Genetic mapping of barley genes.

1<p>Forward (F) and reverse (R) primer; primers highlighted in bold were used for direct sequencing for detection of polymorphisms.</p>2<p>A/GC = anealing temperature/GC buffer (+ = GC buffer added, − = no GC buffer added).</p>3<p>GenBank accessions for genomic sequences from OWB-D (D) and OWB-R (R) parental lines are indicated. SNP positions are relative to OWB-D.</p>4<p>Barley chromosome arm: short (s), long (l). <i>HvCMF5</i> and <i>HvCMF6b</i> were genetically mapped using presence/absence PCR/agarose gel InDel assays.</p>5<p>Due to lack of polymorphism in the genomic fragment investigated, <i>HvCO15</i> was mapped using a SNP in an adjacent predicted gene (<i>HvOs08g42430</i>) located on the same sequence contig (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045307#pone.0045307.s008" target="_blank">Table S4</a>).</p

Phylogenetic analysis of CMF, COL, PRR and ZCCT proteins, based on the CCT domain.

<p>Dashed lines enclose predominantly CMF (red), COL (blue) and PRR (black) families, within which the predominant protein domain configurations within full length proteins are indicated (proteins lacking the predominant pattern are indicated in brackets). Numbered triangles indicate CCT domain intron position (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045307#pone-0045307-g001" target="_blank">Figure 1</a>). COL clades [following 11,13] are indicated with COL I and II proteins possessing two or one B-box domains, respectively. Genes are prefixed with genus and species initials. Gene synonyms as listed in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045307#pone.0045307.s005" target="_blank">Tables S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045307#pone.0045307.s009" target="_blank">S5</a>. Although Group III <i>COL</i> genes are previously described as posessing two B-boxes, domain prediction software used here identified variously one or two B-boxes. Arabidopsis CMF (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045307#pone.0045307.s005" target="_blank">Table S1</a>), COL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045307#pone.0045307-Griffiths1" target="_blank">[13]</a> and PRR <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045307#pone.0045307-Higgins1" target="_blank">[19]</a> members are included, as are ZCCT1 (also termed VRN2, AAS60241) and ZCCT2 (AAS60248) from <i>T. monococcum</i>. Black asterisks indicate nodes with bootstrap values >60%. SiCMF1, BdCMF4, HvCMF7, SbCMF7 and SiCMF7 are excluded, due to deletions within the CCT domain.</p

Location of Poaceae <i>CMF, COL, PRR</i> and <i>ZCCT</i> genes within the framework of Poaceae inter- and intra-specific colinearity.

<p>Rice chromosomal regions duplicated during the ancestral WGD are linked by coloured bands. Paralogous gene pairs arising from WGD are connected by black dashed lines, while colinear genes lacking paralogues are connected by grey dashed lines. As instances where full-length barley orthologues were not identified in the draft genomic sequence and fl-CDNAs does not mean they are absent in barley, their predicted positions are indicated tentatively here as unknown (UK) genes. Protein domain configurations are indicated. Localised segmental chromosomal inversions are not shown. ^a Poaceae orthologues for <i>HvCO2</i> orthologous are present in all genomes except rice <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045307#pone.0045307-Higgins1" target="_blank">[19]</a>. ^b<i>SbCMF16</i>. ^c<i>SbPRR37</i>. ^d<i>SbCMF8</i>. ^e Si011862m, truncated CCT motif. ^f<i>SiCMF8</i>. ^g<i>BdCMF15</i>.</p

Structures of the indole-diterpene biosynthesis loci (<i>IDT/LTM</i>) in sequenced genomes.

<p><i>IDT/LTM</i> genes are indicated by single letters, whereby <i>Q = idtQ</i> or <i>ltmQ</i> (in <i>E. festucae</i>), and so forth. Tracks from top to bottom of each map represent the following: genes, repeats, MITEs, and graphs of AT (red) and GC (blue) contents. Each gene is represented by a filled arrow indicating its direction of transcription. Closed circles indicate telomeres, and distances from the telomere on the <i>E. festucae</i> map are indicated in kilobasepairs (kb). Cyan bars representing repeat sequences are labeled with names or numbers to indicate relationships between repeats in the different species. Vertical bars beneath the repeat maps indicate MITEs. Genes for the first fully cyclized intermediate, paspaline, are indicated in blue, those for subsequent chemical decorations are shown in red, and <i>idt/ltmS</i>, with undetermined function, is in purple. Identifiable genes flanking the clusters are indicated in gray, and unfilled arrows indicate pseudogenes. The major pathway end-product for each strain is listed at the right of its map, abbreviated as indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003323#pgen-1003323-g003" target="_blank">Figure 3</a>, and in bold for those confirmed in this study.</p

Phylogenies of <i>rpbA</i> from sequenced isolates and other Clavicipitaceae.

<p>The phylogenetic tree is based on nucleotide alignment for a portion of the RNA polymerase II largest subunit gene, <i>rpbA</i>. This tree is rooted with <i>Fusarium graminearum</i> as the outgroup. Epichloae are indicated in green, <i>Claviceps</i> species are indicated in blue, <i>Periglandula</i> species are indicated in red, and <i>Aciculosporium take</i> is in black. Species for which genomes were sequenced in this study are shown in bold type, and asterisks indicate plant-associated fungi. Alkaloids listed are the major pathway end-products predicted from the genome sequences, abbreviated as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003323#pgen-1003323-g002" target="_blank">Figure 2</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003323#pgen-1003323-g003" target="_blank">Figure 3</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003323#pgen-1003323-g004" target="_blank">Figure 4</a>. Other abbreviations: (−) = some genes or remnants present, but not predicted to make alkaloids of this class, – = no genes present for this alkaloid class, EA = ergot alkaloids may be produced; IDT = indole-diterpenes may be produced, (ΔR*) = deletion of terminal reductase domain of <i>perA</i>.</p

Structures of the ergot alkaloid biosynthesis loci (<i>EAS</i>) in sequenced genomes.

<p>Tracks from top to bottom of each map represent the following: genes, repeats, MITEs, and graphs of AT (red) and GC (blue) contents. Each gene is represented by one or more boxes representing the coding sequences in exons, and an arrow indicating the direction of transcription. Double-slash marks (//) indicate sequence gaps within scaffolds of the assembled <i>E. festucae</i> genome sequences. Closed circles indicate telomeres, and distances from the telomere on the <i>E. festucae</i> map are indicated in kilobasepairs (kb). Cyan bars beneath each map represent repeat sequences, and are labeled with names or numbers to indicate relationships between repeats in the different species. Vertical bars beneath the repeat maps indicate MITEs. Gene names are abbreviated <i>A</i> through <i>P</i> for <i>easA</i> through <i>easP</i>, <i>W</i> for <i>dmaW</i>, and <i>clo</i> for <i>cloA</i>. Genes for synthesis of the ergoline ring system (skeleton) are shown in dark blue for the steps to chanoclavine-I (<i>W</i>, <i>F</i>, <i>E</i>, and <i>C</i>), and in light blue (<i>D</i>, <i>A</i>, and <i>G</i>) for steps to agroclavine. Genes for subsequent chemical decorations are shown in red (<i>clo</i>, <i>H</i>, <i>O</i>, <i>P</i>, <i>lpsA</i>, <i>lpsB</i>, and <i>lpsC</i>). Identifiable genes flanking the clusters are indicated in gray, and unfilled arrows indicate pseudogenes. The major pathway end-products for each strain are listed below each species name, abbreviated as indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003323#pgen-1003323-g002" target="_blank">Figure 2</a>, and in bold for those confirmed in this study. Note that LAH is a reported product of <i>C. paspali</i>, but the sequenced strain is predicted not to synthesize it due to a defective <i>easE</i> gene.</p

GC proportions in genic and repeat DNA of sequenced genomes.^a

a<p>Abbreviations: CDS = coding sequence, GC = proportion of sequence that is G or C, non-Rpt-IG = nonrepetitive intergenic DNA, Rpt = repetitive DNA.</p>b<p>Statistics for <i>P. ipomoeae</i> are tentative because the assembly was filtered by selecting only contigs containing tBLASTx matches to genome sequences from the other Clavicipitaceae.</p

Symbiosis of meadow fescue with <i>Epichloë festucae</i>, a heritable symbiont.

<p>Single optical slice confocal micrographs of <i>E. festucae</i> expressing enhanced cyan-fluorescent protein were overlain with DIC bright field images of (A) ovules (bar = 100 µm), (B) embryos (bar = 200 µm), and (C) shoot apical meristem and surrounding new leaves (bar = 200 µm). (D) Asymptomatic (left) and “choked” (right) inflorescences simultaneously produced on a single grass plant infected with a single <i>E. festucae</i> genotype. Vertical (seed) transmission of the symbiont occurs via the asymptomatic inflorescence, whereas the choked inflorescence bears the <i>E. festucae</i> fruiting structure (stroma), which produces sexually derived spores (ascospores) that mediate horizontal transmission.</p

Genic and repeat DNA contents of sequenced genomes.^a

a<p>Abbreviations: CDS = coding sequence, MT = mating type, non-Rpt-IG = nonrepetitive intergenic DNA, Rpt = repetitive DNA.</p>b<p>Based on total of contigs ≥500 bp. These sizes differ slightly from total scaffold lengths given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003323#pgen-1003323-t001" target="_blank">Table 1</a> for <i>C. purpurea</i> 20.1, <i>E. festucae</i> E2368, and <i>E. festucae</i> Fl1.</p>c<p><i>C. fusiformis</i> PRL 1980 mating type genes include <i>mtBA</i> and <i>mtAC</i>. <i>P. ipomoeae</i> IasaF13 mating type genes <i>mtAA</i> and <i>mtAC</i> appear to have premature stop codons.</p>d<p>Statistics for <i>P. ipomoeae</i> are tentative because the assembly was filtered by selecting only contigs containing tBLASTx matches to genome sequences from the other Clavicipitaceae.</p

Alkaloid profiles of sequenced isolates.^a

a<p>Strains are abbreviated as follow: <i>Cpu</i> = <i>Claviceps purpurea</i> 20.1, <i>Cfu</i> = <i>C. fusiformis</i> PRL 1980, Cpa = <i>C. paspali</i> RRC-1481, <i>Eam</i> = <i>Epichloë amarillans</i> E57, <i>Ebe</i> = <i>E. brachyelytri</i> E4804, <i>Eel</i> = <i>E. elymi</i> E56, <i>Ef</i>1 = <i>E. festucae</i> Fl1, <i>Ef</i>2 = <i>E. festucae</i> E2368, <i>Egl</i> = <i>E. glyceriae</i> E2772, <i>Et</i>8 = <i>E. typhina</i> E8, <i>Et</i>5 = <i>E. typhina</i> E5819, <i>Nga</i> = <i>N. gansuense</i> E7080, <i>Ngi</i> = <i>N. gansuense</i> var. <i>inebrians</i> E818, <i>Nun</i> = <i>N. uncinatum</i> E167, <i>Pip</i> = <i>P. ipomoeae</i> IasaF13. Symbols: + = present, (+) = intermediate inferred to be synthesized because downstream product is present, − = not predicted and not detected, (−) = predicted but not detected, nt = predicted but not tested, ERA = ergotamine, ERB = ergobalansine, ERC = ergocryptine, ERV = ergovaline. Blank cells indicate compounds not predicted from genotype, and not tested.</p>b<p>Identification of IDT-436 and terpendoles E, I, J, K, M, M, and A are tentative because authentic standards are unavailable.</p