Comparative Analysis of <em>FLC</em> Homologues in Brassicaceae Provides Insight into Their Role in the Evolution of Oilseed Rape

<div><p>We identified nine <em>FLOWERING LOCUS C</em> homologues (<em>BnFLC</em>) in <em>Brassica napus</em> and found that the coding sequences of all <em>BnFLCs</em> were relatively conserved but the intronic and promoter regions were more divergent. The <em>BnFLC</em> homologues were mapped to six of 19 chromosomes. All of the <em>BnFLC</em> homologues were located in the collinear region of <em>FLC</em> in the <em>Arabidopsis</em> genome except <em>BnFLC.A3b</em> and <em>BnFLC.C3b,</em> which were mapped to noncollinear regions of chromosome A3 and C3, respectively. Four of the homologues were associated significantly with quantitative trait loci for flowering time in two mapping populations. The <em>BnFLC</em> homologues showed distinct expression patterns in vegetative and reproductive organs, and at different developmental stages. <em>BnFLC.A3b</em> was differentially expressed between the winter-type and semi-winter-type cultivars. Microsynteny analysis indicated that <em>BnFLC.A3b</em> might have been translocated to the present segment in a cluster with other flowering-time regulators, such as a homologue of <em>FRIGIDA</em> in <em>Arabidopsis.</em> This cluster of flowering-time genes might have conferred a selective advantage to <em>Brassica</em> species in terms of increased adaptability to diverse environments during their evolution and domestication process.</p> </div

Quantitative real-time PCR analysis of expression patterns for four <i>BnFLC</i> homologues.

<p>Different letters above a bar indicate a significant difference (<i>P</i><0.05). Expression levels at the cotyledon stage were considered to be the control. Relative expression values of each <i>BnFLC</i> homologue were normalized with the reference gene <i>β-Actin.</i> (A) Comparison of the relative expression levels of <i>BnFLC</i> homologues in different tissues of the winter cultivar Tapidor. Samples underlined with a solid or dashed line were collected from nonvernalized and vernalized plants, respectively. (B) Relative expression levels of <i>BnFLCs</i> in leaves and cotyledons at different developmental stages in the semi-winter cultivar Ningyou7. No cold treatment was applied throughout all of the developmental stages. (C) Vernalization responsiveness of <i>BnFLC</i> homologues in Tapidor and Ningyou7. The relative fold change between four-week-old leaves (without vernalization) and seven-week-old leaves (four-week-old plants followed by three weeks of cold treatment) was measured.</p

Additional file 1 of Resuscitation room management of patients with non-traumatic critical illness in the emergency department (OBSERvE-DUS-study)

Supplementary Material

<i>Brassica napus FLC</i> homologues, their map positions, and sequence identities compared with their orthologues in <i>B. rapa</i> or <i>B. oleracea.</i>

a<p>The sequences isolated from BACs are indicated by an asterisk, and those isolated from PCR-amplified genomic DNA are designated ‘gDNA’ with the parent indicated in parentheses (T, <i>B. napus</i> cv. Tapidor; N, <i>B. napus</i> cv. Ningyou7).</p>b<p>The obtained sequences were compared with that of <i>AtFLC</i> to define the regions of each <i>BnFLC</i> gene that were isolated.</p>c<p>To calculate sequence identities, indels (insertions or deletions) were excluded and for partial sequences of <i>BnFLC.C2</i> and <i>BnFLC.C3a,</i> the available region was used.</p>d<p>The letter and numeral in parentheses represent the linkage group followed by the ancestral block in which the <i>FLC</i> homologues are located.</p>e<p>The number of nonsynonymous nucleotide substitutions is shown in parentheses.</p>f<p>There was a fragment with higher-order structure in intron 1 of <i>BnFLC.A3a</i> for which we failed to obtain the sequence.</p

Correlation coefficients between <i>BnFLC</i> and <i>BnFRI</i> loci in the Tapidor and Ningyou7 during different developmental stages.

a<p>Pearson’s correlation coefficients were calculated by relative expression values within different <i>BnFLC</i> and <i>BnFRI</i> gene pairs in cotyledons or leaves which harvested from the cotyledon, the stem elongation and the flowering stages of nonvernalized Ningyou7, or from the cotyledon, the first-, two-, and four-leaf stages of nonvernalized Tapidor plants.</p>b<p>The value in parentheses represents the <i>P</i>-value.</p

Differential expression of <i>BnFLCs</i> in four-week-old nonvernalized leaves from <i>B. napus</i> cultivars Tapidor and Ningyou7.

<p>(A) Relative expression levels for four <i>BnFLC</i> homologues. <i>β-Actin</i> was used as a reference gene. Error bars represent standard errors among the three biological replicates; **represents significantly different (<i>p</i><0.05). (B) PCR products of <i>BnFLC.A3b</i> amplified from total RNA of Tapidor (T) and Ningyou7 (N). The major band amplified from Tapidor comprises correctly spliced <i>BnFLC.A3b</i> transcripts, whereas incompletely (unfilled arrow) and incorrectly spliced <i>BnFLC.A3b</i> transcripts (usually lack exon 3; filled arrow) were amplified in Ningyou7. The schematic diagram of a <i>BnFLC.A3b</i> transcript shows exons (E1 to E7) and untranslated regions (UTR). <i>BnFLC.A3b_T, N</i> represents correctly spliced transcript in Tapidor and Ningyou7, <i>BnFLC.A3b_N-1</i> and <i>BnFLC.A3b_N-2</i> represent the two kinds of alternative spliced transcripts in Ningyou7 which lacked partial or complete exon 3, respectively. Shadow box in <i>BnFLC.A3b_N-1</i> indicates the last four nucleotides of exon 3 which were retained. I, II, III and IV, which represent four amplifications from different transcriptional regions of correctly spliced <i>BnFLC.A3b_T, N</i> transcript, are shown below the schematic diagram.</p

Phylogenetic tree of <i>FLC</i> homologues from <i>Brassica</i>, <i>Arabidopsis</i>, <i>Raphanus</i>, and <i>Sinapis</i> species.

<p><i>BnFLC</i> homologues are highlighted in bold, and <i>AtMAF1</i> (<i>MADS AFFECTING FLOWERING 1</i> of <i>A. thaliana</i>, an <i>AtFLC</i>-like gene) was used as the outgroup. <i>Br</i>, <i>Brassica rapa</i>; <i>Bo</i>, <i>B. oleracea</i>; <i>Rs</i>, <i>Raphanus sativus</i> (radish); <i>Sa</i>, <i>Sinapis alba</i> (white mustard); <i>At</i>, <i>Arabidopsis thaliana</i>; <i>Al</i>, <i>A. lyrata</i>; <i>Ah</i>, <i>A. halleri</i>; <i>Aa</i>, <i>A. arenosa; As, A. suecica</i>. GenBank accession numbers are given in parentheses. Bootstrap support values are shown beside the branches.</p

Unraveling the Genetic Basis of Seed Tocopherol Content and Composition in Rapeseed (<em>Brassica napus </em>L.)

<div><h3>Background</h3><p>Tocopherols are important antioxidants in vegetable oils; when present as vitamin E, tocopherols are an essential nutrient for humans and livestock. Rapeseed (<em>Brassica napus</em> L, AACC, 2 n = 38) is one of the most important oil crops and a major source of tocopherols. Although the tocopherol biosynthetic pathway has been well elucidated in the model photosynthetic organisms <em>Arabidopsis thaliana</em> and <em>Synechocystis</em> sp. PCC6803, knowledge about the genetic basis of tocopherol biosynthesis in seeds of rapeseed is scant. This project was carried out to dissect the genetic basis of seed tocopherol content and composition in rapeseed through quantitative trait loci (QTL) detection, genome-wide association analysis, and homologous gene mapping.</p> <h3>Methodology/Principal Findings</h3><p>We used a segregating Tapidor × Ningyou7 doubled haploid (TNDH) population, its reconstructed F₂ (RC-F₂) population, and a panel of 142 rapeseed accessions (association panel). Genetic effects mainly contributed to phenotypic variations in tocopherol content and composition; environmental effects were also identified. Thirty-three unique QTL were detected for tocopherol content and composition in TNDH and RC-F₂ populations. Of these, seven QTL co-localized with candidate sequences associated with tocopherol biosynthesis through <em>in silico</em> and linkage mapping. Several near-isogenic lines carrying introgressions from the parent with higher tocopherol content showed highly increased tocopherol content compared with the recurrent parent. Genome-wide association analysis was performed with 142 <em>B. napus</em> accessions. Sixty-one loci were significantly associated with tocopherol content and composition, 11 of which were localized within the confidence intervals of tocopherol QTL.</p> <h3>Conclusions/Significance</h3><p>This joint QTL, candidate gene, and association mapping study sheds light on the genetic basis of seed tocopherol biosynthesis in rapeseed. The sequences presented here may be used for marker-assisted selection of oilseed rape lines with superior tocopherol content and composition.</p> </div

Analysis of the upstream regions of <i>Brassica FLC</i> homologues and <i>AtFLC.</i>

<p>(A) Comparison of the potential conserved <i>cis</i>-blocks upstream of <i>AtFLC</i> and <i>Brassica FLC</i> (0 to −2880 bp) homologues. Different conserved segments (more than 75% sequence identity, and referred to as <i>cis</i>-blocks in the text) are shown as boxes with different shading and are numbered for comparison. Arrows at the 5′ end indicate the approximate position of the neighbour gene (<i>At5g10150</i> and its homologues; unfilled) or the gene fragment (grey) upstream of <i>FLC.</i> Dashed lines represent upstream sequences that were not determined. (B) Alignment of 30 bp sequences that contain putative <i>cis</i>-regulatory elements (G-box and CAAT-box) in <i>cis</i>-block 4 among <i>FLC</i> homologues. (C) Alignment for the sequences in <i>cis</i>-block 5 among <i>FLC</i> homologues. The position of the putative CAAT-box (in the minus strand) is shown.</p

A high MELD-XI score identified sicker patients with multiple preconditions.

<p>Normally distributed data points are expressed as mean ± standard deviation.</p