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Phylogenetic trees for segments comprising unique sequences in the genomic <i>HMA4</i> region of <i>A. halleri</i>.

<p>Shown are maximum likelihood trees for amplicons (A) S2, (B) S9, and (C) S12. Alleles are named according to <i>A. halleri</i> individuals ([collection site].[individual], see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003707#pgen-1003707-t001" target="_blank">Table 1</a>) and are color-coded based on the population of their origin: blue, Harz Mountains (1 to 5); violet, Thuringian Forest (6 and 7, BAC); brown, Auby (8.1); red, <i>A. halleri</i> ssp. <i>gemmifera</i> (9.1). Published sequences from <i>A. halleri</i> BACs were included (Genbank accession numbers EU382073.1 and EU382072.1) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003707#pgen.1003707-Hanikenne1" target="_blank">[8]</a>, and <i>A. lyrata</i> ssp. <i>lyrata</i> sequences <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003707#pgen.1003707-Hu1" target="_blank">[39]</a> are shown as outgroup (green) where possible. Percentages of bootstrap support (1000 replicates) of a minimum of 75% are given at the corresponding nodes. Branch lengths are scaled by the number of substitutions per site. The datasets were as follows (number of sequences × number of aligned positions in bp): S2: 42×1464 (A); S9: 39×3710 (B); S12: 42×2480 (C). Asterisks (*) denote the second alleles that were inferred in individuals from which only a single sequence was obtained and which were thus concluded to be homozygous for the respective locus. Note that in <i>A. halleri</i> ssp. <i>gemmifera</i> (individual 9.1), primer pairs designed to obtain S6 and S9 (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003707#pgen.1003707.s008" target="_blank">Table S1</a>) both yielded the same set of four highly similar sequences (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003707#pgen.1003707.s001" target="_blank">Figure S1D</a>).</p

<i>HMA4</i> gene copy number in field-collected individuals of <i>A.</i>

<p><b>halleri<i> </i></b><b>.</b><b> </b> Values (arithmetic means ± s.e.m., <i>n</i> = 16 technical replicates) are given relative to single-copy control loci <i>FRD3 </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003707#pgen.1003707-Talke1" target="_blank">[12]</a> and S13 (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003707#pgen-1003707-g001" target="_blank">Figure 1</a>). <i>A. thaliana</i> (<i>At</i>) genomic DNA served as calibrator. Note that one of the two <i>HMA4</i> gene copies detected in <i>A. lyrata</i> (<i>Al</i>) corresponds to a truncated pseudogene (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003707#pgen.1003707.s001" target="_blank">Figure S1B</a>).</p

Leaf <i>HMA4</i> transcript levels in <i>A. halleri</i> individuals.

<p>Values (arithmetic means ± s.e.m. of two independent biological experiments) are given relative to <i>HMA4</i> transcript levels in <i>A. thaliana</i> (<i>At</i>). Nucleic acids were extracted from leaves of vegetative plants, including <i>A. lyrata</i> (<i>Al</i>), grown on soil in a greenhouse.</p

Nucleotide sequence diversity across the <i>HMA4</i> genomic region in <i>A. halleri</i>.

<p>(A) Average pairwise nucleotide sequence diversity π across the <i>HMA4</i> genomic region in <i>A. halleri</i> ssp. <i>halleri</i>. (B) Molecular population genetic statistical tests for the deviation from neutrality, Tajima's <i>D</i>, Fu and Li's <i>D</i>*, and Fu and Li's <i>F</i>* in <i>A. halleri</i> ssp. <i>halleri</i>. Differently colored symbols are defined by concordantly colored axis titles. Vertical arrows indicate positions of sequenced segments. The region of <i>HMA4</i> triplication is shaded in grey. Marked in red color are stretches of sequence that are present in several, almost identical copies (horizontal bars; detected using NCBI MEGABLAST program with default settings and a word size of 256; see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003707#pgen.1003707.s010" target="_blank">Table S3</a>), and sequenced segments therein (arrows, fonts, red open symbols instead of closed black symbols in panel (A)). Direction of transcription (triangular arrow) is given for each gene (rectangle, Arabidopsis Genome Identifier code of <i>A. thaliana</i> ortholog), with genes of unknown position in <i>A. halleri</i> shown in grey. For additional information see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003707#pgen.1003707.s009" target="_blank">Table S2</a>.</p

Origin of <i>Arabidopsis halleri</i> individuals and other plants used in this study.

<p>Countries of sampling outside Germany are specified. Extractable soil and total leaf concentrations are shown relative to dry mass. M, metalliferous site; NM, non-metalliferous site. Sources of genotypes:</p>a<p>BC1 parent <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003707#pgen.1003707-Courbot1" target="_blank">[17]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003707#pgen.1003707-Bert1" target="_blank">[72]</a>;</p>b<p>Fujita Co., Japan;</p>c<p>Nottingham Arabidopsis Stock Centre.</p><p>n.a.: not analyzed.</p

Recurrent sequence exchange between coding regions of <i>A.</i>

<p><b>halleri HMA4<i> </i></b><b> gene copies.</b><b> </b> (A) Network analysis of consensus sequences of 3′ <i>HMA4</i> coding regions of <i>A. halleri</i> ssp. <i>halleri</i>. Each node represents one mutational step. Node size corresponds to the number of alleles, as specified inside nodes ([collection site].[individual], see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003707#pgen-1003707-t001" target="_blank">Table 1</a>), constituting the respective consensus (h01 to h25). Alleles were assigned to <i>HMA4</i>-<i>1</i> (green, S5), -<i>2</i> (yellow, S7) or -<i>3</i> (orange, S10, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003707#pgen-1003707-g001" target="_blank">Figure 1</a>), with multiple coloration for both ambiguous assignments and assignments to multiple gene copies. Note that assignment to <i>HMA4-2</i> was based directly on sequence data, whereas for all sequences not derived from BAC (B) data, assignment to <i>HMA4-1</i> or <i>HMA4-3</i> was inferred (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003707#s3" target="_blank">Materials and Methods</a>). (B) Polymorphic positions in consensus sequences. Header row refers to the alignment of consensi of segments S5/S7/S10 from <i>A. halleri</i>. SNPs present in single (blue fonts) or multiple (red fonts) consensi are highlighted. For indels, each color marks one allele.</p