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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Abundant Degenerate Miniature Inverted-Repeat Transposable Elements in Genomes of Epichloid Fungal Endophytes of Grasses</title><author><name sortKey="Fleetwood, Damien J" sort="Fleetwood, Damien J" uniqKey="Fleetwood D" first="Damien J." last="Fleetwood">Damien J. Fleetwood</name><affiliation><nlm:aff id="aff1">Forage Biotechnology Section, AgResearch, Palmerston North, New Zealand</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">School of Biological Sciences, University of Auckland, Auckland, New Zealand</nlm:aff></affiliation></author><author><name sortKey="Khan, Anar K" sort="Khan, Anar K" uniqKey="Khan A" first="Anar K." last="Khan">Anar K. Khan</name><affiliation><nlm:aff id="aff3">Bioinformatics, Mathematics and Statistics Section, AgResearch, Dunedin, New Zealand</nlm:aff></affiliation></author><author><name sortKey="Johnson, Richard D" sort="Johnson, Richard D" uniqKey="Johnson R" first="Richard D." last="Johnson">Richard D. Johnson</name><affiliation><nlm:aff id="aff1">Forage Biotechnology Section, AgResearch, Palmerston North, New Zealand</nlm:aff></affiliation></author><author><name sortKey="Young, Carolyn A" sort="Young, Carolyn A" uniqKey="Young C" first="Carolyn A." last="Young">Carolyn A. Young</name><affiliation><nlm:aff id="aff4">Forage Improvement, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma</nlm:aff></affiliation></author><author><name sortKey="Mittal, Shipra" sort="Mittal, Shipra" uniqKey="Mittal S" first="Shipra" last="Mittal">Shipra Mittal</name><affiliation><nlm:aff id="aff4">Forage Improvement, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma</nlm:aff></affiliation></author><author><name sortKey="Wrenn, Ruth E" sort="Wrenn, Ruth E" uniqKey="Wrenn R" first="Ruth E." last="Wrenn">Ruth E. Wrenn</name><affiliation><nlm:aff id="aff5">Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand</nlm:aff></affiliation></author><author><name sortKey="Hesse, Uljana" sort="Hesse, Uljana" uniqKey="Hesse U" first="Uljana" last="Hesse">Uljana Hesse</name><affiliation><nlm:aff id="aff6">Department of Plant Pathology, University of Kentucky, Lexington</nlm:aff></affiliation></author><author><name sortKey="Foster, Simon J" sort="Foster, Simon J" uniqKey="Foster S" first="Simon J." last="Foster">Simon J. Foster</name><affiliation><nlm:aff id="aff5">Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand</nlm:aff></affiliation><affiliation><nlm:aff id="aff7">Present address: Sainsbury Laboratory, John Innes Research Centre, Norwich, United Kingdom</nlm:aff></affiliation></author><author><name sortKey="Schardl, Christopher L" sort="Schardl, Christopher L" uniqKey="Schardl C" first="Christopher L." last="Schardl">Christopher L. Schardl</name><affiliation><nlm:aff id="aff6">Department of Plant Pathology, University of Kentucky, Lexington</nlm:aff></affiliation></author><author><name sortKey="Scott, Barry" sort="Scott, Barry" uniqKey="Scott B" first="Barry" last="Scott">Barry Scott</name><affiliation><nlm:aff id="aff5">Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">21948396</idno><idno type="pmc">3227409</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3227409</idno><idno type="RBID">PMC:3227409</idno><idno type="doi">10.1093/gbe/evr098</idno><date when="2011">2011</date><idno type="wicri:Area/Pmc/Corpus">000181</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000181</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Abundant Degenerate Miniature Inverted-Repeat Transposable Elements in Genomes of Epichloid Fungal Endophytes of Grasses</title><author><name sortKey="Fleetwood, Damien J" sort="Fleetwood, Damien J" uniqKey="Fleetwood D" first="Damien J." last="Fleetwood">Damien J. Fleetwood</name><affiliation><nlm:aff id="aff1">Forage Biotechnology Section, AgResearch, Palmerston North, New Zealand</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">School of Biological Sciences, University of Auckland, Auckland, New Zealand</nlm:aff></affiliation></author><author><name sortKey="Khan, Anar K" sort="Khan, Anar K" uniqKey="Khan A" first="Anar K." last="Khan">Anar K. Khan</name><affiliation><nlm:aff id="aff3">Bioinformatics, Mathematics and Statistics Section, AgResearch, Dunedin, New Zealand</nlm:aff></affiliation></author><author><name sortKey="Johnson, Richard D" sort="Johnson, Richard D" uniqKey="Johnson R" first="Richard D." last="Johnson">Richard D. Johnson</name><affiliation><nlm:aff id="aff1">Forage Biotechnology Section, AgResearch, Palmerston North, New Zealand</nlm:aff></affiliation></author><author><name sortKey="Young, Carolyn A" sort="Young, Carolyn A" uniqKey="Young C" first="Carolyn A." last="Young">Carolyn A. Young</name><affiliation><nlm:aff id="aff4">Forage Improvement, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma</nlm:aff></affiliation></author><author><name sortKey="Mittal, Shipra" sort="Mittal, Shipra" uniqKey="Mittal S" first="Shipra" last="Mittal">Shipra Mittal</name><affiliation><nlm:aff id="aff4">Forage Improvement, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma</nlm:aff></affiliation></author><author><name sortKey="Wrenn, Ruth E" sort="Wrenn, Ruth E" uniqKey="Wrenn R" first="Ruth E." last="Wrenn">Ruth E. Wrenn</name><affiliation><nlm:aff id="aff5">Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand</nlm:aff></affiliation></author><author><name sortKey="Hesse, Uljana" sort="Hesse, Uljana" uniqKey="Hesse U" first="Uljana" last="Hesse">Uljana Hesse</name><affiliation><nlm:aff id="aff6">Department of Plant Pathology, University of Kentucky, Lexington</nlm:aff></affiliation></author><author><name sortKey="Foster, Simon J" sort="Foster, Simon J" uniqKey="Foster S" first="Simon J." last="Foster">Simon J. Foster</name><affiliation><nlm:aff id="aff5">Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand</nlm:aff></affiliation><affiliation><nlm:aff id="aff7">Present address: Sainsbury Laboratory, John Innes Research Centre, Norwich, United Kingdom</nlm:aff></affiliation></author><author><name sortKey="Schardl, Christopher L" sort="Schardl, Christopher L" uniqKey="Schardl C" first="Christopher L." last="Schardl">Christopher L. Schardl</name><affiliation><nlm:aff id="aff6">Department of Plant Pathology, University of Kentucky, Lexington</nlm:aff></affiliation></author><author><name sortKey="Scott, Barry" sort="Scott, Barry" uniqKey="Scott B" first="Barry" last="Scott">Barry Scott</name><affiliation><nlm:aff id="aff5">Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand</nlm:aff></affiliation></author></analytic><series><title level="j">Genome Biology and Evolution</title><idno type="eISSN">1759-6653</idno><imprint><date when="2011">2011</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Miniature inverted-repeat transposable elements (MITEs) are abundant repeat elements in plant and animal genomes; however, there are few analyses of these elements in fungal genomes. Analysis of the draft genome sequence of the fungal endophyte <italic>Epichloë festucae</italic> revealed 13 MITE families that make up almost 1% of the <italic>E. festucae</italic> genome, and relics of putative autonomous parent elements were identified for three families. Sequence and DNA hybridization analyses suggest that at least some of the MITEs identified in the study were active early in the evolution of <italic>Epichloë</italic> but are not found in closely related genera. Analysis of MITE integration sites showed that these elements have a moderate integration site preference for 5′ genic regions of the <italic>E. festucae</italic> genome and are particularly enriched near genes for secondary metabolism. Copies of the EFT-3m/Toru element appear to have mediated recombination events that may have abolished synthesis of two fungal alkaloids in different epichloae. This work provides insight into the potential impact of MITEs on <italic>epichloae</italic> evolution and provides a foundation for analysis in other fungal genomes.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Benjak, A" uniqKey="Benjak A">A Benjak</name></author><author><name sortKey="Boue, S" uniqKey="Boue S">S Boue</name></author><author><name sortKey="Forneck, A" uniqKey="Forneck A">A Forneck</name></author><author><name sortKey="Casacuberta, Jm" uniqKey="Casacuberta J">JM Casacuberta</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Benson, G" uniqKey="Benson G">G Benson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bergemann, M" uniqKey="Bergemann M">M Bergemann</name></author><author><name sortKey="Lespinet, O" uniqKey="Lespinet O">O Lespinet</name></author><author><name sortKey="M Arek, Sb" uniqKey="M Arek S">SB M’Barek</name></author><author><name 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Genome Biol Evol</journal-id><journal-id journal-id-type="hwp">gbe</journal-id><journal-id journal-id-type="publisher-id">gbe</journal-id><journal-title-group><journal-title>Genome Biology and Evolution</journal-title></journal-title-group><issn pub-type="epub">1759-6653</issn><publisher><publisher-name>Oxford University Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">21948396</article-id><article-id pub-id-type="pmc">3227409</article-id><article-id pub-id-type="doi">10.1093/gbe/evr098</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Articles</subject></subj-group></article-categories><title-group><article-title>Abundant Degenerate Miniature Inverted-Repeat Transposable Elements in Genomes of Epichloid Fungal Endophytes of Grasses</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Fleetwood</surname><given-names>Damien J.</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="author-notes" rid="fn1">†</xref></contrib><contrib contrib-type="author"><name><surname>Khan</surname><given-names>Anar K.</given-names></name><xref ref-type="aff" rid="aff3">3</xref><xref ref-type="author-notes" rid="fn1">†</xref></contrib><contrib contrib-type="author"><name><surname>Johnson</surname><given-names>Richard D.</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Young</surname><given-names>Carolyn A.</given-names></name><xref ref-type="aff" rid="aff4">4</xref></contrib><contrib contrib-type="author"><name><surname>Mittal</surname><given-names>Shipra</given-names></name><xref ref-type="aff" rid="aff4">4</xref></contrib><contrib contrib-type="author"><name><surname>Wrenn</surname><given-names>Ruth E.</given-names></name><xref ref-type="aff" rid="aff5">5</xref></contrib><contrib contrib-type="author"><name><surname>Hesse</surname><given-names>Uljana</given-names></name><xref ref-type="aff" rid="aff6">6</xref></contrib><contrib contrib-type="author"><name><surname>Foster</surname><given-names>Simon J.</given-names></name><xref ref-type="aff" rid="aff5">5</xref><xref ref-type="aff" rid="aff7">7</xref></contrib><contrib contrib-type="author"><name><surname>Schardl</surname><given-names>Christopher L.</given-names></name><xref ref-type="aff" rid="aff6">6</xref></contrib><contrib contrib-type="author"><name><surname>Scott</surname><given-names>Barry</given-names></name><xref ref-type="aff" rid="aff5">5</xref></contrib></contrib-group><aff id="aff1"><label>1</label>Forage Biotechnology Section, AgResearch, Palmerston North, New Zealand</aff><aff id="aff2"><label>2</label>School of Biological Sciences, University of Auckland, Auckland, New Zealand</aff><aff id="aff3"><label>3</label>Bioinformatics, Mathematics and Statistics Section, AgResearch, Dunedin, New Zealand</aff><aff id="aff4"><label>4</label>Forage Improvement, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma</aff><aff id="aff5"><label>5</label>Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand</aff><aff id="aff6"><label>6</label>Department of Plant Pathology, University of Kentucky, Lexington</aff><aff id="aff7"><label>7</label>Present address: Sainsbury Laboratory, John Innes Research Centre, Norwich, United Kingdom</aff><author-notes><corresp id="cor1"><label>*</label>Corresponding author: E-mail: <email>d.fleetwood@auckland.ac.nz</email>.</corresp><fn id="fn1"><label>†</label><p>These authors contributed equally to this work.</p></fn><fn><p><bold>Associate editor:</bold> Ya -Ping Zhang</p></fn></author-notes><pub-date pub-type="ppub"><year>2011</year></pub-date><pub-date pub-type="epub"><day>26</day><month>9</month><year>2011</year></pub-date><pub-date pub-type="collection"><year>2011</year></pub-date><pub-date pub-type="pmc-release"><day>26</day><month>9</month><year>2011</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on the
<volume>3</volume><fpage>1253</fpage><lpage>1264</lpage><history><date date-type="accepted"><day>20</day><month>9</month><year>2011</year></date></history><permissions><copyright-statement>© The Author(s) 2011. Published by Oxford University Press on behalf of the <italic>Society for Molecular Biology and Evolution.</italic></copyright-statement><copyright-year>2011</copyright-year><license license-type="creative-commons" xlink:href="http://creativecommons.org/licenses/by-nc/3.0"><license-p><pmc-comment>CREATIVE COMMONS</pmc-comment>This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc/3.0">http://creativecommons.org/licenses/by-nc/3.0</ext-link>), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><abstract><p>Miniature inverted-repeat transposable elements (MITEs) are abundant repeat elements in plant and animal genomes; however, there are few analyses of these elements in fungal genomes. Analysis of the draft genome sequence of the fungal endophyte <italic>Epichloë festucae</italic> revealed 13 MITE families that make up almost 1% of the <italic>E. festucae</italic> genome, and relics of putative autonomous parent elements were identified for three families. Sequence and DNA hybridization analyses suggest that at least some of the MITEs identified in the study were active early in the evolution of <italic>Epichloë</italic> but are not found in closely related genera. Analysis of MITE integration sites showed that these elements have a moderate integration site preference for 5′ genic regions of the <italic>E. festucae</italic> genome and are particularly enriched near genes for secondary metabolism. Copies of the EFT-3m/Toru element appear to have mediated recombination events that may have abolished synthesis of two fungal alkaloids in different epichloae. This work provides insight into the potential impact of MITEs on <italic>epichloae</italic> evolution and provides a foundation for analysis in other fungal genomes.</p></abstract><kwd-group><kwd><italic>Epichloë</italic></kwd><kwd>transposable element</kwd><kwd>endophyte</kwd><kwd>genome evolution</kwd><kwd>fungi</kwd></kwd-group></article-meta></front><body><sec><title>Introduction</title><p>Transposable elements are characterized by their ability to move, or transpose, within genomes and are ubiquitous in all kingdoms of life. Transposons have a substantial impact on genome function and evolution: transposition of these “selfish” elements can lead to mutation by insertion within genes and can alter transcription by removal or addition of <italic>cis</italic> elements or by epigenetic mechanisms (<xref ref-type="bibr" rid="bib26">Kidwell and Lisch 1997</xref>; <xref ref-type="bibr" rid="bib16">Feschotte 2008</xref>). The repeat sequences generated by transposon movement and expansion can also be responsible for local and global genome rearrangements (<xref ref-type="bibr" rid="bib18">Fierro and Martin 1999</xref>; <xref ref-type="bibr" rid="bib32">Mieczkowski et al. 2006</xref>).</p><p>Transposable elements have been divided into two classes. Type 1 elements, or retroelements, transpose through an RNA intermediate, whereas type 2, or DNA transposons, mostly utilize a “cut and paste” mechanism of transposition. Miniature inverted-repeat transposable elements (MITEs) are nonautonomous DNA (type 2) transposable elements that require the transposase from an autonomous parent element for transposition (<xref ref-type="bibr" rid="bib17">Feschotte et al. 2002</xref>). Like autonomous DNA transposons, MITEs are characterized by terminal inverted repeats (TIRs) and a target site duplication (TSD). However, unlike autonomous elements, MITEs have no coding capacity, and unlike other deleted elements, MITEs amplify to high copy number and copies are homogeneous in size (usually <500 bp) (<xref ref-type="bibr" rid="bib17">Feschotte et al. 2002</xref>).</p><p>Some MITEs appear to be direct deletion derivatives of autonomous copies (<xref ref-type="bibr" rid="bib22">Jiang et al. 2003</xref>), whereas in many other cases, MITEs appear to evolve independently by recombination events that lead to a pair of TIRs sufficiently similar to those of an autonomous element to be able to be mobilized by its transposase (<xref ref-type="bibr" rid="bib23">Jiang et al. 2004</xref>). It has long been a puzzle as to how these deleted elements are able to amplify to a much higher copy number than their parents. A recent landmark study showed that the <italic>Stowaway</italic> MITE in rice does not contain a repressor element present in the autonomous <italic>Mariner</italic> elements (<xref ref-type="bibr" rid="bib50">Yang et al. 2009</xref>). In addition, the <italic>Stowaway</italic> MITE has an enhancer of transposition that further facilitates its ability to amplify over <italic>Mariner</italic>. Although MITEs arising from simple deletion are unlikely to contain an enhancer, deletion of repressor elements may be a common mode of copy number amplification of these elements.</p><p>In higher eukaryotes, MITEs can make up a large proportion of the genome repeat content, especially in plants where a substantial proportion of the genome can consist of these elements (<xref ref-type="bibr" rid="bib39">Santiago et al. 2002</xref>; <xref ref-type="bibr" rid="bib23">Jiang et al. 2004</xref>; <xref ref-type="bibr" rid="bib24">Juretic et al. 2004</xref>; <xref ref-type="bibr" rid="bib1">Benjak et al. 2009</xref>). In plants thus far examined, MITEs have an integration site bias for genic regions of the genome (e.g., <xref ref-type="bibr" rid="bib6">Bureau and Wessler 1994a</xref>, <xref ref-type="bibr" rid="bib7">1994b</xref>; <xref ref-type="bibr" rid="bib28">Mao et al. 2000</xref>) and thus likely influence expression of associated genes. MITEs in fungi have received little attention, with just two families being characterized: <italic>Guest</italic> in <italic>Neurospora crassa</italic> (<xref ref-type="bibr" rid="bib51">Yeadon and Catcheside 1995</xref>; <xref ref-type="bibr" rid="bib38">Ramussen et al. 2004</xref>) and <italic>mimp</italic> in <italic>Fusarium oxysporum</italic> (<xref ref-type="bibr" rid="bib21">Hua-Van et al. 2000</xref>; <xref ref-type="bibr" rid="bib13">Dufresne et al. 2007</xref>; <xref ref-type="bibr" rid="bib3">Bergemann et al. 2008</xref>). However, recently, a number of uncharacterized MITEs have been reported in fungal genome sequences (<xref ref-type="bibr" rid="bib31">Martin et al. 2008</xref>; <xref ref-type="bibr" rid="bib42">Spanu et al. 2010</xref>).</p><p>We previously identified five MITE-like elements present within secondary metabolite gene clusters in epichloid fungi (<italic>Epichloë</italic> and <italic>Neotyphodium</italic> species: Ascomycota, Sordariomycetes, Hypocreales, Clavicipitaceae). These fungi are endophytic symbionts of grasses, producing alkaloids that protect the host plant from herbivory by insects and grazing animals. Annotation of the <italic>EAS</italic> gene cluster for ergot alkaloid synthesis in <italic>Neotyphodium lolii</italic> identified two MITEs, Toru and Rima (<xref ref-type="bibr" rid="bib20">Fleetwood et al. 2007</xref>). Examination of the <italic>LTM</italic> gene cluster for lolitrem B biosynthesis revealed three futher MITEs, labeled EFT-14, EFT-24, and EFT-25 (<xref ref-type="bibr" rid="bib54">Young et al. 2009</xref>). The presence of five putative MITEs in such a restricted sequence analysis led to the hypothesis that these elements are abundant components of epichloae genomes.</p><p>Here we describe the presence of 13 families of degenerate MITEs in the 34.4-Mb draft genome sequence of <italic>Epichloë</italic><italic> festucae</italic> E2368. We show that at least some of these families were present in the common ancestor of the epichloae lineage, that overall MITEs show a bias for integration within 5′ regions of genes, and are particularly enriched near secondary metabolism genes. We further describe the probable impact of EFT-3m elements on rearrangements and deletions at two secondary metabolite gene loci, highlighting the possibly large impact of these elements on genome evolution of epichloid fungi.</p></sec><sec sec-type="materials|methods"><title>Materials and Methods</title><sec><title>Fungal Strains</title><p>Strains used for computational, sequence, and Southern blot analysis are described in <xref ref-type="table" rid="tbl1">table 1</xref>. Fungi were grown in potato dextrose broth or agar at 22 °C.</p><table-wrap id="tbl1" position="float"><label>Table 1</label><caption><p>Fungal Strains Used in This Study</p></caption><table frame="hsides" rules="groups"><thead><tr><td rowspan="1" colspan="1">Fungal Strain</td><td align="center" rowspan="1" colspan="1">Parentage<xref ref-type="table-fn" rid="tblfn1">a</xref></td><td align="center" rowspan="1" colspan="1">ATCC# or Reference</td></tr></thead><tbody><tr><td rowspan="1" colspan="1"><italic>Epichloë clarkii</italic> E426</td><td align="center" rowspan="1" colspan="1">n/a</td><td rowspan="1" colspan="1">ATCC 200741(<xref ref-type="bibr" rid="bib33">Moon et al. 2004</xref>)</td></tr><tr><td rowspan="1" colspan="1"><italic>E. festucae</italic> E2368</td><td align="center" rowspan="1" colspan="1">n/a</td><td rowspan="1" colspan="1">C. Schardl, University of Kentucky, KY</td></tr><tr><td rowspan="1" colspan="1"><italic>E. festucae</italic> Fl1</td><td align="center" rowspan="1" colspan="1">n/a</td><td rowspan="1" colspan="1">ATCC MYA-3407 (<xref ref-type="bibr" rid="bib52">Young et al. 2005</xref>; <xref ref-type="bibr" rid="bib34">Moon et al. 1999</xref>)</td></tr><tr><td rowspan="1" colspan="1"><italic>E. sylvatica</italic> E503</td><td align="center" rowspan="1" colspan="1">n/a</td><td rowspan="1" colspan="1">ATCC 200751 (<xref ref-type="bibr" rid="bib33">Moon et al. 2004</xref>)</td></tr><tr><td rowspan="1" colspan="1"><italic>E. typhina</italic> E8</td><td align="center" rowspan="1" colspan="1">n/a</td><td rowspan="1" colspan="1">ATCC 200736 (<xref ref-type="bibr" rid="bib11">Chung et al. 1997</xref>)</td></tr><tr><td rowspan="1" colspan="1"><italic>Neotyphodium coenophialum e19</italic></td><td align="center" rowspan="1" colspan="1">Efe × ETC × LAE</td><td rowspan="1" colspan="1">ATCC 90664 (<xref ref-type="bibr" rid="bib44">Tsai et al. 1992</xref>)</td></tr><tr><td rowspan="1" colspan="1"><italic>N. lolii</italic> Lp19</td><td align="center" rowspan="1" colspan="1">Efe</td><td rowspan="1" colspan="1">(<xref ref-type="bibr" rid="bib10">Christensen et al. 1993</xref>)</td></tr><tr><td rowspan="1" colspan="1"><italic>N. lolii</italic> AR1</td><td align="center" rowspan="1" colspan="1">Efe</td><td rowspan="1" colspan="1">(<xref ref-type="bibr" rid="bib34">Moon et al. 1999</xref>)</td></tr><tr><td rowspan="1" colspan="1"><italic>N. lolii</italic> Lp14 (AKA AR37)</td><td align="center" rowspan="1" colspan="1">Efe</td><td rowspan="1" colspan="1">(<xref ref-type="bibr" rid="bib10">Christensen et al. 1993</xref>)</td></tr><tr><td rowspan="1" colspan="1"><italic>Neotyphodium</italic> sp. Lp1</td><td align="center" rowspan="1" colspan="1">Efe × ETC</td><td rowspan="1" colspan="1">(<xref ref-type="bibr" rid="bib10">Christensen et al. 1993</xref>)</td></tr><tr><td rowspan="1" colspan="1"><italic>N. uncinatum</italic> e167</td><td align="center" rowspan="1" colspan="1">Ebr × ETC</td><td rowspan="1" colspan="1">(<xref ref-type="bibr" rid="bib4">Blankenship et al. 2001</xref>)</td></tr><tr><td rowspan="1" colspan="1"><italic>Claviceps cynodontis</italic> Haskell</td><td align="center" rowspan="1" colspan="1">n/a</td><td rowspan="1" colspan="1">(<xref ref-type="bibr" rid="bib30">Marek et al. 2006</xref>)</td></tr><tr><td rowspan="1" colspan="1"><italic>Fusarium graminearum</italic></td><td align="center" rowspan="1" colspan="1">n/a</td><td rowspan="1" colspan="1">Unnamed, K. Craven, Noble Foundation, OK</td></tr><tr><td rowspan="1" colspan="1"><italic>Phymatotrichopsis omnivora</italic> OKAlf8</td><td align="center" rowspan="1" colspan="1">n/a</td><td rowspan="1" colspan="1">(<xref ref-type="bibr" rid="bib29">Marek et al. 2009</xref>)</td></tr></tbody></table><table-wrap-foot><fn><p>N<sc>ote.</sc>—n/a, not applicable.</p></fn><fn id="tblfn1"><label>a</label><p>Closest sexual ancestors to asexual species (<xref ref-type="bibr" rid="bib33">Moon et al. 2004</xref>). Ebr, <italic>E. bromicola</italic>; Ef, <italic>E. festucae</italic>; ETC, <italic>E. typhina</italic> complex (=<italic>E. typhina, E. sylvatica, E. clarkii</italic>); LAE, <italic>Lolium</italic>-associated endophyte (closest extant species = <italic>E. baconii</italic>).</p></fn></table-wrap-foot></table-wrap></sec><sec><title>Identification of MITEs in the <italic>E. festucae</italic> Genome Sequence</title><p>MITEs were computationally mined from the <italic>E. festucae</italic> genome in two parts: 1) identification of seed MITE sequences used to create libraries of hidden Markov models (HMMs) representing distinct MITE families and subfamilies and 2) searching of HMM libraries against the genome to comprehensively identify and classify MITE instances, including degraded, nested, or autonomous elements, and to support analysis of insertion sites. Bioinformatics analyses were implemented using a combination of various software and custom Perl scripts.</p><p>The <italic>E. festucae</italic> E2368 genome contigs (version 200606) were first masked for the following classes of repeats to prevent repetitive regions resulting in spurious MITE candidates: simple repeats and known fungal repeats (RepeatMasker 3.2.8, <ext-link ext-link-type="uri" xlink:href="http://www.repeatmasker.org">http://www.repeatmasker.org</ext-link>, cited 2011 Oct 7), microsatellites (Sputnik, <ext-link ext-link-type="uri" xlink:href="http://espressosoftware.com/sputnik/index.html">http://espressosoftware.com/sputnik/index.html</ext-link>, cited 2011 Oct 7), tandem repeats TRF 4.00 (<xref ref-type="bibr" rid="bib2">Benson 1999</xref>), and low complexity regions (dust; <xref ref-type="bibr" rid="bib35">Morgulis et al. 2006</xref>). Seed MITEs were identified from the masked genome using two successive rounds of Vmatch (<xref ref-type="bibr" rid="bib27">Kurtz et al. 2001</xref>) to identify TIRs (TIR length range 10–65 bp, ≥80% identity, maximum inter-TIR distance 650 bp). TIRs identified by the first Vmatch round were used to demarcate approximate MITE-containing regions; in the second round, each region was submitted to an individual Vmatch search using the same criteria after masking inter-TIR regions with <italic>N</italic>s, which focused the palindrome search to the terminal ends of the regions. The additional search round served two purposes: 1) refinement of the seed MITE boundaries because we observed that Vmatch sometimes extended match regions in the second round and 2) collapsing of multiple palindromes within the same region to a single seed MITE at each locus. Seed MITE sequences were extracted from the genome and clustered into putative families using an all-against-all basic alignment search tool (Blast) with sensitive discontiguous Blast parameters, which forced matches to be seeded within TIRs (-e 1 × 10<sup>−</sup><sup>10</sup> -b 10000 -v 10000 -U T -F “m D” -r 1 -q -1 -G 2 -E 2 -W 9 -m 9), followed by clustering using the Markov Cluster algorithm (MCL; <xref ref-type="bibr" rid="bib46">van Dongen 2000</xref>) using Blast similarity scores (normalized bit scores) as the similarity criterion. Clusters with <10 members were discarded, and clusters remaining were deemed to represent putative MITE families. Seed MITE sequences within each family were aligned using TCoffee (<xref ref-type="bibr" rid="bib36">Notredame et al. 2000</xref>) and visualized using JalView (<xref ref-type="bibr" rid="bib47">Waterhouse et al. 2009</xref>). To focus on conservation within TIRs and improve alignment in these regions, additional alignments containing seed MITEs with masked inter-TIR regions (replaced by 5 <italic>N</italic>s) were generated.</p><p>To identify subfamily structure, we attempted sequence-based clustering within families using MCL or hierarchical agglomerative clustering (hclust in the R statistical package; <ext-link ext-link-type="uri" xlink:href="http://www.R-project.org">http://www.R-project.org</ext-link>, cited 2011 Oct 7) but did not recover the manually identified subfamily structure of the Toru family, most likely due to degeneracy of subfamily members. Therefore, we manually partitioned families into putative subfamilies based on element lengths. The subfamilies were then aligned using Muscle (<xref ref-type="bibr" rid="bib15">Edgar 2004</xref>) for visualization in JalView. Flanking regions (50 bp either side) were extracted and used to create separate alignments to examine similarity within their genome contexts. Those subfamilies with marginal conservation or conserved flanking regions were discarded.</p><p>To comprehensively identify MITE instances in the genome, libraries of subfamily HMMs were constructed from the seed MITE subfamily alignments using hmmbuild from the HMMER2 package (<xref ref-type="bibr" rid="bib14">Eddy 1998</xref>). Two libraries were created: “global” containing global HMMs aimed at finding complete elements and “local” containing local HMMs aimed at finding deleted instances (fragments of elements which have most likely arisen from deletions occurring within full-length elements over time). We included deleted instances in our analysis and also instances that did not contain both putative TSD sequences. This means there may be a low level of false-positive instances for some families; however, we were willing to accept this in order to perform a comprehensive analysis of the highly degenerate MITEs in the <italic>E. festucae</italic> genome. The libraries were compared with the masked <italic>E. festucae</italic> genome contigs using hmmpfam (from HMMER2), explicitly searching both forward and reverse strands in separate searches; positive, nonzero scoring hits were flagged as candidate MITE instances. Multiple, overlapping MITEs at a single genomic locus were reduced to a single representative by selecting the instance with the best score (note that a global match always trumped a local match). We used hmmalign (from HMMER2) to build subfamily alignments using the appropriate model to guide the alignment so that it best represented any structural characteristics of that subfamily. Alignments including flanking sequences were used to manually correct element boundaries. These corrected elements represented the final collection of MITE instances, used for subsequent analyses. In addition, subfamily consensus sequences were identified using hmmemit (from HMMER2), and TIR coordinates on consensus were found using Vmatch.</p><p>The collection of MITE instances were postprocessed to identify nested MITEs and possible parent autonomous elements because criteria used in the original Vmatch search would not identify TIRs separated by extraordinary distances, which could arise due to displacement of TIRs by nested elements or an autonomous state prior to any significant deletion of the inter-TIR region. Proximal MITE TIRs were linked (assumed to be derived from a single element) if they belonged to the same subfamily and lay within 4 kb in the correct orientation relative to one another.</p><p>A track showing MITE positions in the <italic>E. festucae</italic> Gbrowse is available at the <italic>E. festucae</italic> Genome Project webpage (<ext-link ext-link-type="uri" xlink:href="http://www.endophyte.uky.edu/">http://www.endophyte.uky.edu/</ext-link>, cited 2011 Oct 7).</p></sec><sec><title>Integration Site Analysis</title><p>To identify whether MITEs are preferentially located near genes, insertion sites were compared with locations of open reading frames (ORFs) extracted from messenger RNA models (version 2, available on request), first converting genome contig coordinates of MITEs to positions on scaffolded supercontigs (<italic>E. festucae</italic> genome assembly version 200606). Some spurious computationally generated ORFs were removed by manual curation. An instance was classified as “near” an ORF if it was within 500 bp of an ORF boundary, excluding ranges occupied by other proximal MITEs to simulate the state of the genome prior to any insertion events and avoid penalizing MITEs clustered near ORFs. Counts of MITEs observed near an ORF, near an ORF 5′ end, and near an ORF 3′ end were submitted to two-tailed binomial tests to determine the significance of these observations compared with random insertion. The null probability (<italic>P</italic>) for random insertion was derived by dividing the number of genome positions in near regions by the total number of positions in the genome, excluding positions spanned by MITEs and other ORFs in both cases. Proximity of MITE insertion sites to 41 manually annotated nonribosomal peptide synthetase (NRPS) and polyketide synthetase (PKS) genes was examined in a similar way. Fungal secondary metabolite genes are usually found in tight gene clusters and thus in the absence of other annotated secondary metabolism genes “near” was defined as within 10 kb of an NRPS/PKS ORF, as an approximation of any secondary metabolite gene cluster regions.</p></sec><sec><title>DNA Extraction and Southern Blot Analysis</title><p>Genomic DNA from <italic>Epichloë</italic> spp., <italic>Neotyphodium</italic> spp., <italic>N. crassa</italic>, <italic>Fusarium graminearum</italic>, <italic>Claviceps cynodontis</italic>, and <italic>Phymatotrichopsis omnivora</italic> was isolated from freeze-dried mycelium using ZR Fungal/Bacterial DNA kit (Zymo Research), Plant DNeasy kit (Qiagen, Hilden, Germany), or a published method (<xref ref-type="bibr" rid="bib8">Byrd et al. 1990</xref>). Genomic DNA (2 μg) was digested overnight at 37 °C with 48 units of <italic>Eco</italic>RI (Promega). Digested genomic DNA was separated overnight in 0.7% agarose gel and transferred overnight to nylon membranes (Zeta probe blotting membrane, BioRad) by capillary transfer. Membranes were UV cross-linked (120,000 μJ/cm<sup>2</sup>) in UV stratalinker 2400 (Stratagene). The EFT-14 MITE element was amplified from <italic>E. festucae</italic> genomic DNA using primers EFT-14F, 5′-GTGAGACAGATATATCAGGCACA-3′, and EFT-14R, 5′-GATTTAAGACGGATTGGAATGATG-3′. Sequence-specific polymerase chain reaction (PCR) was carried out in a reaction volume of 50 μl containing 5 ng <italic>E. festucae</italic> genomic DNA, 1× green reaction buffer (Promega), 200 μM of each deoxyribonucleotide triphosphate, 200 nM of each primer, and 1 U GoTaq (Promega). Thermocycling conditions were 94 °C for 2 min, followed by 35 cycles of 94 °C for 15 s, 55 °C for 30 s, 72 °C for 1 min, and then a final extension at 72 °C for 10 min. PCR products were purified using a PCR purification kit (Qiagen). Probe labeling and hybridization were performed using Amersham gene images AlkPhos direct labeling and detection system (GE Healthcare). Hybridizations were carried out overnight at 50 °C in AlkPhos Direct hybridization buffer. Posthybridization washes were performed according to the manufacturer’s instructions (GE Healthcare) using CDP-Star (GE Healthcare) for chemiluminescent signal generation. Blots were exposed to Biomax XAR film (Kodak) for 30 min to 24 h depending on signal strength.</p></sec><sec><title>PCR and DNA Sequencing</title><p>For targeted sequencing of repeat regions, PCR products were amplified using either Pfx50 (Invitrogen) for the <italic>N. lolii</italic> AR1 and <italic>Neotyphodium</italic> sp. Lp1 <italic>easA</italic><italic>–easG</italic> region or Triplemaster (Eppendorf) for the <italic>N. lolii</italic> Lp14 <italic>perA</italic> 3′ region. PCR products were either sequenced directly or cloned into p-GEMT and sequenced by M13 and custom primers. Dye terminator sequencing was performed using BigDye v3.1 (Applied Biosystems) and separated on either an ABI3130XL or ABI3730 capillary sequencer (Applied Biosystems) at the University of Auckland Centre for Genomics and Proteomics or the Massey University Alan Wilson Centre Genome Service, respectively.</p><p>Sequences obtained in this study are available at GenBank under accessions JF494831 (AR1 <italic>easA</italic>–<italic>easG</italic>) and GU966659 (Lp14 <italic>perA</italic>).</p></sec></sec><sec><title>Results</title><sec><title>Identification and Characterization of 13 MITE Families</title><p>Five MITEs previously identified in epichloid endophytes were found to be highly degenerate. For this reason, we predicted that existing algorithms for MITE identification, which rely on high similarity between copies (<xref ref-type="bibr" rid="bib45">Tu 2001</xref>) or known TIR sequences (<xref ref-type="bibr" rid="bib39">Santiago et al. 2002</xref>; <xref ref-type="bibr" rid="bib3">Bergemann et al. 2008</xref>), were not suitable for use in <italic>E. festucae</italic>. We thus developed a computational pipeline utilizing various programs to identify MITEs ab initio.</p><p>A list of candidate MITEs was identified from the <italic>E. festucae</italic> genome by searching for inverted-repeat sequences using Vmatch (<ext-link ext-link-type="uri" xlink:href="http://www.gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evr098/-/DC1">supplementary data</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evr098/-/DC1">Supplementary Material</ext-link> online). Candidates were then sorted into 79 putative MITE families based on similarity in inverted-repeat regions. Alignments of clustered MITE sequences were examined manually, and 66 of these were discarded due to poor overall similarity or due to extensive similarity in the sequences flanking the inverted repeats, indicative of the inverted repeat being within a larger repeat element. We thus examined 13 putative MITE families further. These were named EFT-[number]m, with “m” standing for MITE. Previously identified MITEs, Toru and Rima, were labeled EFT-3m/Toru and EFT-5m/Rima, respectively. Characteristics of the MITE families are described in <xref ref-type="table" rid="tbl2">table 2</xref>; consensus sequences are available as <ext-link ext-link-type="uri" xlink:href="http://www.gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evr098/-/DC1">supplementary data</ext-link> (<ext-link ext-link-type="uri" xlink:href="http://www.gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evr098/-/DC1">Supplementary Material</ext-link> online). Mean pairwise identity within each family varied but was in most cases low, varying between 49% and 90%. Accurate genomic copy numbers of elements were obtained, including counting of degraded and deleted elements which were likely to be missed by the Vmatch search, by searching a MITE library of HMMs representing MITE families/subfamilies against the <italic>E. festucae</italic> genome. Copy number varied, with the highest copy subfamily, EFT-3A, containing 97 full-length elements. Two families contained fewer than 10 full-length copies; however, we considered them as MITEs alongside the high copy elements as in each case there were substantially more deleted instances in the genome sequence and fungal genomes are relatively small in comparison to the plants for which the 10-copy criterion was considered applicable for MITE categorization (<xref ref-type="bibr" rid="bib17">Feschotte et al. 2002</xref>). Combining all families, a total of 1,249 MITE instances were identified (415 full length and 834 deleted).</p><table-wrap id="tbl2" position="float"><label>Table 2</label><caption><p>Characteristics of MITEs in the <italic>Epichloë</italic><italic> festucae</italic> Genome</p></caption><table frame="hsides" rules="groups"><thead><tr><td rowspan="1" colspan="1">Family</td><td align="center" rowspan="1" colspan="1">Mean Length (range)</td><td align="center" rowspan="1" colspan="1">Mean Pairwise %ID</td><td align="center" rowspan="1" colspan="1">TSD</td><td align="center" rowspan="1" colspan="1">TIR Length (%ID)</td><td align="center" rowspan="1" colspan="1">#Full Copies (#Deleted)<xref ref-type="table-fn" rid="tblfn2">a</xref></td><td align="center" rowspan="1" colspan="1">Putative Superfamily</td><td align="center" rowspan="1" colspan="1">Parent Element?</td></tr></thead><tbody><tr><td rowspan="1" colspan="1">EFT-3mA/Toru</td><td align="char" char="(" rowspan="1" colspan="1">135 (109–177)</td><td align="char" char="." rowspan="1" colspan="1">75</td><td align="center" rowspan="1" colspan="1">AT</td><td align="char" char="(" rowspan="1" colspan="1">29 (96)</td><td align="char" char="(" rowspan="1" colspan="1">97 (47)</td><td rowspan="1" colspan="1">Unknown</td><td align="center" rowspan="1" colspan="1">No</td></tr><tr><td rowspan="1" colspan="1">EFT-3mB</td><td align="char" char="(" rowspan="1" colspan="1">256 (218–389)</td><td align="char" char="." rowspan="1" colspan="1">67</td><td align="center" rowspan="1" colspan="1">AT</td><td align="char" char="(" rowspan="1" colspan="1">29 (96)</td><td align="char" char="(" rowspan="1" colspan="1">24 (63)</td><td rowspan="1" colspan="1">Unknown</td><td align="center" rowspan="1" colspan="1">No</td></tr><tr><td rowspan="1" colspan="1">EFT-5m/Rima</td><td align="char" char="(" rowspan="1" colspan="1">294 (259–356)</td><td align="char" char="." rowspan="1" colspan="1">62</td><td align="center" rowspan="1" colspan="1">TA<xref ref-type="table-fn" rid="tblfn3">b</xref></td><td align="char" char="(" rowspan="1" colspan="1">61 (86)</td><td align="char" char="(" rowspan="1" colspan="1">7 (14)</td><td rowspan="1" colspan="1">Tc1/<italic>mariner</italic></td><td align="center" rowspan="1" colspan="1">No</td></tr><tr><td rowspan="1" colspan="1">EFT-8m</td><td align="char" char="(" rowspan="1" colspan="1">401 (364–567)</td><td align="char" char="." rowspan="1" colspan="1">76</td><td align="center" rowspan="1" colspan="1">TWY</td><td align="char" char="(" rowspan="1" colspan="1">34 (91)</td><td align="char" char="(" rowspan="1" colspan="1">12 (98)</td><td rowspan="1" colspan="1">Pif/<italic>Harbinger</italic></td><td align="center" rowspan="1" colspan="1">Yes</td></tr><tr><td rowspan="1" colspan="1">EFT-9m</td><td align="char" char="(" rowspan="1" colspan="1">246 (139–535)</td><td align="char" char="." rowspan="1" colspan="1">70</td><td align="center" rowspan="1" colspan="1">TA</td><td align="char" char="(" rowspan="1" colspan="1">34 (88)</td><td align="char" char="(" rowspan="1" colspan="1">61 (101)</td><td rowspan="1" colspan="1">Tc1/<italic>mariner</italic></td><td align="center" rowspan="1" colspan="1">No</td></tr><tr><td rowspan="1" colspan="1">EFT-11m</td><td align="char" char="(" rowspan="1" colspan="1">406 (393–410)</td><td align="char" char="." rowspan="1" colspan="1">86</td><td align="center" rowspan="1" colspan="1">8 bp</td><td align="char" char="(" rowspan="1" colspan="1">10 (100)</td><td align="char" char="(" rowspan="1" colspan="1">29 (106)</td><td rowspan="1" colspan="1"><italic>hAT</italic></td><td align="center" rowspan="1" colspan="1">No</td></tr><tr><td rowspan="1" colspan="1">EFT-14m</td><td align="char" char="(" rowspan="1" colspan="1">291 (193–434)</td><td align="char" char="." rowspan="1" colspan="1">68</td><td align="center" rowspan="1" colspan="1">TA</td><td align="char" char="(" rowspan="1" colspan="1">36 (91)</td><td align="char" char="(" rowspan="1" colspan="1">63 (116)</td><td rowspan="1" colspan="1">Tc1/<italic>mariner</italic></td><td align="center" rowspan="1" colspan="1">No</td></tr><tr><td rowspan="1" colspan="1">EFT-24m</td><td align="char" char="(" rowspan="1" colspan="1">380 (297–653)</td><td align="char" char="." rowspan="1" colspan="1">49</td><td align="center" rowspan="1" colspan="1">9 bp</td><td align="char" char="(" rowspan="1" colspan="1">115 (81)</td><td align="char" char="(" rowspan="1" colspan="1">31 (89)</td><td rowspan="1" colspan="1">Mutator</td><td align="center" rowspan="1" colspan="1">No</td></tr><tr><td rowspan="1" colspan="1">EFT-25m</td><td align="char" char="(" rowspan="1" colspan="1">81 (77–86)</td><td align="char" char="." rowspan="1" colspan="1">90</td><td align="center" rowspan="1" colspan="1">TA</td><td align="char" char="(" rowspan="1" colspan="1">24 (95)</td><td align="char" char="(" rowspan="1" colspan="1">14 (27)</td><td rowspan="1" colspan="1">Tc1/<italic>mariner</italic></td><td align="center" rowspan="1" colspan="1">Yes</td></tr><tr><td rowspan="1" colspan="1">EFT-26m</td><td align="char" char="(" rowspan="1" colspan="1">364 (287–470)</td><td align="char" char="." rowspan="1" colspan="1">69</td><td align="center" rowspan="1" colspan="1">9 bp</td><td align="char" char="(" rowspan="1" colspan="1">116 (93)</td><td align="char" char="(" rowspan="1" colspan="1">13 (83)</td><td rowspan="1" colspan="1">Mutator</td><td align="center" rowspan="1" colspan="1">Yes</td></tr><tr><td rowspan="1" colspan="1">EFT-27mA</td><td align="char" char="(" rowspan="1" colspan="1">148 (130–188)</td><td align="char" char="." rowspan="1" colspan="1">84</td><td align="center" rowspan="1" colspan="1">AT</td><td align="char" char="(" rowspan="1" colspan="1">38 (97)</td><td align="char" char="(" rowspan="1" colspan="1">14 (20)</td><td rowspan="1" colspan="1">Unknown</td><td align="center" rowspan="1" colspan="1">No</td></tr><tr><td rowspan="1" colspan="1">EFT-27mB</td><td align="char" char="(" rowspan="1" colspan="1">267 (254–402)</td><td align="char" char="." rowspan="1" colspan="1">67</td><td align="center" rowspan="1" colspan="1">AT</td><td align="char" char="(" rowspan="1" colspan="1">40 (87)</td><td align="char" char="(" rowspan="1" colspan="1">19 (14)</td><td rowspan="1" colspan="1">Unknown</td><td align="center" rowspan="1" colspan="1">No</td></tr><tr><td rowspan="1" colspan="1">EFT-28m</td><td align="char" char="(" rowspan="1" colspan="1">259 (207–292)</td><td align="char" char="." rowspan="1" colspan="1">65</td><td align="center" rowspan="1" colspan="1">AT<xref ref-type="table-fn" rid="tblfn4">c</xref></td><td align="char" char="(" rowspan="1" colspan="1">35 (91)</td><td align="char" char="(" rowspan="1" colspan="1">10 (30)</td><td rowspan="1" colspan="1">Unknown</td><td align="center" rowspan="1" colspan="1">No</td></tr><tr><td rowspan="1" colspan="1">EFT-29m<xref ref-type="table-fn" rid="tblfn5">d</xref></td><td align="char" char="(" rowspan="1" colspan="1">83 (57–108)</td><td align="char" char="." rowspan="1" colspan="1">83</td><td align="center" rowspan="1" colspan="1">TA</td><td align="char" char="(" rowspan="1" colspan="1">25 (88)</td><td align="char" char="(" rowspan="1" colspan="1">16 (13)</td><td rowspan="1" colspan="1">Tc1/<italic>mariner</italic></td><td align="center" rowspan="1" colspan="1">No</td></tr><tr><td rowspan="1" colspan="1">EFT-30m</td><td align="char" char="(" rowspan="1" colspan="1">524 (509–540)</td><td align="char" char="." rowspan="1" colspan="1">80</td><td align="center" rowspan="1" colspan="1">TA</td><td align="char" char="(" rowspan="1" colspan="1">43 (95)</td><td align="char" char="(" rowspan="1" colspan="1">5 (13)</td><td rowspan="1" colspan="1">Tc1/<italic>mariner</italic></td><td align="center" rowspan="1" colspan="1">No</td></tr></tbody></table><table-wrap-foot><fn><p>N<sc>ote.</sc>—ID, identity.</p></fn><fn id="tblfn2"><label>a</label><p>Copy number data are from publicly available genome assembly (EF201006); other data are from the version 200606 assembly that all other analysis was performed on.</p></fn><fn id="tblfn3"><label>b</label><p>Putative—degenerate at ends.</p></fn><fn id="tblfn4"><label>c</label><p>Only two with putative AT TSD although frequent AT at one end.</p></fn><fn id="tblfn5"><label>d</label><p>Closely related to EFT-25, internal 30–40 bp dissimilar, and TIRs imperfect.</p></fn></table-wrap-foot></table-wrap><p>To further categorize the MITE elements, we attempted to identify subfamilies within the family designations. The high level of degeneracy precluded identification of relationships from multiple sequence alignments; we thus, used a size-based criterion to identify subfamily relationships. Based on manual size separation and analysis of alignments, two families were identified that contained two subfamilies each, EFT-3m and EFT-27m. EFT-3mA and EFT-3mB differed only in size, whereas EFT-27mA and EFT-27mB shared only the 34 bp of the TIR with no similarity between intervening sequences.</p><p>In the absence of transposase sequences, superfamilies were predicted for each family based on TIR and putative TSD characteristics (<xref ref-type="bibr" rid="bib48">Wicker et al. 2007</xref>) (<xref ref-type="table" rid="tbl2">table 2</xref>). Six families belonged to the Tc1/<italic>mariner</italic> superfamily, consistent with Tc1/<italic>mariner</italic> being a common type 2 superfamily in fungi. Other MITE families were characterized as Mutator, Pif/<italic>Harbinger</italic>, and hAT superfamilies. Three families, EFT-3m, EFT-27m, and EFT-28m were classified as unknown. These families most closely resemble CACTA elements although in the case of EFT-3m and EFT-27m, the TIR terminal sequences (CTCC) did not exactly match the consensus found in either fungi (CCC, <xref ref-type="bibr" rid="bib12">DeMarco et al. 2006</xref>) or plants (CMCWR, <xref ref-type="bibr" rid="bib16">Feschotte 2008</xref>). EFT-28m TIRs do terminate in CCC, but a 2-bp TSD was not always present.</p></sec><sec><title>Degenerate Autonomous Elements EFT-8, EFT-25, and EFT-26</title><p>Considering that MITEs require autonomous elements for transposition, we analyzed the <italic>E. festucae</italic> genome for the presence of putative parent elements. Analysis of linked instances of adjacent deleted MITE sequences containing single TIRs in the correct orientation (<ext-link ext-link-type="uri" xlink:href="http://www.gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evr098/-/DC1">supplementary table S1</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evr098/-/DC1">Supplementary Material online</ext-link>) revealed putative autonomous element relics that are the likely progenitor sequences of EFT-8m, EFT-25m, and EFT-26m. Analysis of linked EFT-8m TIRs revealed a 3076-bp autonomous element relic (EFT-8) with two full-length copies and another four sequences longer than 50% of full length in the genome assembly. BlastX analysis of the GenBank nonredundant databases showed the predicted translation of a 794-bp region of EFT-8 to have 29% identity (<italic>E</italic> = 2 × 10<sup>−</sup><sup>13</sup>) with a hypothetical protein Os08g0459400 from <italic>Oryza sativa</italic> (rice) and similar percent identity but over a smaller region to numerous Pif-like transposases from various plants and fungi. This sequence contained 25 stop codons, and the high AT percentage of the full EFT-8 sequence (71%) suggested that the element had undergone repeat-induced point mutation (RIP; <xref ref-type="bibr" rid="bib9">Cambareri et al. 1989</xref>). RIP results in C:G to T:A transitions in repeat sequences and was observed for other identified autonomous elements in epichloae (<xref ref-type="bibr" rid="bib52">Young et al. 2005</xref>; <xref ref-type="bibr" rid="bib20">Fleetwood et al. 2007</xref>). Alignment of full-length and deleted sequences showed a strong bias for C to T and G to A transitions, supporting RIP as the main cause of the degeneration.</p><p>A putative autonomous EFT-25 relic was identified on contig 1003 as two deleted sequences of EFT-25m separated by 1,313 bp but not containing other nested elements. All three reading frames of the sequence between the TIRs contained numerous stop codons, and BlastX analysis did not match any transposase sequences in the databases; however, the sequence was highly AT rich (78%) and likely to be the degenerate product of RIP. Only a single copy of this putative autonomous relic of EFT-25 was found in the <italic>E. festucae</italic> genome, but eight sequences corresponding to greater than 10% of the full-length sequence were present. In all but one instance, these were truncated at the end of a small contig. Alignment of the EFT-25 sequence with EFT-25m showed the MITE to be a direct deletion derivative of the full-length element.</p><p>A putative autonomous EFT-26 was identified on contig 754, which contained 2709 bp between TIRs. BlastX analysis of this sequence revealed a 1,386-bp region sharing 29% identity (<italic>E</italic> = 3 × 10<sup>−</sup><sup>39</sup>) to a hypothetical protein in <italic>Chaetomium globosum</italic> (EAQ85500) and 32% identity over a smaller region to the <italic>Hop</italic> mutator transposase from <italic>F. oxysporum</italic> (AAP31248). This sequence contained far fewer stop codons (5) than the other two autonomous elements. This result and the lower AT percentage (55%) suggest this element has not been subjected to RIP to the same extent as the other two elements, perhaps suggesting a more recent origin. There was only a single full-length EFT-26 in the assembly with only one other non-MITE sequence aligning over 593 bp of the full-length element at the end of a small contig (contig 2450). Alignment of the full-length EFT-26 sequence with the EFT-26m consensus sequence showed that, as for the other two autonomous relics, the MITE is likely to be a deletion derivative of the full-length element.</p></sec><sec><title>Early MITE Invasion of Epichloid Genomes</title><p>The high sequence variation and large number of indels between copies of individual elements suggest that MITEs are ancient features of epichloid genomes. To determine the taxonomic extent of MITE colonization, we first searched the public databases for the presence of different families in published sequences from different <italic>Epichloë</italic> and <italic>Neotyphodium</italic> species not containing <italic>E. festucae</italic> parentage. We identified EFT-3m, EFT-11m, EFT-14m, EFT-24m, and EFT-26m in one or both of the two <italic>LOL</italic> gene clusters in <italic>N. uncinatum</italic> (<italic>E. typhina</italic> × <italic>E. bromicola</italic>) and EFT-11m in the <italic>LOL</italic> cluster of <italic>Neotyphodium</italic> sp. PauTG-1 (<italic>E. typhina</italic> × <italic>E. elymi</italic>) (<ext-link ext-link-type="uri" xlink:href="http://www.gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evr098/-/DC1">supplementary table S2</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evr098/-/DC1">Supplementary Material</ext-link> online).</p><p>To further examine the distribution of MITEs, we performed Southern blot analysis of a range of epichloae with EFT-14m (<xref ref-type="fig" rid="fig1">fig. 1</xref>). This element was found in similar copy number across the range of <italic>Epichloë</italic> species tested, further supporting an early origin in this genus. Weak nonspecific hybridization was observed for <italic>A. fumigatus</italic> and <italic>N. crassa</italic> (data not shown), but no hybridization was seen for <italic>C. cynodontis</italic>, a member of a clavicipitaceous genus closely related to <italic>Epichloë/Neotyphodium</italic> in recent analyses (<xref ref-type="bibr" rid="bib43">Sung et al. 2007</xref>). Due to the degeneracy of the MITE sequences and the subsequent low stringency hybridization conditions required, we were unable to obtain specific hybridization data for other MITEs tested, EFT-3m and EFT-9m.</p><fig id="fig1" position="float"><label>F<sc>IG</sc>. 1.—</label><caption><p>Taxonomic distribution of EFT-14m. Southern blot analysis was performed using genomic DNA extracted from various epichloae and closely related fungal species, transferred to a nylon membrane and hybridized with an AlkPhos direct labeled (GE Healthcare) EFT-14m probe amplified by PCR using primers EFT-14F and EFT-14R. 1, <italic>Fusarium graminearum</italic>; 2, <italic>Claviceps cynodontis</italic>; 3, <italic>Neotyphodium uncinatum</italic> E167; 4, <italic>Epichloe clarkii</italic> E426; 5, <italic>E. sylvatica</italic> E503, 6, <italic>E.</italic><italic> festucae</italic> E2368; 7, <italic>E. festucae</italic> Fl1, 8, <italic>E. typhina</italic> E8; 9, <italic>N. coenophialum</italic> E19; 10, <italic>Phymatotrichosis omnivora</italic>.</p></caption><graphic xlink:href="gbeevr098f01_ht"></graphic></fig></sec><sec><title>MITEs Are Enriched Upstream of Genes and Near Secondary Metabolite Genes</title><p>MITEs are often found in genic regions of genomes. To determine the precise integration sites of <italic>E. festucae</italic> MITEs, MITE locations on supercontigs, as annotated by the MITE library genome-wide search, were compared with the locations of ORFs. MITE instances were classified as to whether they had inserted near (within 500 bp) to an ORF and near to 5′ or 3′ ends of ORFs (<xref ref-type="table" rid="tbl3">table 3</xref>). MITEs were frequently found within 500 bp of a predicted ORF (<xref ref-type="table" rid="tbl3">table 3</xref>), consistent with similar analyses for other organisms. To further analyze how frequently MITEs are found upstream of ORFs in putative regulatory regions, MITE instances were classified as to whether they had inserted near (within 500 bp) to 5′ or 3′ ends of ORFs (<xref ref-type="table" rid="tbl3">table 3</xref>). Binomial tests (using random insertion in the genome not including MITEs as the null distribution) on these data indicated MITE insertions were somewhat enriched 5′ of ORFs (<italic>x</italic> = 357, <italic>n</italic> = 1,077, <italic>P</italic> = 0.3038, <italic>P</italic> value = 0.051) and were strongly depleted at 3′ ends of ORFs (<italic>x</italic> = 207, <italic>n</italic> = 1,077, <italic>P</italic> = 0.2771, <italic>P</italic> value = 1.34 × 10<sup>−</sup><sup>10</sup>).</p><table-wrap id="tbl3" position="float"><label>Table 3</label><caption><p>Integration Site Data for <italic>Epichloë</italic><italic> festucae</italic> MITEs</p></caption><table frame="hsides" rules="groups"><thead><tr><td rowspan="1" colspan="1">Family</td><td align="center" rowspan="1" colspan="1">Total Copies</td><td align="center" rowspan="1" colspan="1">% Within 500 bp of ORF</td><td align="center" rowspan="1" colspan="1">% Within 500 bp 5′ of ORF</td><td align="center" rowspan="1" colspan="1">% Within 500 bp 3′ of ORF</td><td align="center" rowspan="1" colspan="1">% Near SM Gene</td></tr></thead><tbody><tr><td rowspan="1" colspan="1">EFT-3mA/Toru</td><td align="char" char="." rowspan="1" colspan="1">126</td><td align="char" char="." rowspan="1" colspan="1">44</td><td align="char" char="." rowspan="1" colspan="1">33</td><td align="char" char="." rowspan="1" colspan="1">14</td><td align="char" char="." rowspan="1" colspan="1">5</td></tr><tr><td rowspan="1" colspan="1">EFT-3mB</td><td align="char" char="." rowspan="1" colspan="1">87</td><td align="char" char="." rowspan="1" colspan="1">44</td><td align="char" char="." rowspan="1" colspan="1">21</td><td align="char" char="." rowspan="1" colspan="1">28</td><td align="char" char="." rowspan="1" colspan="1">3</td></tr><tr><td rowspan="1" colspan="1">EFT-5m/Rima</td><td align="char" char="." rowspan="1" colspan="1">19</td><td align="char" char="." rowspan="1" colspan="1">47</td><td align="char" char="." rowspan="1" colspan="1">32</td><td align="char" char="." rowspan="1" colspan="1">26</td><td align="char" char="." rowspan="1" colspan="1">11</td></tr><tr><td rowspan="1" colspan="1">EFT-8m</td><td align="char" char="." rowspan="1" colspan="1">110</td><td align="char" char="." rowspan="1" colspan="1">32</td><td align="char" char="." rowspan="1" colspan="1">18</td><td align="char" char="." rowspan="1" colspan="1">17</td><td align="char" char="." rowspan="1" colspan="1">4</td></tr><tr><td rowspan="1" colspan="1">EFT-9m</td><td align="char" char="." rowspan="1" colspan="1">162</td><td align="char" char="." rowspan="1" colspan="1">49</td><td align="char" char="." rowspan="1" colspan="1">38</td><td align="char" char="." rowspan="1" colspan="1">16</td><td align="char" char="." rowspan="1" colspan="1">3</td></tr><tr><td rowspan="1" colspan="1">EFT-11m</td><td align="char" char="." rowspan="1" colspan="1">18</td><td align="char" char="." rowspan="1" colspan="1">28</td><td align="char" char="." rowspan="1" colspan="1">17</td><td align="char" char="." rowspan="1" colspan="1">11</td><td align="char" char="." rowspan="1" colspan="1">0</td></tr><tr><td rowspan="1" colspan="1">EFT-14m</td><td align="char" char="." rowspan="1" colspan="1">185</td><td align="char" char="." rowspan="1" colspan="1">50</td><td align="char" char="." rowspan="1" colspan="1">35</td><td align="char" char="." rowspan="1" colspan="1">24</td><td align="char" char="." rowspan="1" colspan="1">2</td></tr><tr><td rowspan="1" colspan="1">EFT-24m</td><td align="char" char="." rowspan="1" colspan="1">124</td><td align="char" char="." rowspan="1" colspan="1">44</td><td align="char" char="." rowspan="1" colspan="1">35</td><td align="char" char="." rowspan="1" colspan="1">15</td><td align="char" char="." rowspan="1" colspan="1">2</td></tr><tr><td rowspan="1" colspan="1">EFT-25m</td><td align="char" char="." rowspan="1" colspan="1">40</td><td align="char" char="." rowspan="1" colspan="1">33</td><td align="char" char="." rowspan="1" colspan="1">25</td><td align="char" char="." rowspan="1" colspan="1">15</td><td align="char" char="." rowspan="1" colspan="1">0</td></tr><tr><td rowspan="1" colspan="1">EFT-26m</td><td align="char" char="." rowspan="1" colspan="1">96</td><td align="char" char="." rowspan="1" colspan="1">47</td><td align="char" char="." rowspan="1" colspan="1">32</td><td align="char" char="." rowspan="1" colspan="1">18</td><td align="char" char="." rowspan="1" colspan="1">1</td></tr><tr><td rowspan="1" colspan="1">EFT-27mA</td><td align="char" char="." rowspan="1" colspan="1">34</td><td align="char" char="." rowspan="1" colspan="1">68</td><td align="char" char="." rowspan="1" colspan="1">44</td><td align="char" char="." rowspan="1" colspan="1">35</td><td align="char" char="." rowspan="1" colspan="1">0</td></tr><tr><td rowspan="1" colspan="1">EFT-27mB</td><td align="char" char="." rowspan="1" colspan="1">31</td><td align="char" char="." rowspan="1" colspan="1">42</td><td align="char" char="." rowspan="1" colspan="1">39</td><td align="char" char="." rowspan="1" colspan="1">13</td><td align="char" char="." rowspan="1" colspan="1">0</td></tr><tr><td rowspan="1" colspan="1">EFT-28m</td><td align="char" char="." rowspan="1" colspan="1">40</td><td align="char" char="." rowspan="1" colspan="1">38</td><td align="char" char="." rowspan="1" colspan="1">30</td><td align="char" char="." rowspan="1" colspan="1">10</td><td align="char" char="." rowspan="1" colspan="1">0</td></tr><tr><td rowspan="1" colspan="1">EFT-29m</td><td align="char" char="." rowspan="1" colspan="1">30</td><td align="char" char="." rowspan="1" colspan="1">47</td><td align="char" char="." rowspan="1" colspan="1">33</td><td align="char" char="." rowspan="1" colspan="1">20</td><td align="char" char="." rowspan="1" colspan="1">3</td></tr><tr><td rowspan="1" colspan="1">EFT-30m</td><td align="char" char="." rowspan="1" colspan="1">18</td><td align="char" char="." rowspan="1" colspan="1">39</td><td align="char" char="." rowspan="1" colspan="1">39</td><td align="char" char="." rowspan="1" colspan="1">6</td><td align="char" char="." rowspan="1" colspan="1">6</td></tr><tr><td rowspan="1" colspan="1">All MITEs</td><td align="char" char="." rowspan="1" colspan="1">1,120</td><td align="char" char="." rowspan="1" colspan="1">47<sup>***</sup></td><td align="char" char="." rowspan="1" colspan="1">33<sup>*</sup></td><td align="char" char="." rowspan="1" colspan="1">19<sup>***</sup></td><td align="char" char="." rowspan="1" colspan="1">3<sup>**</sup></td></tr></tbody></table><table-wrap-foot><fn><p>N<sc>ote.</sc>—SM, secondary metabolism. % 5′ + % 3′ ≥% within 500 bp of ORF due to some elements being near adjacent ORFs.</p></fn><fn><p><sup>*</sup><italic>P</italic> ≤ 0.1 (enriched), <sup>**</sup><italic>P</italic> ≤ 0.05 (enriched), <sup>***</sup><italic>P</italic> ≤ 0.005 (depleted).</p></fn></table-wrap-foot></table-wrap><p>As MITEs in epichloae were initially discovered within gene clusters for secondary metabolite production, we next looked at whether MITE integrations were significantly enriched near secondary metabolite genes within the <italic>E. festucae</italic> genome. We first examined previously annotated <italic>LOL</italic>, <italic>EAS</italic>, and <italic>LTM</italic> gene clusters (for loline, ergot alkaloid, and indole diterpene synthesis, respectively) for MITE insertions. (Note: Whereas initial analysis was performed on an early assembly of the <italic>E. festucae</italic> E2368 genome, contig numbers quoted in this section relate to the more complete 201006 assembly, which is publicly accessible.) Within the <italic>LOL</italic> cluster, which contains 11 genes on three contigs in the <italic>E. festucae</italic> E2368 assembly (contigs 1349, 1659, and 4990), we identified eight MITE insertions (3 × EFT-3m, 2 × EFT-11m, 2 × EFT-14m, and 1 × EFT-24). The <italic>EAS</italic> cluster consists of 11 genes and is found on contig 1654. Nine MITEs were found in this cluster, seven EFT-3m, one EFT-5m, and one EFT-9m, which was nested within one of the EFT-3m copies. Half of the 10-gene <italic>LTM</italic> cluster is absent from the genome assembly (<italic>E. festucae</italic> E2368 is a non–indole diterpene-producing strain); however, a truncated cluster of <italic>ltmP</italic>, <italic>ltmQ</italic>, <italic>ltmF</italic>, <italic>ltmC</italic>, and <italic>ltmB</italic> genes is found on contig 597. Four MITEs, two EFT-14m, one EFT-3m, and one EFT-8m are found in this cluster.</p><p>Given the number of MITE insertions in these gene clusters for previously characterized secondary metabolites, we wished to extend this analysis to include uncharacterized secondary metabolite gene loci. As secondary metabolite gene clusters other than those for previously known alkaloids were not yet annotated, we analyzed whether insertions were enriched within 10 kb of genes predicted to encode the secondary metabolite biosynthetic proteins NRPSs and PKSs, based on preliminary annotations derived using a combination of SMURF (<xref ref-type="bibr" rid="bib25">Khaldi et al. 2010</xref>), Blast, and manual annotation (<italic>Epichloë</italic> Genome Consortium, unpublished data). This analysis indicated a strong preference for integration site bias near this class of gene (<italic>x</italic> = 31, <italic>n</italic> = 1,077, <italic>P</italic> = 0.0183, <italic>P</italic> value = 0.016) (<xref ref-type="table" rid="tbl3">table 3</xref>).</p></sec><sec><title>Rearrangements Mediated by EFT-3 Recombination</title><p>Recombination between repeat sequences provided by transposable elements can cause genomic rearrangements. Sequence analysis at the <italic>easA</italic>–<italic>easG</italic> and <italic>perA</italic> loci lead us to examine the effect of the EFT-3m element on local rearrangements in <italic>E. festucae</italic>. Comparison of the <italic>E. festucae</italic> E2368 <italic>easA</italic>–<italic>easG</italic> intergenic region with that of the previously sequenced <italic>N. lolii</italic> Lp19 (<italic>N. lolii</italic> = <italic>E. festucae</italic> anamorph) locus (accession EF125025) revealed a third EFT-3m integration in E2368 directly adjacent to one of the two found in this region in Lp19 (<xref ref-type="fig" rid="fig2">fig. 2</xref>). Comparison of the sequence of these two EFT-3m elements revealed that the “second” element in Lp19 was not 100% identical to either of the adjacent elements at the E2368 locus. Interestingly, the arbitrary left 44 bp were identical to the left side of the second E2368 element (b in <xref ref-type="fig" rid="fig2">fig. 2<italic>A</italic></xref>), whereas the right 90 bp of the Lp19 element were identical to the right side of the “third” E2368 element (c in <xref ref-type="fig" rid="fig2">fig. 2<italic>A</italic></xref>), with the intervening 18 bp identical in both E2368 elements. This indicated that the common ancestral locus likely contained the arrangement found in E2368 and that recombination between the adjacent elements led to a deletion event that recapitulated a single EFT-3m element as observed in Lp19.</p><fig id="fig2" position="float"><label>F<sc>IG.</sc> 2.—</label><caption><p>EFT-3–mediated rearrangements at secondary metabolite gene loci. (<italic>A</italic>) Rearrangements in <italic>easA</italic>–<italic>easG</italic> intergenic region in two <italic>Neotyphodium</italic> strains compared with the <italic>Epichloë</italic><italic> festucae</italic> E2368 locus. 1. Recombination between adjacent EFT-3mB (b) and EFT-3mA (c) MITEs caused a deletion, resulting in the recapitulation of one EFT-3mA copy in <italic>Neotyphodium</italic><italic> lolii</italic> Lp19 (d). 2. Recombination between EFT-3mA copies (a and d) deleted intervening sequence leaving a single copy of EFT-3mA (e—aligned with a and d in <italic>B</italic>) in <italic>N. lolii</italic> AR1. (<italic>B</italic>) Alignment of <italic>N. lolii</italic> AR1 <italic>easA</italic><italic>–easG</italic> EFT-3mA sequence (e in <italic>A</italic>) with the two instances from <italic>N. lolii</italic> Lp19 (a and d in <italic>A</italic>). Regions of 100% sequence identity between the AR1 EFT-3mA sequence and the left and right Lp19 instances are highlighted in yellow and red, respectively. Sequence highlighted in black is identical between all three instances. Lp19 sequence between the two EFT-3mA instances is not shown in the alignment and replaced by 5 × <italic>N</italic>. (<italic>C</italic>) The <italic>perA</italic> locus in <italic>E. festucae</italic> E2368 and Fl1 and <italic>N. lolii</italic> Lp14. The overlined region marked Δ is deleted in E2368 and Lp14 compared with Fl1. The expanded region shows a deleted EFT-3 sequence at the deletion point at the 3′ end of <italic>perA</italic>. EFT-7 is an uncharacterized retrotransposon relic.</p></caption><graphic xlink:href="gbeevr098f02_3c"></graphic></fig><p>This observation led us to examine the <italic>easA</italic>–<italic>easG</italic> locus in two other strains with <italic>E. festucae</italic> lineages. PCR products amplified from the <italic>easA</italic>–<italic>easG</italic> region in the different strains revealed a different length polymorphism in each. Sequencing of these showed one of the strains <italic>Neotyphodium</italic> sp. Lp1 was identical to Lp19 but with an expansion of the simple sequence repeat (SSR) found between the two MITE insertions. In <italic>N. lolii</italic> AR1, a nonproducer of ergot alkaloids, the locus is deleted compared with other strains, with a single EFT-3m insertion and no SSR (<xref ref-type="fig" rid="fig2">fig. 2</xref>). Sequence analysis (<xref ref-type="fig" rid="fig2">fig. 2<italic>B</italic></xref>) revealed that the left 71 bp of this element were identical to the left end of the “left” element found in Lp19 (a in <xref ref-type="fig" rid="fig2">fig. 2<italic>A</italic></xref>), whereas the right 56 bp of the AR1 element were identical to the “right” element in Lp19 (d in <xref ref-type="fig" rid="fig2">fig. 2<italic>A</italic></xref>), with 22 bp of intervening sequence identical in both Lp19 elements. This indicated that recombination between the left and the right elements is likely to have caused a deletion of the 405 bp between the two insertions.</p><p>EFT-3m also appears to have been involved in a deletion event in some epichloid strains at the <italic>perA</italic> locus, a single gene required for synthesis of the anti-insect secondary metabolite peramine. Examination of the sequence of the nonfunctional <italic>perA</italic> gene in the <italic>E. festucae</italic> E2368 genome revealed a deletion of 1,223 bp of the 3′ end of the gene, with a 50-bp deleted instance of an EFT-3m element located at the deletion point adjacent to the remaining <italic>perA</italic> sequence. Previous analysis had shown that the <italic>perA</italic> gene was present in <italic>N. lolii</italic> Lp14, although this strain does not produce peramine (<xref ref-type="bibr" rid="bib40">Scott et al. 2009</xref>). To determine whether the Lp14 <italic>perA</italic> gene also contained a deletion, we sequenced a PCR product amplified from the <italic>perA</italic>-EF104 region and remarkably found an identical deleted <italic>perA</italic> as in E2368, indicative of shared ancestry of these two strains.</p></sec></sec><sec><title>Discussion</title><p>In this study, we identified 13 different families of MITEs, the most diverse collection characterized in fungi to date. We used a computational pipeline, which utilizes several different algorithms and some manual input for the analysis. Previous analysis of two MITE families in epichloae showed that these elements were highly degenerate (<xref ref-type="bibr" rid="bib20">Fleetwood et al. 2007</xref>). This was also the case for the 10 other families characterized in this study, with average pairwise identity ranging from 49% (EFT-24) to 90% (EFT-25) with most around 60–85% identical.</p><p>For three of the MITEs identified, we were able to identify relics of progenitor autonomous transposons. In each case, the MITEs seem to have been derived by deletion of internal sequences. This is one way in which MITEs can arise (<xref ref-type="bibr" rid="bib22">Jiang et al. 2003</xref>), although more commonly they appear to arise by the chance occurrence of sequences related to autonomous element TIRs being found in the appropriate orientation in the genome (<xref ref-type="bibr" rid="bib23">Jiang et al. 2004</xref>). Although we did not identify any MITEs that had been formed in this way, the presence of EFT-27 subfamilies that are dissimilar to each other outside of the TIR sequences suggests that this has occurred in the epichloae. Each of the three autonomous elements identified have been rendered nonfunctional by RIP and contain multiple stop codons and very high AT percentages. Therefore, the MITEs derived from these elements are also highly unlikely to be functional as they require a functioning transposase on a parent element. For most MITEs, we could not identify parent elements. This may mean that the corresponding autonomous elements have degenerated to the extent that we were no longer able to recognize them or may suggest that these MITE families use a transposase from autonomous elements sufficiently dissimilar to the MITEs that they were not able to be recognized by similarity to the MITE TIRs. Whether autonomous elements exist in the genome for these MITEs or not, they are unlikely to be currently mobilizable based on the degeneracy of these families.</p><p>At least half of the MITE families appear to be ancient in the <italic>Epichloë</italic> genus. All are highly degenerate, and this is unlikely to be due to RIP as almost all the families are smaller than the 400 bp identified in <italic>N. crassa</italic> as the minimum length for RIP to function (<xref ref-type="bibr" rid="bib9">Cambareri et al. 1989</xref>). Additionally, alignments show no obvious bias for C:T or G:A transitions characteristic of RIP (<xref ref-type="bibr" rid="bib9">Cambareri et al. 1989</xref>). Thus, this degeneracy is likely to be due to basal levels of mutation over a very long period. The age of the elements could not be estimated due to the lack of a molecular clock; however, the very high degeneracy suggests an ancient origin, and taxonomic distribution data place the invasion of many of the MITEs at least as being early in the evolution of <italic>Epichloë</italic>. The EFT-3m, EFT-11m, EFT-14m, EFT-24m, and EFT-26m MITEs were found in sequences from <italic>Neotyphodium</italic> species that do not have <italic>E. festucae</italic> parentage (asexual <italic>Neotyphodium</italic> species are derivatives of sexual <italic>Epichloë</italic> species and often hybrids), supporting their early invasion. These were all in a single secondary metabolite gene cluster, however, and few epichloae sequences outside of these clusters are present in public gene databases. Thus, the absence of the remaining MITE families in these sequences does not preclude their equally ancient origin. Further evidence was found for EFT-14, which was found in all epichloid species examined (<xref ref-type="fig" rid="fig1">fig. 1</xref>). A number of diverse <italic>Epichloë</italic> and <italic>Neotyphodium</italic> species are currently being sequenced, and analysis of these genomes will confirm whether all the MITEs are as old as EFT-14. If, as seems likely, each of the MITEs were ancient invaders of epichloae, this is in contrast to the other repeat elements thus far identified in this genus. The Tahi and Rua retrotransposons were shown by Southern analysis to have a much more limited taxonomic distribution (<xref ref-type="bibr" rid="bib54">Young et al. 2009</xref>), suggesting they invaded epichloae just prior to the radiation of <italic>E. festucae</italic> and <italic>E. baconii</italic>.</p><p>In other organisms, including the <italic>mimp</italic> element in <italic>F. oxysporum</italic> (<xref ref-type="bibr" rid="bib3">Bergemann et al. 2008</xref>), MITEs are frequently found in genic regions of genomes, sometimes including within introns or 5′ or 3′ UTRs (<xref ref-type="bibr" rid="bib5">Bureau and Wessler 1992</xref>, <xref ref-type="bibr" rid="bib6">1994a</xref>; <xref ref-type="bibr" rid="bib39">Santiago et al. 2002</xref>; <xref ref-type="bibr" rid="bib49">Yang et al. 2005</xref>; <xref ref-type="bibr" rid="bib37">Ohmori et al. 2008</xref>). Our analysis of the genomic location of MITEs in <italic>E. festucae</italic> indicates that <italic>E. festucae</italic> is no exception. Of all MITEs, 47% were found within 500 bp of a gene, some of which may be within 5′ or 3′ UTRs although we did not have sufficient EST data to determine this. Further analysis indicated that for most families, more instances were found within 500 bp of the 5′ end of a gene than the 3′ end (<xref ref-type="table" rid="tbl3">table 3</xref>). This is well within the distance from the ORF start codon that is likely to be <italic>cis</italic> regulatory sequence, and it seems likely that at least a proportion of these integrations have affected expression of downstream genes in some way. Transposons integrated into promoter regions in other organisms have been shown to affect expression by disruption of existing, or provision of new, <italic>cis</italic> regulatory sequences or through alteration of the chromatin environment (<xref ref-type="bibr" rid="bib26">Kidwell and Lisch 1997</xref>; <xref ref-type="bibr" rid="bib16">Feschotte 2008</xref>), although this has rarely been tested for MITEs. How many genes have altered expression and what kind of effects the MITEs have had on expression in epichloae cannot be determined from analysis of a single genome but with multiple diverse <italic>Epichloë</italic> and <italic>Neotyphodium</italic> genomes currently being sequenced; along with complementary transcriptome analyses, these questions may soon be addressed.</p><p>A second aspect of MITE integration site bias of interest in this study was the finding that MITEs are nonrandomly enriched near genes predicted to be involved in secondary metabolite biosynthesis. Secondary metabolite biosynthetic genes are well studied in epichloae due to the protective effects of these natural products on the host grass, and MITEs were initially identified within some of these gene clusters. Further analysis here of gene clusters for the known alkaloids, lolines, indole diterpenes, and ergot alkaloids, showed that these gene clusters contain a striking number of MITE instances from several families. Furthermore, MITEs are significantly more likely than chance to be found near (within 10 kb) a PKS or NRPS gene, which are usually secondary metabolite biosynthetic genes (<xref ref-type="table" rid="tbl3">table 3</xref>). To have so many transposable elements within such a small sequence, as we observe in the <italic>LOL</italic>, <italic>EAS</italic>, and <italic>LTM</italic>, clusters is remarkable. This coclustering of MITEs with secondary metabolite gene clusters is not a phenomenon that has been described in other species to our knowledge. Indeed, the epichloid biosynthetic gene clusters show a level of complexity somewhat higher than that of most characterized gene clusters in other fungi, with “mini-clusters” of genes and MITEs separated by nested autonomous retrotransposons and DNA transposons (<xref ref-type="bibr" rid="bib53">Young et al. 2006</xref>; <xref ref-type="bibr" rid="bib19">Fleetwood 2007</xref>).</p><p>Whether this coclustering has occurred due to an evolutionary benefit to gene clusters containing such a large number of MITEs, by affecting either gene regulation or evolution, or whether this arrangement simply arises passively through a tendency for both gene clusters and transposons to be found in certain genomic regions, particularly telomeres and centromeres, is not easily tested. However, recent work showing a role for transposon sequences in regulation of a secondary metabolite gene cluster in <italic>Aspergillus nidulans</italic> (<xref ref-type="bibr" rid="bib41">Shaaban et al. 2010</xref>) supports a role in regulation. A further hint to the possible role that MITEs may have played in the evolution of gene clusters in epichloae was the finding that MITEs have mediated recombination events at a local level in one and likely two secondary metabolite gene loci. The EFT-3m element has led to deletion events in the <italic>EAS</italic> cluster, whereas the presence of an EFT-3m deleted instance at the deletion point at the 3′ end of the <italic>perA</italic> gene suggests an involvement in that deletion also. The deletion of the 3′ end of the <italic>perA</italic> gene and regulatory sequence of the <italic>easA</italic> and/or <italic>easG</italic> genes has likely led or contributed to the lack of peramine and ergot alkaloid production in the respective strains, a major impact on the grass endophyte symbioses in which these plant-protective natural products play a major role. It seems unlikely that such a restricted analysis would have identified the only instances in which MITEs have mediated local genome rearrangements, and with the large number of deleted instances in the genome, it seems likely that MITE repeat sequences have played a large role in genome evolution of epichloae.</p><p>In this study, we described a large number of MITEs in the <italic>E. festucae</italic> genome and provide evidence for a likely role in genome regulation and evolution in epichloae. Why have MITEs then been so rarely characterized in other fungi? A major reason is likely to be the small size of many of the elements because the cutoff for repeat sequences in repeat searches of fungal genomes is often larger than the size of many MITEs. The epichloid MITEs were also highly degenerate, possibly below the similarity thresholds used by researchers studying other fungal genomes. It seems likely to us that fungal genomes contain more MITEs than currently described, and indeed a preliminary analysis of the <italic>N. crassa</italic>, <italic>F. oxysporum</italic>, and <italic>M. grisea</italic> genomes revealed a number of new MITE families in each genome alongside the previously characterized <italic>Guest</italic> and <italic>mimp</italic> elements (Khan A and Fleetwood D, unpublished data). This class of nonautonomous elements is clearly deserving of more research in fungi, and studies of the impact of the elements on genome evolution and regulation will be of very considerable future interest.</p></sec><sec sec-type="supplementary-material"><title>Supplementary Material</title><p><ext-link ext-link-type="uri" xlink:href="http://www.gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evr098/-/DC1">Supplementary data</ext-link> and tables S1 and S2 are available at <italic>Genome Biology and Evolution</italic> online (<ext-link ext-link-type="uri" xlink:href="http://www.gbe.oxfordjournals.org/">http://www.gbe.oxfordjournals.org/</ext-link>).</p></sec></body><back><ack><p>We thank Mr Alan McCulloch for methodological advice; Mr Craig Miskell for maintaining computational servers and software; Dr Shaun Lott for laboratory resources; Ms Jennifer Webb for help sequencing the <italic>E. festucae</italic> genome; Dr Jolanta Jaromczyk and Mr Charles T. Bullock for genome assemblies; and Ms Elissaveta Arnaoudova, Mr Neil Moore, and Mr Daniel Harris for genome annotations. 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