Genome sequence of the clover symbiont Rhizobium leguminosarum bv. trifolii strain CC275e
Identifieur interne : 000949 ( Pmc/Corpus ); précédent : 000948; suivant : 000950Genome sequence of the clover symbiont Rhizobium leguminosarum bv. trifolii strain CC275e
Auteurs : Clément Delestre ; Aurélie Laugraud ; Hayley Ridgway ; Clive Ronson ; Maureen O Allaghan ; Brent Barrett ; Ross Ballard ; Andrew Griffiths ; Sandra Young ; Celine Blond ; Emily Gerard ; Steve WakelinSource :
- Standards in Genomic Sciences [ 1944-3277 ] ; 2015.
Abstract
The online version of this article (doi:10.1186/s40793-015-0110-1) contains supplementary material, which is available to authorized users.
Url:
DOI: 10.1186/s40793-015-0110-1
PubMed: 26649149
PubMed Central: 4672485
Links to Exploration step
PMC:4672485Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Genome sequence of the clover symbiont <italic>Rhizobium leguminosarum</italic>
bv. <italic>trifolii</italic>
strain CC275e</title>
<author><name sortKey="Delestre, Clement" sort="Delestre, Clement" uniqKey="Delestre C" first="Clément" last="Delestre">Clément Delestre</name>
<affiliation><nlm:aff id="Aff1">University of Bordeaux, IT Science, 351 Cours de la Libération, 33400 Talence, France</nlm:aff>
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<author><name sortKey="Laugraud, Aurelie" sort="Laugraud, Aurelie" uniqKey="Laugraud A" first="Aurélie" last="Laugraud">Aurélie Laugraud</name>
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<author><name sortKey="Ridgway, Hayley" sort="Ridgway, Hayley" uniqKey="Ridgway H" first="Hayley" last="Ridgway">Hayley Ridgway</name>
<affiliation><nlm:aff id="Aff3">Faculty of Agriculture and Life Sciences, Lincoln University, PO Box 84, Christchurch, New Zealand</nlm:aff>
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<author><name sortKey="Ronson, Clive" sort="Ronson, Clive" uniqKey="Ronson C" first="Clive" last="Ronson">Clive Ronson</name>
<affiliation><nlm:aff id="Aff4">Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin, New Zealand</nlm:aff>
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<author><name sortKey="O Allaghan, Maureen" sort="O Allaghan, Maureen" uniqKey="O Allaghan M" first="Maureen" last="O Allaghan">Maureen O Allaghan</name>
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<author><name sortKey="Ballard, Ross" sort="Ballard, Ross" uniqKey="Ballard R" first="Ross" last="Ballard">Ross Ballard</name>
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<author><name sortKey="Blond, Celine" sort="Blond, Celine" uniqKey="Blond C" first="Celine" last="Blond">Celine Blond</name>
<affiliation><nlm:aff id="Aff3">Faculty of Agriculture and Life Sciences, Lincoln University, PO Box 84, Christchurch, New Zealand</nlm:aff>
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<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Genome sequence of the clover symbiont <italic>Rhizobium leguminosarum</italic>
bv. <italic>trifolii</italic>
strain CC275e</title>
<author><name sortKey="Delestre, Clement" sort="Delestre, Clement" uniqKey="Delestre C" first="Clément" last="Delestre">Clément Delestre</name>
<affiliation><nlm:aff id="Aff1">University of Bordeaux, IT Science, 351 Cours de la Libération, 33400 Talence, France</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Laugraud, Aurelie" sort="Laugraud, Aurelie" uniqKey="Laugraud A" first="Aurélie" last="Laugraud">Aurélie Laugraud</name>
<affiliation><nlm:aff id="Aff2">AgResearch Ltd, Lincoln Campus, Private Bag 4749, Christchurch, 8140 New Zealand</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Ridgway, Hayley" sort="Ridgway, Hayley" uniqKey="Ridgway H" first="Hayley" last="Ridgway">Hayley Ridgway</name>
<affiliation><nlm:aff id="Aff3">Faculty of Agriculture and Life Sciences, Lincoln University, PO Box 84, Christchurch, New Zealand</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Ronson, Clive" sort="Ronson, Clive" uniqKey="Ronson C" first="Clive" last="Ronson">Clive Ronson</name>
<affiliation><nlm:aff id="Aff4">Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin, New Zealand</nlm:aff>
</affiliation>
</author>
<author><name sortKey="O Allaghan, Maureen" sort="O Allaghan, Maureen" uniqKey="O Allaghan M" first="Maureen" last="O Allaghan">Maureen O Allaghan</name>
<affiliation><nlm:aff id="Aff2">AgResearch Ltd, Lincoln Campus, Private Bag 4749, Christchurch, 8140 New Zealand</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Barrett, Brent" sort="Barrett, Brent" uniqKey="Barrett B" first="Brent" last="Barrett">Brent Barrett</name>
<affiliation><nlm:aff id="Aff5">AgResearch Ltd, Grasslands Research Centre, Private Bag 11008, Palmerston North, New Zealand</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Ballard, Ross" sort="Ballard, Ross" uniqKey="Ballard R" first="Ross" last="Ballard">Ross Ballard</name>
<affiliation><nlm:aff id="Aff6">South Australian Research and Development Institute, Urrbrae, South Australia Australia</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Griffiths, Andrew" sort="Griffiths, Andrew" uniqKey="Griffiths A" first="Andrew" last="Griffiths">Andrew Griffiths</name>
<affiliation><nlm:aff id="Aff5">AgResearch Ltd, Grasslands Research Centre, Private Bag 11008, Palmerston North, New Zealand</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Young, Sandra" sort="Young, Sandra" uniqKey="Young S" first="Sandra" last="Young">Sandra Young</name>
<affiliation><nlm:aff id="Aff2">AgResearch Ltd, Lincoln Campus, Private Bag 4749, Christchurch, 8140 New Zealand</nlm:aff>
</affiliation>
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<author><name sortKey="Blond, Celine" sort="Blond, Celine" uniqKey="Blond C" first="Celine" last="Blond">Celine Blond</name>
<affiliation><nlm:aff id="Aff3">Faculty of Agriculture and Life Sciences, Lincoln University, PO Box 84, Christchurch, New Zealand</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Gerard, Emily" sort="Gerard, Emily" uniqKey="Gerard E" first="Emily" last="Gerard">Emily Gerard</name>
<affiliation><nlm:aff id="Aff2">AgResearch Ltd, Lincoln Campus, Private Bag 4749, Christchurch, 8140 New Zealand</nlm:aff>
</affiliation>
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<author><name sortKey="Wakelin, Steve" sort="Wakelin, Steve" uniqKey="Wakelin S" first="Steve" last="Wakelin">Steve Wakelin</name>
<affiliation><nlm:aff id="Aff2">AgResearch Ltd, Lincoln Campus, Private Bag 4749, Christchurch, 8140 New Zealand</nlm:aff>
</affiliation>
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<series><title level="j">Standards in Genomic Sciences</title>
<idno type="eISSN">1944-3277</idno>
<imprint><date when="2015">2015</date>
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<front><div type="abstract" xml:lang="en"><p><italic>Rhizobium leguminosarum</italic>
bv. <italic>trifolii</italic>
strain CC275e is a highly effective, N<sub>2</sub>
-fixing microsymbiont of white clover (<italic>Trifolium repens</italic>
L.). The bacterium has been widely used in both Australia and New Zealand as a clover seed inoculant and, as such, has delivered the equivalent of millions of dollars of nitrogen into these pastoral systems. <italic>R. leguminosarum</italic>
strain CC275e is a rod-shaped, motile, Gram-negative, non-spore forming bacterium. The genome was sequenced on an Illumina MiSeq instrument using a 2 × 150 bp paired end library and assembled into 29 scaffolds. The genome size is 7,077,367 nucleotides, with a GC content of 60.9 %. The final, high-quality draft genome contains 6693 protein coding genes, close to 85 % of which were assigned to COG categories. This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession JRXL00000000. The sequencing of this genome will enable identification of genetic traits associated with host compatibility and high N<sub>2</sub>
fixation characteristics in <italic>Rhizobium leguminosarum.</italic>
The sequence will also be useful for development of strain-specific markers to assess factors associated with environmental fitness, competiveness for host nodule occupancy, and survival on legume seeds (New Zealand Ministry of Business, Innovation and Employment program, ‘Improving forage legume-rhizobia performance’ contract C10X1308 and DairyNZ Ltd.).</p>
<sec><title>Electronic supplementary material</title>
<p>The online version of this article (doi:10.1186/s40793-015-0110-1) contains supplementary material, which is available to authorized users.</p>
</sec>
</div>
</front>
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<author><name sortKey="Selengut, J" uniqKey="Selengut J">J Selengut</name>
</author>
<author><name sortKey="Sterk, P" uniqKey="Sterk P">P Sterk</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Woese, Cr" uniqKey="Woese C">CR Woese</name>
</author>
<author><name sortKey="Kandler, O" uniqKey="Kandler O">O Kandler</name>
</author>
<author><name sortKey="Wheelis, Ml" uniqKey="Wheelis M">ML Wheelis</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Garrity, Gm" uniqKey="Garrity G">GM Garrity</name>
</author>
<author><name sortKey="Bell, Ja" uniqKey="Bell J">JA Bell</name>
</author>
<author><name sortKey="Lilburn, T" uniqKey="Lilburn T">T Lilburn</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Garrity, Gm" uniqKey="Garrity G">GM Garrity</name>
</author>
<author><name sortKey="Bell, Ja" uniqKey="Bell J">JA Bell</name>
</author>
<author><name sortKey="Lilburn, T" uniqKey="Lilburn T">T Lilburn</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Kuykendall, Ld" uniqKey="Kuykendall L">LD Kuykendall</name>
</author>
</analytic>
</biblStruct>
<biblStruct></biblStruct>
</listBibl>
</div1>
</back>
</TEI>
<pmc article-type="case-report"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Stand Genomic Sci</journal-id>
<journal-id journal-id-type="iso-abbrev">Stand Genomic Sci</journal-id>
<journal-title-group><journal-title>Standards in Genomic Sciences</journal-title>
</journal-title-group>
<issn pub-type="epub">1944-3277</issn>
<publisher><publisher-name>BioMed Central</publisher-name>
<publisher-loc>London</publisher-loc>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">26649149</article-id>
<article-id pub-id-type="pmc">4672485</article-id>
<article-id pub-id-type="publisher-id">110</article-id>
<article-id pub-id-type="doi">10.1186/s40793-015-0110-1</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Short Genome Report</subject>
</subj-group>
</article-categories>
<title-group><article-title>Genome sequence of the clover symbiont <italic>Rhizobium leguminosarum</italic>
bv. <italic>trifolii</italic>
strain CC275e</article-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name><surname>Delestre</surname>
<given-names>Clément</given-names>
</name>
<xref ref-type="aff" rid="Aff1"></xref>
</contrib>
<contrib contrib-type="author"><name><surname>Laugraud</surname>
<given-names>Aurélie</given-names>
</name>
<xref ref-type="aff" rid="Aff2"></xref>
</contrib>
<contrib contrib-type="author"><name><surname>Ridgway</surname>
<given-names>Hayley</given-names>
</name>
<xref ref-type="aff" rid="Aff3"></xref>
</contrib>
<contrib contrib-type="author"><name><surname>Ronson</surname>
<given-names>Clive</given-names>
</name>
<xref ref-type="aff" rid="Aff4"></xref>
</contrib>
<contrib contrib-type="author"><name><surname>O’Callaghan</surname>
<given-names>Maureen</given-names>
</name>
<xref ref-type="aff" rid="Aff2"></xref>
</contrib>
<contrib contrib-type="author"><name><surname>Barrett</surname>
<given-names>Brent</given-names>
</name>
<xref ref-type="aff" rid="Aff5"></xref>
</contrib>
<contrib contrib-type="author"><name><surname>Ballard</surname>
<given-names>Ross</given-names>
</name>
<xref ref-type="aff" rid="Aff6"></xref>
</contrib>
<contrib contrib-type="author"><name><surname>Griffiths</surname>
<given-names>Andrew</given-names>
</name>
<xref ref-type="aff" rid="Aff5"></xref>
</contrib>
<contrib contrib-type="author"><name><surname>Young</surname>
<given-names>Sandra</given-names>
</name>
<xref ref-type="aff" rid="Aff2"></xref>
</contrib>
<contrib contrib-type="author"><name><surname>Blond</surname>
<given-names>Celine</given-names>
</name>
<xref ref-type="aff" rid="Aff3"></xref>
</contrib>
<contrib contrib-type="author"><name><surname>Gerard</surname>
<given-names>Emily</given-names>
</name>
<xref ref-type="aff" rid="Aff2"></xref>
</contrib>
<contrib contrib-type="author" corresp="yes"><name><surname>Wakelin</surname>
<given-names>Steve</given-names>
</name>
<address><email>Steve.Wakelin@agresearch.co.nz</email>
</address>
<xref ref-type="aff" rid="Aff2"></xref>
</contrib>
<aff id="Aff1"><label></label>
University of Bordeaux, IT Science, 351 Cours de la Libération, 33400 Talence, France</aff>
<aff id="Aff2"><label></label>
AgResearch Ltd, Lincoln Campus, Private Bag 4749, Christchurch, 8140 New Zealand</aff>
<aff id="Aff3"><label></label>
Faculty of Agriculture and Life Sciences, Lincoln University, PO Box 84, Christchurch, New Zealand</aff>
<aff id="Aff4"><label></label>
Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin, New Zealand</aff>
<aff id="Aff5"><label></label>
AgResearch Ltd, Grasslands Research Centre, Private Bag 11008, Palmerston North, New Zealand</aff>
<aff id="Aff6"><label></label>
South Australian Research and Development Institute, Urrbrae, South Australia Australia</aff>
</contrib-group>
<pub-date pub-type="epub"><day>8</day>
<month>12</month>
<year>2015</year>
</pub-date>
<pub-date pub-type="pmc-release"><day>8</day>
<month>12</month>
<year>2015</year>
</pub-date>
<pub-date pub-type="collection"><year>2015</year>
</pub-date>
<volume>10</volume>
<elocation-id>121</elocation-id>
<history><date date-type="received"><day>6</day>
<month>8</month>
<year>2015</year>
</date>
<date date-type="accepted"><day>2</day>
<month>11</month>
<year>2015</year>
</date>
</history>
<permissions><copyright-statement>© Delestre et al. 2015</copyright-statement>
<license license-type="OpenAccess"><license-p><bold>Open Access</bold>
This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link>
), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/publicdomain/zero/1.0/">http://creativecommons.org/publicdomain/zero/1.0/</ext-link>
) applies to the data made available in this article, unless otherwise stated.</license-p>
</license>
</permissions>
<abstract id="Abs1"><p><italic>Rhizobium leguminosarum</italic>
bv. <italic>trifolii</italic>
strain CC275e is a highly effective, N<sub>2</sub>
-fixing microsymbiont of white clover (<italic>Trifolium repens</italic>
L.). The bacterium has been widely used in both Australia and New Zealand as a clover seed inoculant and, as such, has delivered the equivalent of millions of dollars of nitrogen into these pastoral systems. <italic>R. leguminosarum</italic>
strain CC275e is a rod-shaped, motile, Gram-negative, non-spore forming bacterium. The genome was sequenced on an Illumina MiSeq instrument using a 2 × 150 bp paired end library and assembled into 29 scaffolds. The genome size is 7,077,367 nucleotides, with a GC content of 60.9 %. The final, high-quality draft genome contains 6693 protein coding genes, close to 85 % of which were assigned to COG categories. This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession JRXL00000000. The sequencing of this genome will enable identification of genetic traits associated with host compatibility and high N<sub>2</sub>
fixation characteristics in <italic>Rhizobium leguminosarum.</italic>
The sequence will also be useful for development of strain-specific markers to assess factors associated with environmental fitness, competiveness for host nodule occupancy, and survival on legume seeds (New Zealand Ministry of Business, Innovation and Employment program, ‘Improving forage legume-rhizobia performance’ contract C10X1308 and DairyNZ Ltd.).</p>
<sec><title>Electronic supplementary material</title>
<p>The online version of this article (doi:10.1186/s40793-015-0110-1) contains supplementary material, which is available to authorized users.</p>
</sec>
</abstract>
<kwd-group xml:lang="en"><title>Keywords</title>
<kwd>Root-nodule bacteria</kwd>
<kwd>Microsymbiont</kwd>
<kwd>Nitrogen fixation</kwd>
<kwd>Rhizobia</kwd>
<kwd><italic>Alphaproteobacteria</italic>
</kwd>
</kwd-group>
<funding-group><award-group><funding-source><institution>MBIE</institution>
</funding-source>
<award-id>C10X1308</award-id>
</award-group>
</funding-group>
<custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name>
<meta-value>© The Author(s) 2015</meta-value>
</custom-meta>
</custom-meta-group>
</article-meta>
</front>
<body><sec id="Sec1"><title>Introduction</title>
<p>White clover (<ext-link ext-link-type="uri" xlink:href="http://www.theplantlist.org/tpl1.1/record/ild-8135"><italic>Trifolium repens</italic>
</ext-link>
) is the most widely established and important legume in pastures in New Zealand [<xref ref-type="bibr" rid="CR1">1</xref>
] and globally [<xref ref-type="bibr" rid="CR2">2</xref>
]. In symbiosis with nodule-forming <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>Rhizobium leguminosarum</italic>
</ext-link>
bacteria of the biovar <italic>trifolii</italic>
(hereafter <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
<italic>bv trifolii</italic>
), clover plants fix atmospheric nitrogen into a plant-available, thus providing an economically and environmentally sustainable method of maintaining soil fertility and pasture production. Across New Zealand there are 11,400+ farms using pastures containing forage legumes (mostly white clover), covering 7.88 million hectares [<xref ref-type="bibr" rid="CR3">3</xref>
]. This constitutes about 29 % of the total land area and excludes hill country/tussock grasslands. Estimates of nitrogen input from legumes vary, however average at 185 kg N ha<sup>−1</sup>
yr<sup>−1</sup>
for pastures with a slope less than 12° [<xref ref-type="bibr" rid="CR4">4</xref>
]. Based on recent average costs of urea fertilizer (2013–14 average), the value of N<sub>2</sub>
fixation into New Zealand pastures is 1.8 billion per year; this is highly conservative as it does not encompass the value of increased forage quality, N<sub>2</sub>
fixation in extensive hill country systems, and reduced environmental costs.</p>
<p><ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
<italic>bv trifolii</italic>
strains vary extensively in their ability to form nodules with white clover [<xref ref-type="bibr" rid="CR5">5</xref>
], and also their effectiveness at fixing nitrogen during symbiosis [<xref ref-type="bibr" rid="CR6">6</xref>
]. As such, dedicated selection and screening programs have played a vital role in ensuring clover (and, of course, other legume species) are matched with an optimal rhizobia symbiont [<xref ref-type="bibr" rid="CR7">7</xref>
]. These are most commonly delivered into farming systems as rhizobia-inoculated seed [<xref ref-type="bibr" rid="CR8">8</xref>
].</p>
<p>The inoculation of white clover seed with rhizobia commenced in New Zealand in the early 20th century [<xref ref-type="bibr" rid="CR8">8</xref>
]. In addition to New Zealand produced inoculant strains, <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
<italic>bv trifolii</italic>
strain CC275e was sourced from Australia [<xref ref-type="bibr" rid="CR9">9</xref>
]. From 1974, the inoculant production in New Zealand industry was phased-out and the sole commercial strain for inoculation of white clover seed was strain CC275e, which was then replaced with <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
<italic>bv trifolii</italic>
strain TA1 (also from Australia) around 2005. Thus, <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
<italic>bv trifolii</italic>
strain CC275e was in widespread use in New Zealand for approximately three decades, and is likely to have contributed billions of dollars of nitrogen into New Zealand’s pastoral systems. On white clover, <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
<italic>bv trifolii</italic>
strain CC275e has been reported to fix more nitrogen than strain TA1 and has greater persistence in soils [<xref ref-type="bibr" rid="CR9">9</xref>
]. The decision by the inoculant industry to replace strain CC275e with strain TA1 was based on ease of production.</p>
<p>A number of synonyms of strain <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
<italic>bv trifolii</italic>
strain CC275e exist. In New Zealand, a culture of strain CC275e was received by the Plant Diseases Division of the Department of Scientific and Industrial Research in 1974 and a re-isolate of this culture is referred to as strain <ext-link ext-link-type="uri" xlink:href="http://doi.org/10.1601/strainfinder?urlappend=%3Fid%3DPDD2163">PDD2163</ext-link>
. Furthermore, in New Zealand, strain CC275e has also been referred to as strain W16 [<xref ref-type="bibr" rid="CR10">10</xref>
], but when used commercially was most commonly known as strain NZP561 [<xref ref-type="bibr" rid="CR11">11</xref>
]. In Australia, where the bacterium originates, early work referred to it as strain W16 or Strain Hastings T71 [<xref ref-type="bibr" rid="CR10">10</xref>
]. However, strain CC275e was the designation used when the bacterium was deposited in the CSIRO (Canberra) culture collection [<xref ref-type="bibr" rid="CR12">12</xref>
], and this is the most commonly used synonym. In the American Type Culture Collection, the bacterium is referred to as <ext-link ext-link-type="uri" xlink:href="http://doi.org/10.1601/strainfinder?urlappend=%3Fid%3DATCC+35181">ATCC 35181</ext-link>
. For this study, an original <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
<italic>bv trifolii</italic>
strain CC275e culture was obtained from the Australian Inoculant Research Group (Gosford, NSW, Australia). These sequence data complements those of <ext-link ext-link-type="uri" xlink:href="http://plants.usda.gov/core/profile?symbol=TRIFO"><italic>Trifolium</italic>
</ext-link>
-nodulating <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
<italic>bv trifolii</italic>
strain WSM1325 (GenBank ID 241202755), strain WSM2304 (GenBank ID 209547612), strain WSM1689 (GenBank ID 752843554), and strain TA1 (GenBank ID 653806106).</p>
</sec>
<sec id="Sec2"><title>Organism information</title>
<sec id="Sec3"><title>Classification and features</title>
<p><ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>Rhizobium leguminosarum</italic>
</ext-link>
bv. <italic>trifolii</italic>
strain CC275e is a Gram-negative, motile, non-spore forming, non-encapsulated, rod shaped bacterium (Fig. <xref rid="Fig1" ref-type="fig">1</xref>
). Colonies of <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
<italic>bv trifolii</italic>
strain CC275e form within 4 to 5 days when grown on yeast mannitol agar (YMA; [<xref ref-type="bibr" rid="CR13">13</xref>
]) at 25 °C. Colonies are white-opaque, domed and glassy in appearance, with smooth margins.<fig id="Fig1"><label>Fig. 1</label>
<caption><p>TEM micrograph of three <italic>Rhizobium leguminosarum</italic>
bv. <italic>trifolii</italic>
CC275e cells. The length of the bar = 1 um</p>
</caption>
<graphic xlink:href="40793_2015_110_Fig1_HTML" id="MO1"></graphic>
</fig>
</p>
<p><ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>Rhizobium leguminosarum</italic>
</ext-link>
and closely related species are generally regarded as non-fastidious, chemo-organotrophic bacteria [<xref ref-type="bibr" rid="CR14">14</xref>
]. Although the wider substrate requirements for strain CC275e have not been formally described, the authors support this classification based on personal experience in the handling, cultivation and fermentation of <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
<italic>bv trifolii</italic>
strain CC275e.</p>
<p>The <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
<italic>bv trifolii</italic>
strain CC275e genome contains three (100 % identical) copies of the 16S rRNA gene. Alignment of these nucleotide sequences against other species supports close 16S rRNA phylogeny with <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
originating from other legume hosts (Fig. <xref rid="Fig2" ref-type="fig">2</xref>
). The 16S rRNA gene sequence has highest similarity to other accessions of <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
biovars <italic>trifolii</italic>
(99.8 %) and <italic>phaseoli</italic>
(99.6 %) (Fig. <xref rid="Fig2" ref-type="fig">2</xref>
) - the GenBank accession numbers for these are provided in Additional file <xref rid="MOESM1" ref-type="media">1</xref>
: Table S1. The species is placed within the order <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1277"><italic>Rhizobiales</italic>
</ext-link>
of the class <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.809"><italic>Alphaproteobacteria</italic>
</ext-link>
[<xref ref-type="bibr" rid="CR15">15</xref>
]. Minimum information about the Genome Sequence (MIGS) is provided in Table <xref rid="Tab1" ref-type="table">1</xref>
.<fig id="Fig2"><label>Fig. 2</label>
<caption><p>Phylogenetic tree showing relationship of <italic>R. leguminosarum bv trifolii</italic>
CC275e with closely and distantly related taxa in the order <italic>Rhizobiales</italic>
. The tree is based on 1498 bp length alignment of the 16S rRNA gene using MUSCLE with default parameters [<xref ref-type="bibr" rid="CR31">31</xref>
]. The tree was constructed using maximum likelihood method, with the General Time Reversible model (rate 4 classes; [<xref ref-type="bibr" rid="CR32">32</xref>
]). Nodes with bootstrap (1000 repetitions) support > 50 % are shown [<xref ref-type="bibr" rid="CR33">33</xref>
]. Accession numbers relating to the nucleotide sequences for each of the strains are listed in Additional file <xref rid="MOESM1" ref-type="media">1</xref>
: Table S1</p>
</caption>
<graphic xlink:href="40793_2015_110_Fig2_HTML" id="MO2"></graphic>
</fig>
<table-wrap id="Tab1"><label>Table 1</label>
<caption><p>Classification and general features of <italic>Rhizobium leguminosarum</italic>
bv. <italic>trifolii</italic>
strain CC275e according to the MIGS recommendations [<xref ref-type="bibr" rid="CR34">34</xref>
]</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th>MIGS ID</th>
<th>Property</th>
<th>Term</th>
<th>Evidence codes<sup>a</sup>
</th>
</tr>
</thead>
<tbody><tr><td></td>
<td>Current classification</td>
<td>Domain <italic>Bacteria</italic>
</td>
<td>TAS [<xref ref-type="bibr" rid="CR35">35</xref>
]</td>
</tr>
<tr><td></td>
<td></td>
<td>Phylum <italic>Proteobacteria</italic>
</td>
<td>TAS [<xref ref-type="bibr" rid="CR36">36</xref>
]</td>
</tr>
<tr><td></td>
<td></td>
<td>Class <italic>Alphaproteobacteria</italic>
</td>
<td>TAS [<xref ref-type="bibr" rid="CR37">37</xref>
]</td>
</tr>
<tr><td></td>
<td></td>
<td>Order <italic>Rhizobiales</italic>
</td>
<td>TAS [<xref ref-type="bibr" rid="CR38">38</xref>
]</td>
</tr>
<tr><td></td>
<td></td>
<td>Family <italic>Rhizobiaceae</italic>
</td>
<td>TAS [<xref ref-type="bibr" rid="CR15">15</xref>
]</td>
</tr>
<tr><td></td>
<td></td>
<td>Genus <italic>Rhizobium</italic>
</td>
<td>TAS [<xref ref-type="bibr" rid="CR15">15</xref>
]</td>
</tr>
<tr><td></td>
<td></td>
<td>Species <italic>Rhizobium leguminosarum</italic>
</td>
<td>TAS [<xref ref-type="bibr" rid="CR14">14</xref>
]</td>
</tr>
<tr><td></td>
<td></td>
<td>Strain CC275e</td>
<td>TAS [<xref ref-type="bibr" rid="CR12">12</xref>
]</td>
</tr>
<tr><td></td>
<td>Gram Stain</td>
<td>Negative</td>
<td>TAS [<xref ref-type="bibr" rid="CR15">15</xref>
]</td>
</tr>
<tr><td></td>
<td>Cell Shape</td>
<td>Rod</td>
<td>TAS [<xref ref-type="bibr" rid="CR15">15</xref>
]</td>
</tr>
<tr><td></td>
<td>Motility</td>
<td>Motile</td>
<td>TAS [<xref ref-type="bibr" rid="CR15">15</xref>
]</td>
</tr>
<tr><td></td>
<td>Sporulation</td>
<td>Non spore-forming</td>
<td>TAS [<xref ref-type="bibr" rid="CR15">15</xref>
]</td>
</tr>
<tr><td></td>
<td>Temperature range</td>
<td>Mesophile</td>
<td>TAS [<xref ref-type="bibr" rid="CR15">15</xref>
]</td>
</tr>
<tr><td></td>
<td>Optimum temperature</td>
<td>28 °C</td>
<td>NAS</td>
</tr>
<tr><td></td>
<td>pH range; optimum</td>
<td>Unknown</td>
<td>NAS</td>
</tr>
<tr><td></td>
<td>Carbon source</td>
<td>Varied, chemoorganotrophic</td>
<td>TAS [<xref ref-type="bibr" rid="CR15">15</xref>
]</td>
</tr>
<tr><td>MIGS-6</td>
<td>Habitat</td>
<td>Soil, root nodule</td>
<td>TAS [<xref ref-type="bibr" rid="CR12">12</xref>
]</td>
</tr>
<tr><td>MIGS–6.3</td>
<td>Salinity</td>
<td>Non–halophile</td>
<td>NAS</td>
</tr>
<tr><td>MIGS–22</td>
<td>Oxygen requirement</td>
<td>Aerobic</td>
<td>TAS [<xref ref-type="bibr" rid="CR15">15</xref>
]</td>
</tr>
<tr><td>MIGS–15</td>
<td>Biotic relationship</td>
<td>Free living, legume symbiotic</td>
<td>TAS [<xref ref-type="bibr" rid="CR15">15</xref>
]</td>
</tr>
<tr><td>MIGS–14</td>
<td>Pathogenicity</td>
<td>Non–pathogen</td>
<td>TAS [<xref ref-type="bibr" rid="CR15">15</xref>
, <xref ref-type="bibr" rid="CR39">39</xref>
]</td>
</tr>
<tr><td>MIGS–4</td>
<td>Geographic location</td>
<td>Tasmania, Australia</td>
<td>TAS [<xref ref-type="bibr" rid="CR12">12</xref>
]</td>
</tr>
<tr><td>MIGS–5</td>
<td>Sample collection date</td>
<td>1966</td>
<td>TAS [<xref ref-type="bibr" rid="CR12">12</xref>
]</td>
</tr>
<tr><td>MIGS–4.1</td>
<td>Latitude</td>
<td>Not recorded</td>
<td></td>
</tr>
<tr><td>MIGS–4.2</td>
<td>Longitude</td>
<td>Not recorded</td>
<td></td>
</tr>
<tr><td>MIGS–4.3</td>
<td>Depth</td>
<td>Not recorded</td>
<td></td>
</tr>
<tr><td>MIGS–4.4</td>
<td>Altitude</td>
<td>Not recorded</td>
<td></td>
</tr>
</tbody>
</table>
<table-wrap-foot><p><sup>a</sup>
Evidence codes – <italic>IDA</italic>
Inferred from Direct Assay, <italic>TAS</italic>
Traceable Author Statement (i.e., a direct report exists in the literature), <italic>NAS</italic>
Non–traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [<xref ref-type="bibr" rid="CR34">34</xref>
]</p>
</table-wrap-foot>
</table-wrap>
</p>
<sec id="Sec4"><title>Symbiotaxonomy</title>
<p><ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
<italic>bv trifolii</italic>
strain CC275e is nodule forming (Nod<sup>+</sup>
) and N<sub>2</sub>
fixing (Fix<sup>+</sup>
) on a range of annual and perennial clover host species. The original isolation of <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
<italic>bv trifolii</italic>
strain CC275e was from <ext-link ext-link-type="uri" xlink:href="http://www.theplantlist.org/tpl1.1/record/ild-8135"><italic>Trifolium repens</italic>
</ext-link>
L. collected from Montague, North Western Tasmania [<xref ref-type="bibr" rid="CR12">12</xref>
], and has been used commercially due to its efficacy at forming symbioses and fixation of nitrogen on white clover hosts [<xref ref-type="bibr" rid="CR9">9</xref>
]. The strain is also moderately effective (<italic>sensu</italic>
Brockwell et al. [<xref ref-type="bibr" rid="CR12">12</xref>
]) on <italic>T. fragiferum</italic>
L. (strawberry clover; perennial), and <italic>T. michelianum</italic>
Savi<italic>,</italic>
(balansa clover; annual). On <italic>T. subterraneum</italic>
L. (subterranean clover; annual), <italic>T. purpureum</italic>
Lois. (purple clover; annual), and <italic>T. hirtum</italic>
All. (rose clover; annual), strain CC275e has been described as effective [<xref ref-type="bibr" rid="CR12">12</xref>
].</p>
</sec>
</sec>
</sec>
<sec id="Sec5"><title>Genome sequencing information</title>
<sec id="Sec6"><title>Genome project history</title>
<p><ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
<italic>bv trifolii</italic>
strain CC275e was selected for sequencing based on its long history of commercial use as an inoculant for various clover (<italic>Trifolium</italic>
spp.) hosts in Australia and New Zealand. In symbiosis with clover, this strain of bacteria has provided biologically-fixed nitrogen into soils for several decades, and thereby contributed to the fertility and productivity of pastoral agricultural systems in two countries. As part of a New Zealand MBIE-funded program, ‘Improving forage legume-rhizobia performance’ (C10X1308), the genomics of elite host nodulating (nod<sup>+</sup>
) and N<sub>2</sub>
fixing (fix<sup>+</sup>
) strains are being compared with closely related, ineffective strains. The aim is to identify markers to facilitate rhizobia selection programs, and to provide experimental tools for host colonization/competition experiments. Based on efforts in other <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
<italic>bv trifolii</italic>
strains (see accessions listed in the introduction) a sequencing strategy was developed using a predicted genome size of approximately 7 Mb. The genome sequencing and assembly was completed in 2014; summary information on the project is given in Table <xref rid="Tab2" ref-type="table">2</xref>
. The final <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
<italic>bv trifolii</italic>
CC275e genome assembly is a high-quality draft on 29 scaffolds, and resulted from approximately 150× sequencing coverage.<table-wrap id="Tab2"><label>Table 2</label>
<caption><p>Genome sequencing project information for <italic>Rhizobium leguminosarum</italic>
bv. trifolii strain CC275e</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th>MIGS ID</th>
<th>Property</th>
<th>Term</th>
</tr>
</thead>
<tbody><tr><td>MIGS-31</td>
<td>Finishing quality</td>
<td>High-quality draft</td>
</tr>
<tr><td>MIGS-28</td>
<td>Libraries Used</td>
<td>Illumina TruSeq™ DNA Sample Preparation Kit V2, 2 × 150 bp paired end library</td>
</tr>
<tr><td>MIGS-29</td>
<td>Sequencing platform</td>
<td>Illumina MiSeq™</td>
</tr>
<tr><td>MIGS-31.2</td>
<td>Fold coverage</td>
<td>3.75 million reads, ≈150 × genome coverage</td>
</tr>
<tr><td>MIGS-30</td>
<td>Assemblers</td>
<td>A5, SSPACE, Velvet Optimiser</td>
</tr>
<tr><td>MIGS-32</td>
<td>Gene calling method</td>
<td>Glimmer 3</td>
</tr>
<tr><td></td>
<td>Locus Tag</td>
<td></td>
</tr>
<tr><td></td>
<td>Genbank ID</td>
<td>JRXL00000000</td>
</tr>
<tr><td></td>
<td>Genbank Date of Release</td>
<td>27st October, 2014</td>
</tr>
<tr><td></td>
<td>GOLD ID</td>
<td>Gp0113226</td>
</tr>
<tr><td></td>
<td>BIOPROJECT</td>
<td>259682</td>
</tr>
<tr><td>MIGS-13</td>
<td>Source Material Identifier</td>
<td>ATCC 35181</td>
</tr>
<tr><td></td>
<td>Project relevance</td>
<td>Symbiotic N<sub>2</sub>
fixation, agriculture</td>
</tr>
</tbody>
</table>
</table-wrap>
</p>
</sec>
<sec id="Sec7"><title>Growth conditions and genomic DNA preparation</title>
<p>A loop of a single colony of <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
<italic>bv trifolii</italic>
CC275e was inoculated into YM broth [<xref ref-type="bibr" rid="CR13">13</xref>
] and grown to mid-log phase via incubation at 28 °C at 200 rpm for 12 h. DNA was extracted from the cell culture using a Gentra Puregene Cell kit (Qiagen). Spectrophotometry was used to quantify the DNA and ensure quality was sufficient for sequencing analysis (Nanodrop Thermo Scientific).</p>
</sec>
<sec id="Sec8"><title>Genome sequencing and assembly</title>
<p>Genome sequencing was conducted through NZGL (contract NZGL00940) at Massey University (MGS). Sequencing was performed on an Illumina MiSeq<sup>TM</sup>
instrument (details in Table <xref rid="Tab2" ref-type="table">2</xref>
), using 2 × 150 bp paired-end (PE) library with an average insert size of 420 bp. The sequencing run generated 3,751,285 reads totaling 1088 Mb of data.</p>
<p>Reads were assembled using the Java Assembling and Scaffolding Tool (JAST; [<xref ref-type="bibr" rid="CR16">16</xref>
]). Quality control of the sequence reads was conducted in Flexbar [<xref ref-type="bibr" rid="CR17">17</xref>
], and initial <italic>de novo</italic>
assembly in A5 [<xref ref-type="bibr" rid="CR18">18</xref>
]; this resulted in 52 contigs. Bowtie2 [<xref ref-type="bibr" rid="CR19">19</xref>
] and Velvet [<xref ref-type="bibr" rid="CR20">20</xref>
] were further used to optimize the assembly, using the genome of the closely strain <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
strain <ext-link ext-link-type="uri" xlink:href="http://doi.org/10.1601/strainfinder?urlappend=%3Fid%3DWSM1325">WSM1325</ext-link>
(Fig. <xref rid="Fig2" ref-type="fig">2</xref>
) as a reference (NCBI accession 241202755). SSPACE [<xref ref-type="bibr" rid="CR21">21</xref>
] was used to assemble the 35 contigs into 29 scaffolds (Table <xref rid="Tab3" ref-type="table">3</xref>
). Summary details of the sequencing process are given in Table <xref rid="Tab2" ref-type="table">2</xref>
.<table-wrap id="Tab3"><label>Table 3</label>
<caption><p>Genome statistics for <italic>Rhizobium leguminosarum</italic>
bv. <italic>trifolii</italic>
strain CC275e</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th>Attribute</th>
<th>Value</th>
<th>% of total</th>
</tr>
</thead>
<tbody><tr><td>Genome size (bp)</td>
<td>7,077,367</td>
<td char="." align="char">100.00</td>
</tr>
<tr><td>DNA coding (bp)</td>
<td>6,201,447</td>
<td char="." align="char">87.62</td>
</tr>
<tr><td>DNA G + C (bp)</td>
<td>4,306,744</td>
<td char="." align="char">60.90</td>
</tr>
<tr><td>DNA scaffolds</td>
<td>29</td>
<td></td>
</tr>
<tr><td>Total genes</td>
<td>6747</td>
<td char="." align="char">100.00</td>
</tr>
<tr><td>Protein coding genes</td>
<td>6693</td>
<td char="." align="char">99.00</td>
</tr>
<tr><td>RNA genes</td>
<td>54</td>
<td char="." align="char">0.80</td>
</tr>
<tr><td>Pseudo genes</td>
<td>not determined</td>
<td char="." align="char">not determined</td>
</tr>
<tr><td>Genes in internal clusters</td>
<td>not determined</td>
<td char="." align="char">not determined</td>
</tr>
<tr><td>Genes with function prediction</td>
<td>5018</td>
<td char="." align="char">74.37</td>
</tr>
<tr><td>Genes assigned to COGs</td>
<td>5722</td>
<td char="." align="char">84.80</td>
</tr>
<tr><td>Genes with Pfam domains</td>
<td>5682</td>
<td char="." align="char">84.22</td>
</tr>
<tr><td>Genes with signal peptides</td>
<td>531</td>
<td char="." align="char">7.87</td>
</tr>
<tr><td>Genes with transmembrane helices</td>
<td>1584</td>
<td char="." align="char">23.48</td>
</tr>
<tr><td>CRISPR repeats</td>
<td>0</td>
<td></td>
</tr>
</tbody>
</table>
</table-wrap>
</p>
</sec>
<sec id="Sec9"><title>Genome annotation</title>
<p>Annotation was added by the NCBI Prokaryotic Genome Annotation Pipeline (<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/genome/annotation_prok/">http://www.ncbi.nlm.nih.gov/genome/annotation_prok/</ext-link>
). Clusters of orthologous groups of proteins (COGs) were predicted using COGnitor [<xref ref-type="bibr" rid="CR22">22</xref>
], and the presence of signal peptides was detected using SignalP [<xref ref-type="bibr" rid="CR23">23</xref>
]. Pfam domains were predicted using HMMER [<xref ref-type="bibr" rid="CR24">24</xref>
] against the Pfam-A database [<xref ref-type="bibr" rid="CR25">25</xref>
]. Transmembrane predictions and CRISPR repeats were found in Genious [<xref ref-type="bibr" rid="CR26">26</xref>
] using the Transmembrane prediction (<ext-link ext-link-type="uri" xlink:href="http://www.geneious.com/plugins/transmembrane-prediction-plugin">http://www.geneious.com/plugins/transmembrane-prediction-plugin</ext-link>
) and CRT plugins [<xref ref-type="bibr" rid="CR27">27</xref>
] respectively.</p>
</sec>
</sec>
<sec id="Sec10"><title>Genome properties</title>
<p>The genome of <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
<italic>bv trifolii</italic>
strain CC275e is estimated to be 7,077,367 nucleotides in size (Table <xref rid="Tab3" ref-type="table">3</xref>
). The GC content is 60.9 % which is similar to closely related strains such as <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
<italic>bv trifolii</italic>
strain TA1 (60.74 %; [<xref ref-type="bibr" rid="CR28">28</xref>
]). The final draft consists of 29 scaffolds, the largest of which is 1,609,666 bp and the smallest 1167 bp. In total, 6747 genes were identified, 99 % of these were protein coding and the rest rRNA genes (Table <xref rid="Tab3" ref-type="table">3</xref>
). The majority of protein coding genes (84.22 %) have functionality predicted against COG categories; these are listed in Table <xref rid="Tab4" ref-type="table">4</xref>
. The remainder are listed as hypothetical.<table-wrap id="Tab4"><label>Table 4</label>
<caption><p>Number of protein coding genes of <italic>Rhizobium leguminosarum</italic>
bv. <italic>trifolii</italic>
strain CC275e associated with the general COG functional categories</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th>Code</th>
<th>Value</th>
<th>% of total</th>
<th>COG category</th>
</tr>
</thead>
<tbody><tr><td>J</td>
<td>189</td>
<td char="." align="char">2.69</td>
<td>Translation</td>
</tr>
<tr><td>A</td>
<td>0</td>
<td char="." align="char">0.00</td>
<td>RNA processing and modification</td>
</tr>
<tr><td>K</td>
<td>624</td>
<td char="." align="char">8.88</td>
<td>Transcription</td>
</tr>
<tr><td>L</td>
<td>186</td>
<td char="." align="char">2.65</td>
<td>Replication</td>
</tr>
<tr><td>B</td>
<td>2</td>
<td char="." align="char">0.03</td>
<td>Chromatin structure and dynamics</td>
</tr>
<tr><td>D</td>
<td>38</td>
<td char="." align="char">0.54</td>
<td>Cell cycle control</td>
</tr>
<tr><td>Y</td>
<td>0</td>
<td char="." align="char">0.00</td>
<td>Nuclear structure</td>
</tr>
<tr><td>V</td>
<td>64</td>
<td char="." align="char">0.91</td>
<td>Defense mechanisms</td>
</tr>
<tr><td>T</td>
<td>361</td>
<td char="." align="char">5.14</td>
<td>Signal transduction mechanisms</td>
</tr>
<tr><td>M</td>
<td>297</td>
<td char="." align="char">4.23</td>
<td>Cell wall/membrane/ biogenesis</td>
</tr>
<tr><td>N</td>
<td>96</td>
<td char="." align="char">1.37</td>
<td>Cell motility</td>
</tr>
<tr><td>Z</td>
<td>0</td>
<td char="." align="char">0.00</td>
<td>Cytoskeleton</td>
</tr>
<tr><td>W</td>
<td>0</td>
<td char="." align="char">0.00</td>
<td>Extracellular structures</td>
</tr>
<tr><td>U</td>
<td>74</td>
<td char="." align="char">1.05</td>
<td>Intracellular trafficking</td>
</tr>
<tr><td>O</td>
<td>185</td>
<td char="." align="char">2.63</td>
<td>Posttranslational modification</td>
</tr>
<tr><td>C</td>
<td>295</td>
<td char="." align="char">4.20</td>
<td>Energy production and conversion</td>
</tr>
<tr><td>G</td>
<td>646</td>
<td char="." align="char">9.19</td>
<td>Carbohydrate transport and metabolism</td>
</tr>
<tr><td>E</td>
<td>672</td>
<td char="." align="char">9.56</td>
<td>Amino acid transport and metabolism</td>
</tr>
<tr><td>F</td>
<td>108</td>
<td char="." align="char">1.54</td>
<td>Nucleotide transport and metabolism</td>
</tr>
<tr><td>H</td>
<td>151</td>
<td char="." align="char">2.15</td>
<td>Coenzyme transport and metabolism</td>
</tr>
<tr><td>I</td>
<td>238</td>
<td char="." align="char">3.39</td>
<td>Lipid transport and metabolism</td>
</tr>
<tr><td>P</td>
<td>234</td>
<td char="." align="char">3.33</td>
<td>Inorganic ion transport and metabolism</td>
</tr>
<tr><td>Q</td>
<td>95</td>
<td char="." align="char">1.35</td>
<td>Secondary metabolites biosynthesis</td>
</tr>
<tr><td>R</td>
<td>623</td>
<td char="." align="char">8.87</td>
<td>General function prediction only</td>
</tr>
<tr><td>S</td>
<td>544</td>
<td char="." align="char">7.74</td>
<td>Function unknown</td>
</tr>
<tr><td>-</td>
<td>1305</td>
<td char="." align="char">18.57</td>
<td>Not in COGs</td>
</tr>
</tbody>
</table>
</table-wrap>
</p>
<p>Analysis of the genome by Eckhart gel electrophoresis [<xref ref-type="bibr" rid="CR29">29</xref>
] (Fig. <xref rid="Fig3" ref-type="fig">3</xref>
) revealed the presence of six mega-plasmids. Mega-plasmids are typical of the ‘ancillary genome’ present in many <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
strains [<xref ref-type="bibr" rid="CR30">30</xref>
] and commonly host many of the recognition factors associated with host compatibility, and nitrogen fixation. Based on the known mega-plasmid profile of <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
<italic>bv trifolii</italic>
strain WSM1325 (Fig. <xref rid="Fig3" ref-type="fig">3</xref>
), the mega-plasmids in <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
<italic>bv trifolii</italic>
strain CC275e are approximately >1000, 500, 280, 280, 150, and 140 kb in size. As yet it is unknown to which scaffolds these mega-plasmids are associated.<fig id="Fig3"><label>Fig. 3</label>
<caption><p>Eckhardt gel electropherogram showing ‘mega-plasmid’ profiles of <italic>R. leguminosarum bv trifolii</italic>
strain CC275e against strains TA1 and WSM1325. The bright central band for strain CC275e represents co-migration of two similarly sized plasmids. Also, note double band at bottom of strain CC275e lane profile. The size of plasmids in reference strain WSM1325 are 294, 350, 516, and 829, 661, 516, 350, and 294 kb</p>
</caption>
<graphic xlink:href="40793_2015_110_Fig3_HTML" id="MO3"></graphic>
</fig>
</p>
</sec>
<sec id="Sec11"><title>Conclusions</title>
<p><ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1279"><italic>Rhizobium</italic>
</ext-link>
<italic>leguminosarium</italic>
bv. <italic>trifolii</italic>
bacteria are an important resource for agricultural production [<xref ref-type="bibr" rid="CR1">1</xref>
, <xref ref-type="bibr" rid="CR2">2</xref>
, <xref ref-type="bibr" rid="CR4">4</xref>
]. In symbiosis with a suitable legume host (legume root nodules), atmospheric nitrogen fixed by these bacteria provides a source of plant nutrition that increases the farming system fertility in an economically and environmentally sustainable manner. Strains of <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
<italic>bv trifolii</italic>
vary in host-compatibility between legume species [<xref ref-type="bibr" rid="CR5">5</xref>
], and their nitrogen fixation efficacy when in symbiosis [<xref ref-type="bibr" rid="CR6">6</xref>
]. Understanding the genetic factors controlling these, and other phenotypes such as saprophytic survival, and desiccation tolerance, will enable increased utilization of <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
<italic>bv trifolii</italic>
for farming systems. The strain <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
<italic>bv trifolii</italic>
strain CC275e has been commercially used as an inoculant for white-clover for several decades [<xref ref-type="bibr" rid="CR9">9</xref>
]. The genome sequencing of this ‘highly efficacious’ bacterium, allows for the identification of genetic factors associated with desirable phenotypes (see previous). This will be achieved by comparison of the <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1601/nm.1280"><italic>R. leguminosarum</italic>
</ext-link>
<italic>bv trifolii</italic>
strain CC275e with closely related stains (e.g. based on 16S rRNA similarity) that differ in one or more phenotypes.</p>
</sec>
</body>
<back><app-group><app id="App1"><sec id="Sec12"><title>Additional file</title>
<p><media position="anchor" xlink:href="40793_2015_110_MOESM1_ESM.xlsx" id="MOESM1"><label>Additional file 1: Table S1.</label>
<caption><p>List of strain names and associated NCBI GenBank accession numbers for bacterial isolates in Fig. <xref rid="Fig2" ref-type="fig">2</xref>
. (XLSX 13 kb)</p>
</caption>
</media>
</p>
</sec>
</app>
</app-group>
<glossary><title>Abbreviations</title>
<def-list><def-item><term>CSIRO</term>
<def><p>Commonwealth scientific and industrial research organisation</p>
</def>
</def-item>
<def-item><term>Fix<sup>+</sup>
</term>
<def><p>Nitrogen fixation positive</p>
</def>
</def-item>
<def-item><term>NZGL</term>
<def><p>New Zealand genomics Ltd</p>
</def>
</def-item>
<def-item><term>Nod<sup>+</sup>
</term>
<def><p>Nodulation positive</p>
</def>
</def-item>
<def-item><term>MGS</term>
<def><p>Massey genome service</p>
</def>
</def-item>
<def-item><term>MBIE</term>
<def><p>Ministry of business, innovation and employment</p>
</def>
</def-item>
<def-item><term><italic>R. leguminosarum bv trifolii</italic>
</term>
<def><p><italic>Rhizobium leguminosarum</italic>
symbiovar <italic>trifolii</italic>
</p>
</def>
</def-item>
<def-item><term>TEM</term>
<def><p>Transmission electron microscopy</p>
</def>
</def-item>
<def-item><term>YM</term>
<def><p>Yeast mannitol</p>
</def>
</def-item>
</def-list>
</glossary>
<fn-group><fn><p><bold>Competing interests</bold>
</p>
<p>The authors declare that they have no competing interests.</p>
</fn>
<fn><p><bold>Authors’ contributions</bold>
</p>
<p>SW, HR, CR, MO, BB, RB, and AG conceived of this study, participated in design, and helped draft the manuscript. CD and AL conducted genome assembly and associated bioinformatic analysis. SY, CB, and EG coordinated and conducted all microbiology, cell handling for TEM, DNA extraction and purification, and Eckhardt gel electrophoresis. All authors read and approved the final manuscript.</p>
</fn>
</fn-group>
<ack><title>Acknowledgements</title>
<p>This work was funded through the New Zealand MBIE and DairyNZ funded programme “Improving forage legume-rhizobia performance” (C10X1308). Clément Delestre acknowledges the AgResearch bioinformatics team for internship funding. Sequencing was performed by MGS, and was coordinated by Lorraine Berry under NZGL contract NZGL00940 within the public-good funding stream. Sample QC, library QC, and library preparation was performed by Xiaoxiao Lin, sequencing by Richard Fong, and data QC by Mauro Truglio. Prof. Michael Hynes (University of Calgary) provided useful knowledge on <italic>R. leguminosarum</italic>
mega-plasmids. TEM was conducted by Dr Duane Harland and James Vernon (AgResearch).</p>
</ack>
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