aff3"><addr-line>Department of Crop Sciences, Sultan Qaboos University, Al Khoud, Oman</addr-line></nlm:aff></affiliation></author><author><name sortKey="Kuo, Chih Horng" sort="Kuo, Chih Horng" uniqKey="Kuo C" first="Chih-Horng" last="Kuo">Chih-Horng Kuo</name><affiliation><nlm:aff id="aff4"><addr-line>Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff5"><addr-line>Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, National Chung Hsing University and Academia Sinica, Taipei, Taiwan</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff6"><addr-line>Biotechnology Center, National Chung Hsing University, Taichung, Taiwan</addr-line></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">25398081</idno><idno type="pmc">4232571</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4232571</idno><idno type="RBID">PMC:4232571</idno><idno type="doi">10.1371/journal.pone.0113049</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">000219</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Complete Chloroplast Genome Sequence of Omani Lime (<italic>Citrus aurantiifolia</italic>) and Comparative Analysis within the Rosids</title><author><name sortKey="Su, Huei Jiun" sort="Su, Huei Jiun" uniqKey="Su H" first="Huei-Jiun" last="Su">Huei-Jiun Su</name><affiliation><nlm:aff id="aff1"><addr-line>Institute of Ecology and Evolutionary Biology, National Taiwan University, Taipei, Taiwan</addr-line></nlm:aff></affiliation></author><author><name sortKey="Hogenhout, Saskia A" sort="Hogenhout, Saskia A" uniqKey="Hogenhout S" first="Saskia A." last="Hogenhout">Saskia A. Hogenhout</name><affiliation><nlm:aff id="aff2"><addr-line>Department of Cell and Developmental Biology, John Innes Centre, Norwich, United Kingdom</addr-line></nlm:aff></affiliation></author><author><name sortKey="Al Sadi, Abdullah M" sort="Al Sadi, Abdullah M" uniqKey="Al Sadi A" first="Abdullah M." last="Al-Sadi">Abdullah M. Al-Sadi</name><affiliation><nlm:aff id="aff3"><addr-line>Department of Crop Sciences, Sultan Qaboos University, Al Khoud, Oman</addr-line></nlm:aff></affiliation></author><author><name sortKey="Kuo, Chih Horng" sort="Kuo, Chih Horng" uniqKey="Kuo C" first="Chih-Horng" last="Kuo">Chih-Horng Kuo</name><affiliation><nlm:aff id="aff4"><addr-line>Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff5"><addr-line>Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, National Chung Hsing University and Academia Sinica, Taipei, Taiwan</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff6"><addr-line>Biotechnology Center, National Chung Hsing University, Taichung, Taiwan</addr-line></nlm:aff></affiliation></author></analytic><series><title level="j">PLoS ONE</title><idno type="eISSN">1932-6203</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The genus <italic>Citrus</italic> contains many economically important fruits that are grown worldwide for their high nutritional and medicinal value. Due to frequent hybridizations among species and cultivars, the exact number of natural species and the taxonomic relationships within this genus are unclear. To compare the differences between the <italic>Citrus</italic> chloroplast genomes and to develop useful genetic markers, we used a reference-assisted approach to assemble the complete chloroplast genome of Omani lime (<italic>C</italic>. <italic>aurantiifolia</italic>). The complete <italic>C</italic>. <italic>aurantiifolia</italic> chloroplast genome is 159,893 bp in length; the organization and gene content are similar to most of the rosids lineages characterized to date. Through comparison with the sweet orange (<italic>C. sinensis</italic>) chloroplast genome, we identified three intergenic regions and 94 simple sequence repeats (SSRs) that are potentially informative markers with resolution for interspecific relationships. These markers can be utilized to better understand the origin of cultivated <italic>Citrus</italic>. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">PLoS One</journal-id><journal-id journal-id-type="iso-abbrev">PLoS ONE</journal-id><journal-id journal-id-type="publisher-id">plos</journal-id><journal-id journal-id-type="pmc">plosone</journal-id><journal-title-group><journal-title>PLoS ONE</journal-title></journal-title-group><issn pub-type="epub">1932-6203</issn><publisher><publisher-name>Public Library of Science</publisher-name><publisher-loc>San Francisco, USA</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">25398081</article-id><article-id pub-id-type="pmc">4232571</article-id><article-id pub-id-type="publisher-id">PONE-D-14-24554</article-id><article-id pub-id-type="doi">10.1371/journal.pone.0113049</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group><subj-group subj-group-type="Discipline-v2"><subject>Biology and Life Sciences</subject><subj-group><subject>Biotechnology</subject><subj-group><subject>Plant Biotechnology</subject><subj-group><subject>Plant Genomics</subject><subj-group><subject>Plant Genomes</subject></subj-group></subj-group></subj-group></subj-group><subj-group><subject>Cell Biology</subject><subj-group><subject>Cellular Structures and Organelles</subject><subj-group><subject>Chloroplasts</subject></subj-group></subj-group><subj-group><subject>Cellular Types</subject><subj-group><subject>Plant Cells</subject></subj-group></subj-group><subj-group><subject>Plant Cell Biology</subject></subj-group></subj-group><subj-group><subject>Computational Biology</subject><subj-group><subject>Genome Analysis</subject><subject>Genome Evolution</subject></subj-group></subj-group><subj-group><subject>Evolutionary Biology</subject><subj-group><subject>Evolutionary Systematics</subject><subj-group><subject>Phylogenetics</subject><subj-group><subject>Plant Phylogenetics</subject></subj-group></subj-group></subj-group><subj-group><subject>Organismal Evolution</subject><subj-group><subject>Plant Evolution</subject></subj-group></subj-group><subj-group><subject>Molecular Evolution</subject></subj-group></subj-group><subj-group><subject>Genetics</subject><subj-group><subject>Genomics</subject><subject>Plant Genetics</subject></subj-group></subj-group><subj-group><subject>Plant Science</subject></subj-group><subj-group><subject>Taxonomy</subject></subj-group></subj-group></article-categories><title-group><article-title>Complete Chloroplast Genome Sequence of Omani Lime (<italic>Citrus aurantiifolia</italic>) and Comparative Analysis within the Rosids</article-title><alt-title alt-title-type="running-head">Chloroplast Genome Sequence of Omani Lime (<italic>Citrus aurantiifolia</italic>)</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Su</surname><given-names>Huei-Jiun</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Hogenhout</surname><given-names>Saskia A.</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Al-Sadi</surname><given-names>Abdullah M.</given-names></name><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Kuo</surname><given-names>Chih-Horng</given-names></name><xref ref-type="aff" rid="aff4"><sup>4</sup></xref><xref ref-type="aff" rid="aff5"><sup>5</sup></xref><xref ref-type="aff" rid="aff6"><sup>6</sup></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib></contrib-group><aff id="aff1"><label>1</label><addr-line>Institute of Ecology and Evolutionary Biology, National Taiwan University, Taipei, Taiwan</addr-line></aff><aff id="aff2"><label>2</label><addr-line>Department of Cell and Developmental Biology, John Innes Centre, Norwich, United Kingdom</addr-line></aff><aff id="aff3"><label>3</label><addr-line>Department of Crop Sciences, Sultan Qaboos University, Al Khoud, Oman</addr-line></aff><aff id="aff4"><label>4</label><addr-line>Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan</addr-line></aff><aff id="aff5"><label>5</label><addr-line>Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, National Chung Hsing University and Academia Sinica, Taipei, Taiwan</addr-line></aff><aff id="aff6"><label>6</label><addr-line>Biotechnology Center, National Chung Hsing University, Taichung, Taiwan</addr-line></aff><contrib-group><contrib contrib-type="editor"><name><surname>Yin</surname><given-names>Tongming</given-names></name><role>Editor</role><xref ref-type="aff" rid="edit1"></xref></contrib></contrib-group><aff id="edit1"><addr-line>Nanjing Forestry University, China</addr-line></aff><author-notes><corresp id="cor1">* E-mail: <email>chk@gate.sinica.edu.tw</email></corresp><fn fn-type="conflict"><p><bold>Competing Interests: </bold>The authors confirm the following: Corresponding author Chih-Horng Kuo is an Academic Editor for PLOS ONE (since Jul. 2014). This does not alter the authors’ adherence to PLOS ONE Editorial policies and criteria.</p></fn><fn fn-type="con"><p>Conceived and designed the experiments: HJS CHK. Performed the experiments: SAH AMA. Analyzed the data: HJS CHK. Contributed reagents/materials/analysis tools: HJS AMA CHK. Contributed to the writing of the manuscript: HJS SAH AMA CHK.</p></fn></author-notes><pub-date pub-type="collection"><year>2014</year></pub-date><pub-date pub-type="epub"><day>14</day><month>11</month><year>2014</year></pub-date><volume>9</volume><issue>11</issue><elocation-id>e113049</elocation-id><history><date date-type="received"><day>5</day><month>6</month><year>2014</year></date><date date-type="accepted"><day>18</day><month>10</month><year>2014</year></date></history><permissions><copyright-year>2014</copyright-year><copyright-holder>Su et al</copyright-holder><license><license-p>This is an open-access article distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><abstract><p>The genus <italic>Citrus</italic> contains many economically important fruits that are grown worldwide for their high nutritional and medicinal value. Due to frequent hybridizations among species and cultivars, the exact number of natural species and the taxonomic relationships within this genus are unclear. To compare the differences between the <italic>Citrus</italic> chloroplast genomes and to develop useful genetic markers, we used a reference-assisted approach to assemble the complete chloroplast genome of Omani lime (<italic>C</italic>. <italic>aurantiifolia</italic>). The complete <italic>C</italic>. <italic>aurantiifolia</italic> chloroplast genome is 159,893 bp in length; the organization and gene content are similar to most of the rosids lineages characterized to date. Through comparison with the sweet orange (<italic>C. sinensis</italic>) chloroplast genome, we identified three intergenic regions and 94 simple sequence repeats (SSRs) that are potentially informative markers with resolution for interspecific relationships. These markers can be utilized to better understand the origin of cultivated <italic>Citrus</italic>. A comparison among 72 species belonging to 10 families of representative rosids lineages also provides new insights into their chloroplast genome evolution.</p></abstract><funding-group><funding-statement>Funding for this work was provided by research grants from Biotechnology and Biological Sciences Research Council (BB/J004553/1) and the Gatsby Charitable Foundation to SAH, Sultan Qaboos University (SR/AGR/CROP/13/01) to AMA, and the Institute of Plant and Microbial Biology at Academia Sinica to CHK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</funding-statement></funding-group><counts><page-count count="11"></page-count></counts><custom-meta-group><custom-meta id="data-availability"><meta-name>Data Availability</meta-name><meta-value>The authors confirm that all data underlying the findings are fully available without restriction. The raw Illumina reads are publicly available from the NCBI SRA under the accession number SRR1611615 (<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/sra/SRR1611615">http://www.ncbi.nlm.nih.gov/sra/SRR1611615</ext-link>). The complete genome sequence is available from the NCBI GenBank under the accession number KJ865401.1 (<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KJ865401.1">http://www.ncbi.nlm.nih.gov/nuccore/KJ865401.1</ext-link>).</meta-value></custom-meta></custom-meta-group></article-meta><notes><title>Data Availability</title><p>The authors confirm that all data underlying the findings are fully available without restriction. The raw Illumina reads are publicly available from the NCBI SRA under the accession number SRR1611615 (<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/sra/SRR1611615">http://www.ncbi.nlm.nih.gov/sra/SRR1611615</ext-link>). The complete genome sequence is available from the NCBI GenBank under the accession number KJ865401.1 (<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KJ865401.1">http://www.ncbi.nlm.nih.gov/nuccore/KJ865401.1</ext-link>).</p></notes></front><body><sec id="s1"><title>Introduction</title><p><italic>Citrus</italic> is in the family of Rutaceae, which is one of the largest families in order Sapindales. Flowers and leaves of <italic>Citrus</italic> are usually strong scented, the extracts from which contain many useful flavonoids and other compounds that are effective insecticides, fungicides and medicinal agents <xref rid="pone.0113049-Mabberley1" ref-type="bibr">[1]</xref>–<xref rid="pone.0113049-Ezeabara1" ref-type="bibr">[3]</xref>. <italic>Citrus</italic> is of great economic importance and contains many fruit crops such as oranges, grapefruit, lemons, limes, and tangerines. However, due to a long cultivation history, wide dispersion, somatic bud mutation, and sexual compatibility among <italic>Citrus</italic> species and related genera, the taxonomy of <italic>Citrus</italic> remains controversial <xref rid="pone.0113049-Nicolosi1" ref-type="bibr">[4]</xref>, <xref rid="pone.0113049-Hynniewta1" ref-type="bibr">[5]</xref> and the origination of many <italic>Citrus</italic> species and hybrids is still unresolved <xref rid="pone.0113049-Liu1" ref-type="bibr">[6]</xref>, <xref rid="pone.0113049-Penjor1" ref-type="bibr">[7]</xref>.</p><p>The chloroplast (cp) genome sequence contains useful information in plant systematics because of its maternal inheritance in most angiosperms <xref rid="pone.0113049-Corriveau1" ref-type="bibr">[8]</xref>, <xref rid="pone.0113049-Zhang1" ref-type="bibr">[9]</xref> and its highly conserved structures for developing promising genetic markers. The only complete cp genome available in <italic>Citrus</italic> is sweet orange (<italic>Citrus sinensis</italic>) <xref rid="pone.0113049-Bausher1" ref-type="bibr">[10]</xref>, which has provided valuable information to the position of Sapindales in rosids. Although a genome sequencing project is in progress for <italic>C. clementine</italic>, its complete chloroplast genome sequence is not available yet. To identify the cp genome regions that are polymorphic and may be used as molecular markers for resolving the evolutionary relationships among <italic>Citrus</italic> species, a second cp genome within the genus is necessary for comparative analysis. For this purpose, the major aim of this study is to determine the complete cp genome sequence of <italic>C. aurantiifolia</italic>.</p><p><italic>C. aurantiifolia</italic>, which is commonly known as Key lime, Mexican lime, Omani lime, Indian lime, or acid lime, is native to Southeast Asia and widely cultivated in tropics and subtropics. Oman is known to be a transit country for lime, from which lime spread to Africa and the New World <xref rid="pone.0113049-Davies1" ref-type="bibr">[11]</xref>. In Oman, Omani lime is considered the fourth most important fruit crop in terms of cultivated area and production. The products of Omani lime can be used for beverage, food additives and cosmetic industries <xref rid="pone.0113049-Vand1" ref-type="bibr">[12]</xref>. Omani lime is sensitive to several biotic agents, the most serious of which is ‘<italic>Candidatus</italic> Phytoplasma aurantifolia’, the cause of witches’ broom disease of lime (WBDL). Recent studies on WBDL focused on effect of genetic diversity of Omani limes on the disease <xref rid="pone.0113049-AlSadi1" ref-type="bibr">[13]</xref>, transcriptome and proteomic analysis of lime response to infection by phytoplasma <xref rid="pone.0113049-Taheri1" ref-type="bibr">[14]</xref>–<xref rid="pone.0113049-Monavarfeshani1" ref-type="bibr">[16]</xref> and effect of phytoplasma on seed germination, growth and metabolite content in lime <xref rid="pone.0113049-Faghihi1" ref-type="bibr">[17]</xref>, <xref rid="pone.0113049-Zafari1" ref-type="bibr">[18]</xref>.</p><p>Here, we present the complete chloroplast genome sequence of Omani lime (<italic>C. aurantiifolia</italic>). To identify loci of potential utility for the molecular identification and phylogenetic analyses of <italic>Citrus</italic> cultivars and species, we compared the intergenic regions and SSRs in the cp genomes of <italic>C</italic>. <italic>aurantiifolia</italic> and <italic>C. sinensis</italic>. Furthermore, we performed phylogenetic analyses to infer the history of gene losses in the cp genome evolution among representative rosids lineages.</p></sec><sec sec-type="materials|methods" id="s2"><title>Materials and Methods</title><sec id="s2a"><title>Sample Preparation and Sequencing</title><p>The Omani lime leaves were collected from a 5-year-old lime tree at a private farm located in the Omani territory of Madha (GPS coordinates: 25.276318, 56.318909). This farm is owned by one of the co-authors of this work, Dr. Abdullah M. Al-Sadi, whom should be contacted for future permissions. This study does not involve endangered or protected species and does not require specific permission from regulatory authority concerned with protection of wildlife. The sample was stored in a cool box and transported to the Plant Pathology Research Laboratory at Sultan Qaboos University (Al Khoud, Oman) for DNA extraction following a protocol of Maixner et al. <xref rid="pone.0113049-Maixner1" ref-type="bibr">[19]</xref>. The leaves were washed with clear water before the isolation procedure. 1 g of leaves were used and crushed in 3 ml CTAB extraction buffer (2% CTAB, 1.4 M NaCl, 500 mM EDTA pH8, 1 M Tris-HCl pH8 and 0.2% beta-mercaptol). 1.5 ml of the leave extract was transferred to a 2 ml tube and incubated in a water-bath at 65°C for 15 min. The tube was turned up and down twice during incubation, centrifuged at 960 g for 5 min, and the supernatant was subsequently transferred to a clean eppendorf tube. An equal volume of chloroform-isoamyl alcohol mix (24∶1) was added and the tube was centrifuged at 21000 g for 20 min. The supernatant was transferred to a new tube and then 0.6 volume of isopropanol was added to the supernatant and incubated at −20°C for 30 min. The DNA pellet was collected by centrifugation at 21000 g for 20 min and then washed with 1 ml of 70% ethanol. The final DNA was resuspended in 100 µl TE (Tris 10 mM, EDTA 1 mM pH8) and was stored at −80°C until used.</p><p>The library construction and sequencing were done at the Genome Analysis Centre (Norwich, UK). The Illumina TruSeq DNA Sample Preparation v2 Kit was used to prepare an indexed library. The DNA sample was sheared to a fragment size of 500–600 bp using a sonicator, followed by end-repair and the addition of a single A base for binding of the indexed adapter. The appropriate sized library (500 bp) was selected by gel electrophoresis, followed by PCR enrichment. The 251 bp paired-end sequencing run was performed on an Illumina MiSeq instrument using the SBS chemistry and Illumina software MCS v2.3.0.3 and RTA v1.18.42. The raw reads were deposited at the NCBI Sequence Read Archive under the accession number SRR1611615.</p></sec><sec id="s2b"><title>Genome Assembly and Analyses</title><p>The procedures for genome assembly and annotation were based on our previous studies of cp genomes <xref rid="pone.0113049-Ku1" ref-type="bibr">[20]</xref>, <xref rid="pone.0113049-Ku2" ref-type="bibr">[21]</xref>. In addition to the standard <italic>de novo</italic> assembly approach by using Velvet v1.2.10 <xref rid="pone.0113049-Zerbino1" ref-type="bibr">[22]</xref> with the k-mer size set to 243, a reference-based approach for assembly as described below was used in parallel. All of the raw reads were initially mapped onto the published cp genome of <italic>C. sinensis</italic><xref rid="pone.0113049-Bausher1" ref-type="bibr">[10]</xref> using BWA v0.6.2 <xref rid="pone.0113049-Li1" ref-type="bibr">[23]</xref>. The sequence variations were identified with SAMtools v0.1.19 <xref rid="pone.0113049-Li2" ref-type="bibr">[24]</xref> and visually inspected using IGV v2.3.25 <xref rid="pone.0113049-Robinson1" ref-type="bibr">[25]</xref>. The variants were corrected with the raw reads and the regions without sufficient coverage were converted into gaps. This corrected sequence was then used as the new draft reference for the next iteration of verification. Gaps were filled using the reads overhang at margins and the process was repeated until the reference was fully supported by all mapped raw reads. The final assembly, which was supported by our <italic>de novo</italic> and reference-based approaches, resulted in an average of 1,441-fold coverage of paired-end reads with a mapping quality of 60 and the region with the lowest coverage is 506-fold.</p><p>The preliminary annotations of the <italic>C</italic>. <italic>aurantiifolia</italic> cp genome were performed online using the automatic annotator DOGMA <xref rid="pone.0113049-Wyman1" ref-type="bibr">[26]</xref> and verified using BLASTN <xref rid="pone.0113049-Altschul1" ref-type="bibr">[27]</xref>, <xref rid="pone.0113049-Camacho1" ref-type="bibr">[28]</xref> searches (e-value cutoff = 1e-10) against other land plant cp genomes. Each annotated gene was manually compared with <italic>C. sinensis</italic> cp genome for start and stop codons or intron junctions to ensure accurate annotation. The codon usage was analyzed by using the seqinr R-cran package <xref rid="pone.0113049-Charif1" ref-type="bibr">[29]</xref>. A circular map of genome was produced using OGDRAW <xref rid="pone.0113049-Lohse1" ref-type="bibr">[30]</xref>.</p><p>To identify the differences between <italic>C</italic>. <italic>aurantiifolia</italic> and <italic>C. sinensis,</italic> the two sequences were aligned using Mauve v2.3.1 <xref rid="pone.0113049-Darling1" ref-type="bibr">[31]</xref> and the result was analyzed using custom Perl scripts. Intergenic gene regions were parsed out from the two <italic>Citrus</italic> cp genomes and aligned using MUSCLE v3.8.31 <xref rid="pone.0113049-Edgar1" ref-type="bibr">[32]</xref> with the default settings. The pairwise distances were calculated using the DNADIST program in the PHYLIP package v3.695 <xref rid="pone.0113049-Felsenstein1" ref-type="bibr">[33]</xref>.</p><p>The positions and types of simple sequence repeats (SSRs) in the two <italic>Citrus</italic> cp genomes were detected using MISA (<ext-link ext-link-type="uri" xlink:href="http://pgrc.ipk-gatersleben.de/misa/">http://pgrc.ipk-gatersleben.de/misa/</ext-link>). The minimum number of repeats were set to 10, 5, 4, 3, 3, and 3 for mono-, di-, tri-, tetra-, penta-, and hexanucleotides, respectively. For long repeats, the program REPuter <xref rid="pone.0113049-Kurtz1" ref-type="bibr">[34]</xref> was used to identify the number and location of direct and inverted (i.e., palindromic) repeats. A minimum repeat size of 30 bp and sequence identity greater than 90% setting were used according to the study of <italic>C. sinensis</italic> cp genome <xref rid="pone.0113049-Bausher1" ref-type="bibr">[10]</xref>. The redundant or overlapping repeats were identified and filtered manually.</p></sec><sec id="s2c"><title>Phylogenetic Inference</title><p>Phylogenetic analysis of the representative rosids lineages with complete cp genomes available was performed using PhyML v20120412 <xref rid="pone.0113049-Guindon1" ref-type="bibr">[35]</xref> with the GTR+I+G model. A total of 72 rosids species were chosen as the ingroups and <italic>Vitis venifera</italic> was included as the outgroup, the accession numbers were provided in <xref ref-type="supplementary-material" rid="pone.0113049.s003">Table S1</xref>. The protein-coding and rRNA genes were parsed from the selected cp genomes and clustered into ortholog groups using OrthoMCL <xref rid="pone.0113049-Li3" ref-type="bibr">[36]</xref>. The presence/absence of orthologous genes in each genome was examined and further verified using TBLASTN <xref rid="pone.0113049-Altschul1" ref-type="bibr">[27]</xref>, <xref rid="pone.0113049-Camacho1" ref-type="bibr">[28]</xref> searches (e-value cutoff = 1e-10). The nucleotide sequences of the conserved genes were aligned individually by using MUSCLE with the default settings. The concatenated alignment was used to infer a maximum likelihood phylogeny as described above. The bootstrap supports were estimated from 1,000 resampled alignments generated by the SEQBOOT program in the PHYLIP package.</p></sec><sec id="s2d"><title>Investigations of <italic>orf56</italic> and <italic>ycf68</italic></title><p>To investigate the presence/absence of <italic>orf56</italic> and <italic>ycf68</italic> in the selected cp genomes, the gene sequences from <italic>C. aurantiifolia</italic> was used as the queries to perform BLASTN <xref rid="pone.0113049-Altschul1" ref-type="bibr">[27]</xref>, <xref rid="pone.0113049-Camacho1" ref-type="bibr">[28]</xref> searches (e-value cutoff = 1e-10). The significant hits were examined to investigate the presence of intact open reading frames (ORFs). Phylogenetic analysis of the cp <italic>orf56</italic> genes and the homologous mitochondrial sequences was performed as described above. The final alignment contains 190 aligned nucleotide sites and a total of 70 sequences, including two sequences of <italic>Amborella</italic> as the outgroup.</p></sec></sec><sec id="s3"><title>Results and Discussion</title><sec id="s3a"><title>General Features of the Omani Lime Chloroplast Genome</title><p>The complete cp genome of <italic>C</italic>. <italic>aurantiifolia</italic> (Christm.) Swingle (GenBank accession number KJ865401.1) is 159,893 bp in length, including a large single copy (LSC) region of 87,148 bp, a small single copy (SSC) region of 18,763 bp, and a pair of inverted repeats (IRa and IRb) of 26,991 bp each (<xref ref-type="fig" rid="pone-0113049-g001">Figure 1</xref> and <xref ref-type="table" rid="pone-0113049-t001">Table 1</xref>). A total of 137 different genes, including 93 protein-coding genes, 30 tRNA genes, and four rRNA genes, were annotated (<xref ref-type="supplementary-material" rid="pone.0113049.s004">Table S2</xref>). Among these, 12 protein-coding genes and 7 tRNA genes are duplicated in the IR regions. Most of the protein-coding genes are composed of a single exon, while 14 contain one intron and three contain two introns. The gene <italic>rps12</italic> was predicted to undergo trans-splicing, with the 5′ exon located in the LSC region and the other two exons located in the IR regions.</p><fig id="pone-0113049-g001" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0113049.g001</object-id><label>Figure 1</label><caption><title>Chloroplast genome map of <italic>Citrus aurantiifolia</italic>.</title><p>Gene drawn inside the circle are transcribed clockwise, whereas those outside are counterclockwise. The within-genome GC content variation is indicated in the middle circles.</p></caption><graphic xlink:href="pone.0113049.g001"></graphic></fig><table-wrap id="pone-0113049-t001" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0113049.t001</object-id><label>Table 1</label><caption><title>Summary of the <italic>Citrus</italic> chloroplast genome characteristics.</title></caption><alternatives><graphic id="pone-0113049-t001-1" xlink:href="pone.0113049.t001"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1">Attribute</td><td align="left" rowspan="1" colspan="1"><italic>C. aurantiifolia</italic> (KJ865401)</td><td align="left" rowspan="1" colspan="1"><italic>C. sinensis</italic> (NC_008334)</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Size (bp)</td><td align="left" rowspan="1" colspan="1">159,893</td><td align="left" rowspan="1" colspan="1">160,129</td></tr><tr><td align="left" rowspan="1" colspan="1">overall GC content (%)</td><td align="left" rowspan="1" colspan="1">38.4</td><td align="left" rowspan="1" colspan="1">38.5</td></tr><tr><td align="left" rowspan="1" colspan="1">LSC size in bp (% total)</td><td align="left" rowspan="1" colspan="1">87,148 (54.5%)</td><td align="left" rowspan="1" colspan="1">87,744 (54.8%)</td></tr><tr><td align="left" rowspan="1" colspan="1">SSC size in bp (% total)</td><td align="left" rowspan="1" colspan="1">18,763 (11.7%)</td><td align="left" rowspan="1" colspan="1">18,393 (11.5%)</td></tr><tr><td align="left" rowspan="1" colspan="1">IR size in bp (% total)<xref ref-type="table-fn" rid="nt101">a</xref></td><td align="left" rowspan="1" colspan="1">26,991 (16.9%)</td><td align="left" rowspan="1" colspan="1">26,996 (16.9%)</td></tr><tr><td align="left" rowspan="1" colspan="1">Protein-coding regions size in bp (% total)</td><td align="left" rowspan="1" colspan="1">81,468 (51.0%)</td><td align="left" rowspan="1" colspan="1">79,773 (49.8%)</td></tr><tr><td align="left" rowspan="1" colspan="1">rRNA and tRNA size in bp (% total)</td><td align="left" rowspan="1" colspan="1">11,850 (7.5%)</td><td align="left" rowspan="1" colspan="1">11,850 (7.4%)</td></tr><tr><td align="left" rowspan="1" colspan="1">Introns size in bp (% total)</td><td align="left" rowspan="1" colspan="1">17,129 (10.7%)</td><td align="left" rowspan="1" colspan="1">18,252 (11.4%)</td></tr><tr><td align="left" rowspan="1" colspan="1">Intergenic spacer size in bp (% total)</td><td align="left" rowspan="1" colspan="1">49,446 (30.9%)</td><td align="left" rowspan="1" colspan="1">50,254 (31.4%)</td></tr><tr><td align="left" rowspan="1" colspan="1">Number of different genes</td><td align="left" rowspan="1" colspan="1">115</td><td align="left" rowspan="1" colspan="1">113<xref ref-type="table-fn" rid="nt102">b</xref></td></tr><tr><td align="left" rowspan="1" colspan="1">Number of different protein-coding genes</td><td align="left" rowspan="1" colspan="1">81</td><td align="left" rowspan="1" colspan="1">79<xref ref-type="table-fn" rid="nt102">b</xref></td></tr><tr><td align="left" rowspan="1" colspan="1">Number of different rRNA genes</td><td align="left" rowspan="1" colspan="1">4</td><td align="left" rowspan="1" colspan="1">4</td></tr><tr><td align="left" rowspan="1" colspan="1">Number of different tRNA genes</td><td align="left" rowspan="1" colspan="1">30</td><td align="left" rowspan="1" colspan="1">30</td></tr><tr><td align="left" rowspan="1" colspan="1">Number of different genes duplicated by IR</td><td align="left" rowspan="1" colspan="1">22</td><td align="left" rowspan="1" colspan="1">20</td></tr><tr><td align="left" rowspan="1" colspan="1">Number of different genes with introns</td><td align="left" rowspan="1" colspan="1">17</td><td align="left" rowspan="1" colspan="1">17</td></tr></tbody></table></alternatives><table-wrap-foot><fn id="nt101"><label>a</label><p>Each cp genome contains two copies of inverted repeats (IRs).</p></fn><fn id="nt102"><label>b</label><p>According to the original annotation, not including <italic>orf56</italic>.</p></fn></table-wrap-foot></table-wrap><p>The protein-coding regions contain a total of 27,159 codons (<xref ref-type="supplementary-material" rid="pone.0113049.s005">Table S3</xref>). Isoleucine and cysteine are the most and least frequent amino acids and have 2,892 (10.7%) and 359 (1.2%) codons, respectively. The codon usage is biased towards a high ratio of A/T at the third position, which is also observed in many land plant cp genomes <xref rid="pone.0113049-Clegg1" ref-type="bibr">[37]</xref>.</p></sec><sec id="s3b"><title>Sequence Comparisons with Sweet Orange</title><p>The general characteristics of the two <italic>Citrus</italic> cp genomes are summarized in <xref ref-type="table" rid="pone-0113049-t001">Table 1</xref>, overall the compositions are quite similar. The GC content of these <italic>Citrus</italic> cp genomes is approximately 38.5%, which is slightly higher than the average of the 72 representative rosids lineages (36.7%). In these two <italic>Citrus</italic> cp genomes, the genic regions, introns, and intergenic regions account for ca. 58%, 11%, and 31%, respectively (<xref ref-type="table" rid="pone-0113049-t001">Table 1</xref>).</p><p>The pairwise sequence alignment between the two <italic>Citrus</italic> cp genomes revealed approximately 1.3% sequence divergence (<xref ref-type="table" rid="pone-0113049-t002">Table 2</xref>), including 1,780 indels (1.11%) and 330 substitutions (0.21%). The LSC region contains more sequence polymorphisms than expected by its size, including 1,360 (76.4%) indels and 235 (71.2%) substitutions. In contrast, the two IR regions account for ca. 34% of the cp genome yet contain only 16 (0.9%) indels and 12 (3.6%) substitutions. The size differences in the LSC and SSC regions between these two cp genomes are mostly explained by one large indel in each region. The LSC sizes differ by 596 bp and a 523-bp indel was found in the spacer between <italic>rps16</italic> and <italic>trnQ-UUG</italic>. The SSC sizes differ by 370 bp and a 354-bp indel was found in the spacer between <italic>rpl32</italic> and <italic>trnL-UAA.</italic></p><table-wrap id="pone-0113049-t002" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0113049.t002</object-id><label>Table 2</label><caption><title>Differences between the <italic>C. aurantiifolia</italic> and <italic>C. sinensis</italic> cp genomes.</title></caption><alternatives><graphic id="pone-0113049-t002-2" xlink:href="pone.0113049.t002"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col></colgroup><tbody><tr><td align="left" rowspan="1" colspan="1"><bold>Indel</bold></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">Length (bp)</td><td align="left" rowspan="1" colspan="1">Count</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">43</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">2–10</td><td align="left" rowspan="1" colspan="1">20</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">11–100</td><td align="left" rowspan="1" colspan="1">18</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">101–1,000</td><td align="left" rowspan="1" colspan="1">3</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">Sum</td><td align="left" rowspan="1" colspan="1">1,780</td><td align="left" rowspan="1" colspan="1">116</td><td align="left" rowspan="1" colspan="1">Percentage<xref ref-type="table-fn" rid="nt103">a</xref>: 1.11%</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Substitution</bold></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">Type</td><td align="left" rowspan="1" colspan="1">Count</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">A <-> T</td><td align="left" rowspan="1" colspan="1">34</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">C <-> G</td><td align="left" rowspan="1" colspan="1">15</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">A <-> C</td><td align="left" rowspan="1" colspan="1">81</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">T <-> C</td><td align="left" rowspan="1" colspan="1">64</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">A <-> G</td><td align="left" rowspan="1" colspan="1">51</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">T <-> G</td><td align="left" rowspan="1" colspan="1">85</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">Sum</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">330</td><td align="left" rowspan="1" colspan="1">Percentage<xref ref-type="table-fn" rid="nt103">a</xref>: 0.21%</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>10 most divergent intergenic regions</bold></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">Region</td><td align="left" rowspan="1" colspan="1">Length<xref ref-type="table-fn" rid="nt104">b</xref> (bp)</td><td align="left" rowspan="1" colspan="1">Pairwise distance</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><italic>rps3</italic> - <italic>rpl22</italic> (LSC)</td><td align="left" rowspan="1" colspan="1">234</td><td align="left" rowspan="1" colspan="1">0.027</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><italic>ndhE</italic> - <italic>ndhG</italic> (SSC)</td><td align="left" rowspan="1" colspan="1">276</td><td align="left" rowspan="1" colspan="1">0.018</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><italic>psaC</italic> - <italic>ndhE</italic> (SSC)</td><td align="left" rowspan="1" colspan="1">231</td><td align="left" rowspan="1" colspan="1">0.017</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><italic>psbH</italic> - <italic>petB</italic> (LSC)</td><td align="left" rowspan="1" colspan="1">118</td><td align="left" rowspan="1" colspan="1">0.017</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">t<italic>rnY-GUA-trnE-UCC</italic> (LSC)</td><td align="left" rowspan="1" colspan="1">59</td><td align="left" rowspan="1" colspan="1">0.017</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><italic>trnH</italic>-<italic>GUG</italic> - <italic>psbA</italic> (LSC)</td><td align="left" rowspan="1" colspan="1">449</td><td align="left" rowspan="1" colspan="1">0.016</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><italic>rpl32</italic> - <italic>trnL</italic>-<italic>UAG</italic> (SSC)</td><td align="left" rowspan="1" colspan="1">1,141</td><td align="left" rowspan="1" colspan="1">0.015</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><italic>psbT-psbN</italic> (LSC)</td><td align="left" rowspan="1" colspan="1">66</td><td align="left" rowspan="1" colspan="1">0.015</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><italic>trnG-GCC-trnR-UCU</italic> (LSC)</td><td align="left" rowspan="1" colspan="1">204</td><td align="left" rowspan="1" colspan="1">0.015</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><italic>trnD-GUC-trnY-GUA</italic> (LSC)</td><td align="left" rowspan="1" colspan="1">469</td><td align="left" rowspan="1" colspan="1">0.013</td></tr></tbody></table></alternatives><table-wrap-foot><fn id="nt103"><label>a</label><p>Relative to the length of <italic>C. aurantiifolia.</italic></p></fn><fn id="nt104"><label>b</label><p>Length in C. aurantiifolia.</p></fn></table-wrap-foot></table-wrap><p>To identify the intergenic regions that may be useful for phylogenic analysis or molecular identification, we searched for the spacers that are >400 bp in length and exhibit above-average sequence divergence between the two <italic>Citrus</italic> species (i.e., >1.3%). A total of three regions satisfied these criteria, including the spacer between <italic>trnH-GUG</italic> and <italic>psbA</italic> (449 bp, 1.6% divergence), the spacer between <italic>rpl32</italic> and <italic>trnL-UAG</italic> (1141 bp, 1.5% divergence), and the spacer between <italic>trnD-GUC</italic> and <italic>trnY-GUA</italic> (469 bp, 1.3% divergence).</p><p>The junctions between the IR, LSC, and SSC regions in <italic>C</italic>. <italic>aurantiifolia</italic> are similar to that of <italic>C. sinensis</italic> except for the LSC-IRb boundary. A total of 23 indels and five substitutions were found at this region, resulting in one copy of <italic>rpl22</italic> spanning across the LSC-IRb junction in <italic>C</italic>. <italic>aurantiifolia</italic>. Comparing the IR junctions of <italic>Citrus</italic> with <italic>Theobroma</italic> and <italic>Gossypium</italic> in Malvaceae <xref rid="pone.0113049-Kane1" ref-type="bibr">[38]</xref>, it was found that the IRs in <italic>Citrus</italic> have expanded to include <italic>rps19</italic> and 252 nt of <italic>rpl22</italic>, whereas in Malvaceae, <italic>rps19</italic> is located in LSC and <italic>rpl22</italic> was missing <xref rid="pone.0113049-Kane1" ref-type="bibr">[38]</xref>–<xref rid="pone.0113049-Xu1" ref-type="bibr">[40]</xref>.</p></sec><sec id="s3c"><title>Analyses of Repetitive Sequences</title><p>A total of 109 SSR loci were found in the cp genome of <italic>C. aurantiifoliaa</italic>, accounting for 1,352 bp of the total sequence (ca. 0.8%). Among these, 94 were also found in <italic>C. sinensis</italic> and 42 exhibit length polymorphism (<xref ref-type="table" rid="pone-0113049-t003">Table 3</xref>). Most SSRs are located in intergenic regions, but some were found in coding genes such as <italic>matK</italic> and <italic>ycf1</italic>. Concerning the controversial status of <italic>Citrus</italic> taxonomy, the SSRs identified in this study may provide new perspective to refine the phylogeny and elucidate the origin of the cultivars. Furthermore, these SSRs may be used as molecular markers for population studies.</p><table-wrap id="pone-0113049-t003" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0113049.t003</object-id><label>Table 3</label><caption><title>List of simple sequence repeats.</title></caption><alternatives><graphic id="pone-0113049-t003-3" xlink:href="pone.0113049.t003"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1">Repeat unit</td><td align="left" rowspan="1" colspan="1">Length (bp)</td><td align="left" rowspan="1" colspan="1">Number of SSRs</td><td align="left" rowspan="1" colspan="1">Start position<xref ref-type="table-fn" rid="nt105">a</xref></td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">A</td><td align="left" rowspan="1" colspan="1">10</td><td align="left" rowspan="1" colspan="1">6</td><td align="left" rowspan="1" colspan="1"><underline>4512</underline>; <underline>47812</underline>; 53871; <bold>72614</bold>; 121748; <bold>159288</bold></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">11</td><td align="left" rowspan="1" colspan="1">6</td><td align="left" rowspan="1" colspan="1"><bold>6866</bold>; <underline>10130</underline>; <underline>69481</underline>; <underline>71892</underline>; <underline>117725</underline>; <bold>134802</bold></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">9</td><td align="left" rowspan="1" colspan="1"><bold>8332</bold>; <underline>31399</underline>; <underline>47307</underline>; <underline>63928</underline>; <bold>111804</bold>; <bold>113977 (</bold><bold><italic>ycf1</italic></bold><bold>)</bold>; 118367; <underline>140302 (<italic>ycf68</italic></underline>); <underline>144255</underline></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">13</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1"><underline>10107</underline>; <underline>84557</underline></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">14</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1"><bold>385</bold></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">15</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1"><underline>32360</underline></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">16</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1"><underline>69965</underline>; 118302</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">17</td><td align="left" rowspan="1" colspan="1">3</td><td align="left" rowspan="1" colspan="1"><underline>7620</underline>; <underline>39139</underline>; <underline>74176</underline></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">19</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1"><bold>12023</bold></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">22</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">70289</td></tr><tr><td align="left" rowspan="1" colspan="1">T</td><td align="left" rowspan="1" colspan="1">10</td><td align="left" rowspan="1" colspan="1">10</td><td align="left" rowspan="1" colspan="1"><bold>2424 (</bold><bold><italic>matK</italic></bold><bold>)</bold>; <bold>19786</bold>; <bold>26964</bold><bold>(</bold><bold><italic>rpoB</italic></bold><bold>)</bold>; <bold>37622</bold>; <underline>46938</underline>; <bold>63632</bold>; <bold>87731</bold>; 117742; <underline>117871</underline>; <underline>118851</underline></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">11</td><td align="left" rowspan="1" colspan="1">11</td><td align="left" rowspan="1" colspan="1">9401; <underline>10416</underline>; <bold>17001</bold>; <underline>30912</underline>; 46021; 63530; <underline>112216</underline>; <underline>117988</underline>; 118224; <bold>121703</bold>; <bold>131189</bold> (<italic>ycf1</italic>)</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">6</td><td align="left" rowspan="1" colspan="1"><bold>14722</bold>; <underline>29024</underline>; 102773; <underline>106715 (<italic>ycf68</italic>)</underline>; <bold>133040 (</bold><bold><italic>ycf1</italic></bold><bold>)</bold>; <bold>135213</bold></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">13</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1"><bold>73946</bold>;<underline> 80423</underline></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">14</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">1776; <underline>85274</underline></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">15</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1"><underline>54209</underline>; <underline>57817</underline></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">17</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">45965</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">18</td><td align="left" rowspan="1" colspan="1">3</td><td align="left" rowspan="1" colspan="1"><underline>52748</underline>; <underline>68339</underline>; <underline>81409</underline></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">20</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1"><underline>49202</underline></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">23</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1"><underline>23694</underline>; <bold>33282</bold></td></tr><tr><td align="left" rowspan="1" colspan="1">C</td><td align="left" rowspan="1" colspan="1">10</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1"><bold>28769</bold>; <bold>104247</bold></td></tr><tr><td align="left" rowspan="1" colspan="1">G</td><td align="left" rowspan="1" colspan="1">10</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1"><bold>142772</bold></td></tr><tr><td align="left" rowspan="1" colspan="1">AT</td><td align="left" rowspan="1" colspan="1">10</td><td align="left" rowspan="1" colspan="1">4</td><td align="left" rowspan="1" colspan="1"><bold>20631 (</bold><bold><italic>rpoC2</italic></bold><bold>)</bold>; <bold>33636</bold>; 11817; <bold>121517 (</bold><bold><italic>ndhD</italic></bold><bold>)</bold></td></tr><tr><td align="left" rowspan="1" colspan="1">AAG</td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1"><bold>97331</bold></td></tr><tr><td align="left" rowspan="1" colspan="1">AAT</td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1"><bold>38604</bold>; <bold>122629</bold></td></tr><tr><td align="left" rowspan="1" colspan="1">ATA</td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">70220</td></tr><tr><td align="left" rowspan="1" colspan="1">ATT</td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">3</td><td align="left" rowspan="1" colspan="1"><bold>10283</bold>; <bold>53810</bold>; <bold>54088</bold></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">18</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1"><bold>1760</bold>;</td></tr><tr><td align="left" rowspan="1" colspan="1">CTT</td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1"><bold>37353 (</bold><bold><italic>psbC</italic></bold><bold>)</bold>; <bold>149686</bold></td></tr><tr><td align="left" rowspan="1" colspan="1">TAA</td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1"><bold>30250</bold>; 61945</td></tr><tr><td align="left" rowspan="1" colspan="1">TAT</td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1"><bold>83297</bold></td></tr><tr><td align="left" rowspan="1" colspan="1">TTC</td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1"><bold>73084</bold></td></tr><tr><td align="left" rowspan="1" colspan="1">TAAA</td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">4866; <bold>45088</bold></td></tr><tr><td align="left" rowspan="1" colspan="1">AAAT</td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">3</td><td align="left" rowspan="1" colspan="1"><bold>30423</bold>; <bold>32502</bold>; <bold>71394</bold></td></tr><tr><td align="left" rowspan="1" colspan="1">ATAC</td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1"><bold>51167</bold></td></tr><tr><td align="left" rowspan="1" colspan="1">ATTT</td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">49193</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">20</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1"><bold>117168</bold></td></tr><tr><td align="left" rowspan="1" colspan="1">TTAA</td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">39175; 39188</td></tr><tr><td align="left" rowspan="1" colspan="1">TTAG</td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1"><bold>61483</bold></td></tr><tr><td align="left" rowspan="1" colspan="1">TTTC</td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1"><bold>14352</bold></td></tr><tr><td align="left" rowspan="1" colspan="1">TCTT</td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1"><bold>46961</bold></td></tr><tr><td align="left" rowspan="1" colspan="1">AATAA</td><td align="left" rowspan="1" colspan="1">20</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">144226</td></tr><tr><td align="left" rowspan="1" colspan="1">TTTTA</td><td align="left" rowspan="1" colspan="1">20</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">102781</td></tr><tr><td align="left" rowspan="1" colspan="1">TTCAAA</td><td align="left" rowspan="1" colspan="1">18</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">63817</td></tr></tbody></table></alternatives><table-wrap-foot><fn id="nt105"><label>a</label><p>The SSR-containing coding regions are indicated in parentheses. SSRs that are identical in the <italic>C. sinensis</italic> chloroplast genome are highlighted in bold; SSRs that are conserved but with different lengths are highlighted by underline.</p></fn></table-wrap-foot></table-wrap><p>In addition, 62 large repeats that are longer than 30 bp were found in the <italic>C. aurantiifolia</italic> cp genome (<xref ref-type="table" rid="pone-0113049-t004">Table 4</xref>). Most of these repeats are located in intergenic spacers, except for three that are located in the coding regions of <italic>rps4</italic>, <italic>psaA</italic> and <italic>psaB.</italic> Twelve of these long repeats were also found in <italic>C. sinensis,</italic> indicating that these repeats might be widespread in the genus.</p><table-wrap id="pone-0113049-t004" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0113049.t004</object-id><label>Table 4</label><caption><title>List of long repeat sequences.</title></caption><alternatives><graphic id="pone-0113049-t004-4" xlink:href="pone.0113049.t004"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1">Repeatsize</td><td align="left" rowspan="1" colspan="1">Type<xref ref-type="table-fn" rid="nt106">a</xref></td><td align="left" rowspan="1" colspan="1">Start position of1st repeat</td><td align="left" rowspan="1" colspan="1">Start position the repeatfound in other region</td><td align="left" rowspan="1" colspan="1">Location<xref ref-type="table-fn" rid="nt107">b</xref></td><td align="left" rowspan="1" colspan="1">Region</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">30</td><td align="left" rowspan="1" colspan="1">D</td><td align="left" rowspan="1" colspan="1">1759</td><td align="left" rowspan="1" colspan="1">1762</td><td align="left" rowspan="1" colspan="1">IGS (<italic>psbA</italic>-<italic>trnK-UUU</italic>)</td><td align="left" rowspan="1" colspan="1">LSC</td></tr><tr><td align="left" rowspan="1" colspan="1">30</td><td align="left" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">1771</td><td align="left" rowspan="1" colspan="1">12015</td><td align="left" rowspan="1" colspan="1">IGS (<italic>psbA</italic>-<italic>trnK-UUU</italic>, <italic>atpA</italic>-<italic>atpF</italic>)</td><td align="left" rowspan="1" colspan="1">LSC</td></tr><tr><td align="left" rowspan="1" colspan="1">30</td><td align="left" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">8231</td><td align="left" rowspan="1" colspan="1">37726, 47606</td><td align="left" rowspan="1" colspan="1"><bold>IGS (</bold><bold><italic>trnS-GCU</italic></bold><bold>, </bold><bold><italic>trnS-UGA</italic></bold><bold>, </bold><bold><italic>trnS-GGA</italic></bold><bold>),</bold></td><td align="left" rowspan="1" colspan="1">LSC</td></tr><tr><td align="left" rowspan="1" colspan="1">30</td><td align="left" rowspan="1" colspan="1">D</td><td align="left" rowspan="1" colspan="1">23226</td><td align="left" rowspan="1" colspan="1">85067</td><td align="left" rowspan="1" colspan="1"><bold>intron (</bold><bold><italic>rpoC1</italic></bold><bold>), IGS (</bold><bold><italic>rpl16</italic></bold><bold>-</bold><bold><italic>rps3</italic></bold><bold>)</bold></td><td align="left" rowspan="1" colspan="1">LSC</td></tr><tr><td align="left" rowspan="1" colspan="1">30</td><td align="left" rowspan="1" colspan="1">D</td><td align="left" rowspan="1" colspan="1">23686</td><td align="left" rowspan="1" colspan="1">52733</td><td align="left" rowspan="1" colspan="1">intron (<italic>rpoC1</italic><bold>)</bold>, IGS (<italic>ndhK</italic>-<italic>ndhC</italic>)</td><td align="left" rowspan="1" colspan="1">LSC</td></tr><tr><td align="left" rowspan="1" colspan="1">30</td><td align="left" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">23687</td><td align="left" rowspan="1" colspan="1">70291</td><td align="left" rowspan="1" colspan="1">intron (<italic>rpoC1</italic>), IGS (<italic>trnP-UGG</italic>-<italic>psaJ</italic>)</td><td align="left" rowspan="1" colspan="1">LSC</td></tr><tr><td align="left" rowspan="1" colspan="1">30</td><td align="left" rowspan="1" colspan="1">D</td><td align="left" rowspan="1" colspan="1">23692</td><td align="left" rowspan="1" colspan="1">33280</td><td align="left" rowspan="1" colspan="1">intron (<italic>rpoC1</italic>), IGS (<italic>trnE-UUC</italic>-<italic>trnT-GGU</italic>)</td><td align="left" rowspan="1" colspan="1">LSC</td></tr><tr><td align="left" rowspan="1" colspan="1">30</td><td align="left" rowspan="1" colspan="1">D</td><td align="left" rowspan="1" colspan="1">49192</td><td align="left" rowspan="1" colspan="1">117171</td><td align="left" rowspan="1" colspan="1">IGS (<italic>psbA</italic>-<italic>trnK-UUU</italic>), IGS (<italic>atpA</italic>-<italic>atpF</italic>)</td><td align="left" rowspan="1" colspan="1">LSC, IR</td></tr><tr><td align="left" rowspan="1" colspan="1">30</td><td align="left" rowspan="1" colspan="1">D, P</td><td align="left" rowspan="1" colspan="1">49197</td><td align="left" rowspan="1" colspan="1">102764, 144233</td><td align="left" rowspan="1" colspan="1">IGS (<italic>trnT-UGU</italic>-<italic>trnL-UAA</italic>), IGS (<italic>rps12</italic>-<italic>trnV-GAC</italic>)</td><td align="left" rowspan="1" colspan="1">LSC, IR</td></tr><tr><td align="left" rowspan="1" colspan="1">30</td><td align="left" rowspan="1" colspan="1">D, P</td><td align="left" rowspan="1" colspan="1">51215</td><td align="left" rowspan="1" colspan="1">102768, 144229</td><td align="left" rowspan="1" colspan="1">IGS (<italic>trnF-GAA</italic>-<italic>ndhJ</italic>), IGS (<italic>rps12</italic>-<italic>trnV-GAC</italic>)</td><td align="left" rowspan="1" colspan="1">LSC, IR</td></tr><tr><td align="left" rowspan="1" colspan="1">30</td><td align="left" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">71344</td><td align="left" rowspan="1" colspan="1">71344</td><td align="left" rowspan="1" colspan="1"><bold>IGS (</bold><bold><italic>rpl33</italic></bold><bold>-</bold><bold><italic>rps18</italic></bold><bold>)</bold></td><td align="left" rowspan="1" colspan="1">LSC</td></tr><tr><td align="left" rowspan="1" colspan="1">30</td><td align="left" rowspan="1" colspan="1">D, P</td><td align="left" rowspan="1" colspan="1">102768</td><td align="left" rowspan="1" colspan="1">102773, 144224</td><td align="left" rowspan="1" colspan="1">IGS (<italic>rps12</italic>-<italic>trnV-GAC</italic>)</td><td align="left" rowspan="1" colspan="1">IR</td></tr><tr><td align="left" rowspan="1" colspan="1">30</td><td align="left" rowspan="1" colspan="1">D</td><td align="left" rowspan="1" colspan="1">144225</td><td align="left" rowspan="1" colspan="1">144230</td><td align="left" rowspan="1" colspan="1">IGS (<italic>trnV</italic>-<italic>GAC</italic>-<italic>rps12</italic>)</td><td align="left" rowspan="1" colspan="1">IR</td></tr><tr><td align="left" rowspan="1" colspan="1">31</td><td align="left" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">4492</td><td align="left" rowspan="1" colspan="1">117868</td><td align="left" rowspan="1" colspan="1">IGS (<italic>trnK-UUU</italic>-<italic>rps16</italic>), IGS (<italic>rpl32</italic>-<italic>trnL-UAG</italic>)</td><td align="left" rowspan="1" colspan="1">LSC, IR</td></tr><tr><td align="left" rowspan="1" colspan="1">31</td><td align="left" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">10106</td><td align="left" rowspan="1" colspan="1">49188</td><td align="left" rowspan="1" colspan="1">IGS (<italic>trnG-GCC</italic>-<italic>trnR-UCU</italic>, <italic>trnT-UGU</italic>-<italic>trnL-UAA</italic>)</td><td align="left" rowspan="1" colspan="1">LSC</td></tr><tr><td align="left" rowspan="1" colspan="1">31</td><td align="left" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">29811</td><td align="left" rowspan="1" colspan="1">29811</td><td align="left" rowspan="1" colspan="1"><bold>IGS (</bold><bold><italic>petN</italic></bold><bold>-</bold><bold><italic>psbM</italic></bold><bold>)</bold></td><td align="left" rowspan="1" colspan="1">LSC</td></tr><tr><td align="left" rowspan="1" colspan="1">31</td><td align="left" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">33281</td><td align="left" rowspan="1" colspan="1">70282</td><td align="left" rowspan="1" colspan="1"><bold>IGS (</bold><bold><italic>trnE-UUC</italic></bold><bold>-</bold><bold><italic>trnT-GGU</italic></bold><bold>, </bold><bold><italic>trnP-UGG</italic></bold><bold>-</bold><bold><italic>psaJ</italic></bold><bold>)</bold></td><td align="left" rowspan="1" colspan="1">LSC</td></tr><tr><td align="left" rowspan="1" colspan="1">31</td><td align="left" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">119977</td><td align="left" rowspan="1" colspan="1">119977</td><td align="left" rowspan="1" colspan="1">intron (<italic>ccsA</italic>)</td><td align="left" rowspan="1" colspan="1">IR</td></tr><tr><td align="left" rowspan="1" colspan="1">32</td><td align="left" rowspan="1" colspan="1">D</td><td align="left" rowspan="1" colspan="1">7615</td><td align="left" rowspan="1" colspan="1">74171</td><td align="left" rowspan="1" colspan="1">IGS (<italic>psbK</italic>-<italic>psbI</italic>), intron (<italic>clpP</italic>)</td><td align="left" rowspan="1" colspan="1">LSC</td></tr><tr><td align="left" rowspan="1" colspan="1">32</td><td align="left" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">39166</td><td align="left" rowspan="1" colspan="1">39166</td><td align="left" rowspan="1" colspan="1">IGS (<italic>trnG-GCC</italic>-<italic>trnfM-CAU</italic>)</td><td align="left" rowspan="1" colspan="1">LSC</td></tr><tr><td align="left" rowspan="1" colspan="1">34</td><td align="left" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">38774</td><td align="left" rowspan="1" colspan="1">38782</td><td align="left" rowspan="1" colspan="1"><bold>IGS (</bold><bold><italic>psbZ</italic></bold><bold>-</bold><bold><italic>trnG-GCC</italic></bold><bold>)</bold></td><td align="left" rowspan="1" colspan="1">LSC</td></tr><tr><td align="left" rowspan="1" colspan="1">34</td><td align="left" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">49186</td><td align="left" rowspan="1" colspan="1">70288</td><td align="left" rowspan="1" colspan="1">rps4, IGS (<italic>trnP-UGG</italic>-<italic>psaJ</italic>)</td><td align="left" rowspan="1" colspan="1">LSC</td></tr><tr><td align="left" rowspan="1" colspan="1">34</td><td align="left" rowspan="1" colspan="1">D, P</td><td align="left" rowspan="1" colspan="1">111432</td><td align="left" rowspan="1" colspan="1">111464, 135529, 135561</td><td align="left" rowspan="1" colspan="1"><bold>IGS (</bold><bold><italic>rrn4.5</italic></bold><bold>-</bold><bold><italic>rrn5</italic></bold><bold>)</bold></td><td align="left" rowspan="1" colspan="1">IR</td></tr><tr><td align="left" rowspan="1" colspan="1">35</td><td align="left" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">10097</td><td align="left" rowspan="1" colspan="1">49193</td><td align="left" rowspan="1" colspan="1">IGS <italic>trnG-GCC</italic>-<italic>trnR-UCU</italic>, <italic>trnT-UGU</italic>-<italic>trnL-UAA</italic>)</td><td align="left" rowspan="1" colspan="1">LSC</td></tr><tr><td align="left" rowspan="1" colspan="1">36</td><td align="left" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">27648</td><td align="left" rowspan="1" colspan="1">27648</td><td align="left" rowspan="1" colspan="1">IGS (<italic>rpoB</italic>-<italic>trnC-GCA</italic>)</td><td align="left" rowspan="1" colspan="1">LSC</td></tr><tr><td align="left" rowspan="1" colspan="1">40</td><td align="left" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">77776</td><td align="left" rowspan="1" colspan="1">77776</td><td align="left" rowspan="1" colspan="1">IGS (<italic>psbT</italic>-<italic>psbN</italic>)</td><td align="left" rowspan="1" colspan="1">LSC</td></tr><tr><td align="left" rowspan="1" colspan="1">41</td><td align="left" rowspan="1" colspan="1">D</td><td align="left" rowspan="1" colspan="1">41294</td><td align="left" rowspan="1" colspan="1">43518</td><td align="left" rowspan="1" colspan="1"><bold><italic>psaB</italic></bold><bold>, </bold><bold><italic>psaA</italic></bold></td><td align="left" rowspan="1" colspan="1">LSC</td></tr><tr><td align="left" rowspan="1" colspan="1">41</td><td align="left" rowspan="1" colspan="1">D, P</td><td align="left" rowspan="1" colspan="1">102353</td><td align="left" rowspan="1" colspan="1">124945, 144633</td><td align="left" rowspan="1" colspan="1"><bold>IGS (</bold><bold><italic>rps12</italic></bold><bold>-</bold><bold><italic>trnV-GAC</italic></bold><bold>), intron (</bold><bold><italic>ndhA</italic></bold><bold>)</bold></td><td align="left" rowspan="1" colspan="1">IR</td></tr><tr><td align="left" rowspan="1" colspan="1">48</td><td align="left" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">30626</td><td align="left" rowspan="1" colspan="1">30626</td><td align="left" rowspan="1" colspan="1"><bold>IGS (</bold><bold><italic>petN</italic></bold><bold>-</bold><bold><italic>psbM</italic></bold><bold>)</bold></td><td align="left" rowspan="1" colspan="1">LSC</td></tr><tr><td align="left" rowspan="1" colspan="1">50</td><td align="left" rowspan="1" colspan="1">D</td><td align="left" rowspan="1" colspan="1">39020</td><td align="left" rowspan="1" colspan="1">39044</td><td align="left" rowspan="1" colspan="1"><bold>IGS (</bold><bold><italic>trnG-GCC</italic></bold><bold>-</bold><bold><italic>trnfM-CAU</italic></bold><bold>)</bold></td><td align="left" rowspan="1" colspan="1">LSC</td></tr><tr><td align="left" rowspan="1" colspan="1">51</td><td align="left" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">9984</td><td align="left" rowspan="1" colspan="1">9984</td><td align="left" rowspan="1" colspan="1">IGS (<italic>trnG-GCC</italic>-<italic>trnR-UCU</italic>)</td><td align="left" rowspan="1" colspan="1">LSC</td></tr><tr><td align="left" rowspan="1" colspan="1">53</td><td align="left" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">8869</td><td align="left" rowspan="1" colspan="1">31095</td><td align="left" rowspan="1" colspan="1"><bold>IGS</bold> (<italic>trnS-GCU</italic>-<italic>trnG-GCC</italic>, <bold><italic>psbM</italic></bold><bold>-</bold><bold><italic>trnD-GUC</italic></bold>)</td><td align="left" rowspan="1" colspan="1">LSC</td></tr><tr><td align="left" rowspan="1" colspan="1">54</td><td align="left" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">441</td><td align="left" rowspan="1" colspan="1">441</td><td align="left" rowspan="1" colspan="1">IGS (<italic>trnH-GUG</italic>-<italic>psbA</italic>)</td><td align="left" rowspan="1" colspan="1">LSC</td></tr></tbody></table></alternatives><table-wrap-foot><fn id="nt106"><label>a</label><p>D: direct repeat; P: palindrome inverted repeat.</p></fn><fn id="nt107"><label>b</label><p>IGS: intergenic spacer region. Sequences conserved in the <italic>C. sinensis</italic> chloroplast genome are highlighted in bold.</p></fn></table-wrap-foot></table-wrap></sec><sec id="s3d"><title>Gene Content Analyses within the Rosids</title><p>A maximum likelihood phylogenetic analysis of 72 representative rosids lineages was conducted based on a concatenated alignment of four rRNA and 58 protein-coding genes with 54,689 sites (<xref ref-type="fig" rid="pone-0113049-g002">Figure 2</xref>). <italic>Citrus</italic> represents Sapindales and is sister to the clade containing Malvales and Brassicales. These relationships are congruent with the previous reports <xref rid="pone.0113049-Bausher1" ref-type="bibr">[10]</xref>, <xref rid="pone.0113049-Bremer1" ref-type="bibr">[41]</xref>–<xref rid="pone.0113049-Ruhfel1" ref-type="bibr">[43]</xref>. Based on this phylogeny and the gene content, we inferred the gene loss events during the cp genome evolution in rosids.</p><fig id="pone-0113049-g002" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0113049.g002</object-id><label>Figure 2</label><caption><title>Maximum likelihood phylogeny of the representative rosids lineages.</title><p>The common grape vine (<italic>Vitis vinifera</italic>) is included as the outgroup to root the tree. The concatenated alignment includes 62 conserved chloroplast genome genes and 54,689 aligned nucleotide sites. Nodes received <70% bootstrap support are indicated by gray arrows. The putative events of gene losses are inferred based on the most parsimonious scenario.</p></caption><graphic xlink:href="pone.0113049.g002"></graphic></fig><p>The translation initiation factor gene <italic>infA</italic> in cp has been lost independently at least 24 times in angiosperms and evidence provided from some cases suggested functional replacement by a nucleus copy <xref rid="pone.0113049-Millen1" ref-type="bibr">[44]</xref>. Although the majority of <italic>infA</italic> in our selected cp genomes were found to be pseudogenized or completely lost, an intact <italic>infA</italic> was found in <italic>Quercus</italic>, <italic>Francoa</italic>, and two <italic>Cuscumis</italic> species.</p><p>The <italic>rpl22</italic> were found to be lost in Fabaceae <xref rid="pone.0113049-Gantt1" ref-type="bibr">[45]</xref> and <italic>Castanea</italic> of Fagaceae <xref rid="pone.0113049-Jansen1" ref-type="bibr">[46]</xref> following independent transfers to nucleus. Furthermore, another putative loss of <italic>rpl22</italic> was detected in <italic>Passiflora</italic><xref rid="pone.0113049-Jansen1" ref-type="bibr">[46]</xref>. The <italic>rpl22</italic> in Malvaceae, including <italic>Theobroma</italic> and three <italic>Gossypium</italic> species, were found to be pseudogenized in our analysis. In <italic>Citrus</italic>, the ORF of <italic>rpl22</italic> was shortened to 252–264 nt compared to the typical length of 399–489 nt in other rosids <xref rid="pone.0113049-Bausher1" ref-type="bibr">[10]</xref>, <xref rid="pone.0113049-Jansen1" ref-type="bibr">[46]</xref>. However, compared with the pseudogenized <italic>rpl22</italic> found in Malvalvace, the <italic>rpl22</italic> homologs in <italic>Citrus</italic> still show high sequence conservation. Additionally, the <italic>rpl22</italic> transcripts can be identified in the EST database for various <italic>Citrus</italic> species (data not shown). Taking account into the above consideration, we did not annotate <italic>rpl22</italic> as a pseudogene in <italic>Citrus</italic>.</p><p>The parallel losses of <italic>rps16</italic> were found in several rosids lineages (<xref ref-type="fig" rid="pone-0113049-g002">Figure 2</xref>), including one time in Salicaceae, two times in Fabaceae and another two times in Brassicaceae. The loss of <italic>rps16</italic> in <italic>Medicago</italic> and <italic>Populus</italic> was found to be substituted by a nuclear-encoded copy that transferred from the mitochondrion (mt) <xref rid="pone.0113049-Ueda1" ref-type="bibr">[47]</xref>. Because the nuclear-encoded RPS16 was found to target both mt and cp in <italic>Arabidopsis</italic>, <italic>Lycopersicon</italic>, and <italic>Oryza</italic><xref rid="pone.0113049-Ueda1" ref-type="bibr">[47]</xref>, it is possible that the cp genome-encoded <italic>rps16</italic> would not be maintained by selection and will eventually become lost in these lineages.</p><p>There are only a few gene loss events of photosynthetic genes found in rosids. In addition to the loss of <italic>psaI</italic> in <italic>Lathyrus sativus</italic><xref rid="pone.0113049-Magee1" ref-type="bibr">[48]</xref>, the <italic>accD</italic> seems to be lost independently in <italic>Trifolium subterraneum</italic> and several Gerantiaceae species except for <italic>Geranium palmatum</italic>. In <italic>Trifolium,</italic> a nuclear-encoded <italic>accD</italic> copy has been reported <xref rid="pone.0113049-Magee1" ref-type="bibr">[48]</xref>, which presented another example of horizontal gene transfer from cp to nucleus. Successful gene transfers from cp to the nucleus in angiosperms are rare and have been only documented for four genes in rosids. Other than the three genes described above (i.e., <italic>infA</italic>, <italic>rpl22</italic>, and <italic>accD</italic>), the <italic>rpl32</italic> in <italic>Populus</italic> (Salicaceae) is the fourth example <xref rid="pone.0113049-Cusack1" ref-type="bibr">[49]</xref>–<xref rid="pone.0113049-Jansen2" ref-type="bibr">[51]</xref>.</p><p>The IR has been reported to be independently lost at least five times among seed plants, two of which are within rosids <xref rid="pone.0113049-Jansen2" ref-type="bibr">[51]</xref>. In addition to the inverted repeat lacking clade (IRLC) of papilionoid Fabaceae <xref rid="pone.0113049-Wojciechowski1" ref-type="bibr">[52]</xref> and <italic>Erodium</italic> of Gerantiaceae <xref rid="pone.0113049-Blazier1" ref-type="bibr">[53]</xref>, <xref rid="pone.0113049-Guisinger1" ref-type="bibr">[54]</xref>, the IR was found to be lost in two lineages of <italic>Fragaria</italic> (Rosaceae), which are <italic>F. vesca</italic> ssp. <italic>bracteatea</italic> and <italic>F. mandschurica</italic> (accession: NC_018767, not shown in <xref ref-type="fig" rid="pone-0113049-g002">Figure 2</xref>). Based on the <italic>Fragaria</italic> phylogeny shown in a previous study <xref rid="pone.0113049-Njuguna1" ref-type="bibr">[55]</xref>, it seems that IR loss was not a single event in <italic>Fragaria</italic>.</p></sec><sec id="s3e"><title>Molecular Evolution of <italic>orf56</italic> and <italic>ycf68</italic> within the Rosids</title><p>In the comparison of gene content between the two <italic>Citrus</italic> cp genomes, <italic>C. aurantiifolia</italic> was found to contain two additional protein-coding genes. The first gene, <italic>orf56</italic>, is located in the <italic>trnA-UGC</italic> intron that contains one sequence homologous to previously recognized mitochondrial <italic>ACRS</italic> (ACR-toxin sensitivity gene) in <italic>Citrus</italic><xref rid="pone.0113049-Ohtani1" ref-type="bibr">[56]</xref>. In addition to the 171-bp identical sequences between cp <italic>orf56</italic> and the ORF sequences of <italic>ACRS</italic> in mt, the full length of 355-bp region of <italic>ACRS</italic> that conferred sensitivity to ACR-toxin in <italic>E. coil</italic> are also identical. Furthermore, the whole <italic>trnA-UGC</italic> among two <italic>Citrus</italic> cp regions and <italic>C. jambhiri</italic> mitochondrial <italic>ACRS</italic> shared more than 96% identity (<xref ref-type="supplementary-material" rid="pone.0113049.s001">Figure S1</xref>), which highlight the conservation of this region between cp and mt.</p><p>The gene <italic>orf56</italic> has also been included in the annotation of complete cp genomes of <italic>Calycanthus</italic><xref rid="pone.0113049-Goremykin1" ref-type="bibr">[57]</xref> and <italic>Pelargonium</italic><xref rid="pone.0113049-Chumley1" ref-type="bibr">[58]</xref>. Our BLAST search against the rosids genome database revealed that in addition to <italic>Citrus</italic> and <italic>Pelargonium</italic>, all of the species examined in Cucurbitaceae and Myrtales also contain an intact <italic>orf56</italic> (<xref ref-type="fig" rid="pone-0113049-g003">Figure 3</xref>). Moreover, an intact <italic>ACRS</italic> ORF is also present in the mt genomes of <italic>Liriodendron</italic><xref rid="pone.0113049-Richardson1" ref-type="bibr">[59]</xref> and <italic>Silene</italic><xref rid="pone.0113049-Sloan1" ref-type="bibr">[60]</xref> and the ORF sequences between cp and mt are identical. Goremykin et al. <xref rid="pone.0113049-Goremykin1" ref-type="bibr">[57]</xref> suggested that the <italic>ACRS</italic> gene was relative recently transferred from cp to mt. Based on the phylogeny containing the cp <italic>orf56</italic> and the mt <italic>ACRS</italic> (<xref ref-type="supplementary-material" rid="pone.0113049.s002">Figure S2</xref>), it appears that <italic>orf56</italic> has been independently transferred from cp to mt in different lineages.</p><fig id="pone-0113049-g003" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0113049.g003</object-id><label>Figure 3</label><caption><title>The phylogenetic distribution patterns of <italic>orf56</italic> and <italic>ycf68</italic>.</title></caption><graphic xlink:href="pone.0113049.g003"></graphic></fig><p>The second gene, <italic>ycf68</italic>, is located in the <italic>trnI-GAU</italic> intron. A nearly identical sequence was found in <italic>C. sinensis</italic> but an additional T insertion near the C-terminus abolished the stop codon at the corresponding position. The intact <italic>ycf68</italic> can be detected in several monocots and Nymphaeaceae <xref rid="pone.0113049-Raubeson1" ref-type="bibr">[61]</xref>, <xref rid="pone.0113049-Ahmed1" ref-type="bibr">[62]</xref>. However, in the majority of other rosids (<xref ref-type="fig" rid="pone-0113049-g003">Figure 3</xref>) and the rest of the eudicots <xref rid="pone.0113049-Raubeson1" ref-type="bibr">[61]</xref>, the <italic>ycf68</italic> homologs all contain premature stop codons. Although Raubeson et al. <xref rid="pone.0113049-Raubeson1" ref-type="bibr">[61]</xref> argued that <italic>ycf68</italic> is not a protein-coding gene based on the lack of intron-folding pattern, the high levels of sequence conservation among the ORFs of identified homologs suggest that the true identity and functionality of this putative gene remains to be further investigated.</p></sec></sec><sec id="s4"><title>Conclusions</title><p>We reported the complete cp genome sequence of <italic>Citrus aurantiifolia</italic> (Rutaceae) in this study. The genome organization and gene content is typical of most angiosperms and highly similar to that of <italic>C. sinensis</italic> (i.e., 98.7% identical at the nucleotide level). The only difference in the gene content between the two <italic>Citrus</italic> cp genomes is the <italic>C. aurantiifolia</italic>-specific presence of a protein-coding gene (<italic>ycf68</italic>) in the <italic>trnI</italic>-<italic>GAU</italic> intron. Notably, three long intergenic spacers with high sequence divergence and 94 shared SSR regions were identified in the <italic>C. aurantiifolia</italic>-<italic>C. sinensis</italic> comparison. These regions may provide phylogenetic utility at low taxonomic levels and could be applied to the molecular identification of <italic>Citrus</italic> cultivars. Finally, our comparative analysis of gene content among 72 representative rosids lineages highlighted multiple events of gene losses within this group.</p></sec><sec sec-type="supplementary-material" id="s5"><title>Supporting Information</title><supplementary-material content-type="local-data" id="pone.0113049.s001"><label>Figure S1</label><caption><p><bold>Alignment of the </bold><bold><italic>orf56</italic></bold><bold>-containing sequences of two </bold><bold><italic>Citrus</italic></bold><bold> cp genomes and </bold><bold><italic>C. jambhiri</italic></bold><bold> mitochondrial </bold><bold><italic>ACRS</italic></bold><bold> sequences.</bold></p><p>(TIF)</p></caption><media xlink:href="pone.0113049.s001.tif"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pone.0113049.s002"><label>Figure S2</label><caption><p><bold>The maximum likelihood phylogeny of the cp </bold><bold><italic>orf56</italic></bold><bold> and mt </bold><bold><italic>ACRS</italic></bold><bold> ORF sequences.</bold></p><p>(TIF)</p></caption><media xlink:href="pone.0113049.s002.tif"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pone.0113049.s003"><label>Table S1</label><caption><p><bold>List of the complete chloroplast genome sequences included in the phylogenetic analysis.</bold></p><p>(XLSX)</p></caption><media xlink:href="pone.0113049.s003.xlsx"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pone.0113049.s004"><label>Table S2</label><caption><p><bold>List of the genes found in the </bold><bold><italic>C. aurantiifolia</italic></bold><bold> cp genome.</bold></p><p>(XLSX)</p></caption><media xlink:href="pone.0113049.s004.xlsx"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pone.0113049.s005"><label>Table S3</label><caption><p><bold>Codon usage of the </bold><bold><italic>C. aurantiifolia</italic></bold><bold> cp genome.</bold></p><p>(XLSX)</p></caption><media xlink:href="pone.0113049.s005.xlsx"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack><p>We thank Sam T. 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