Molecular evolutionary genetics.

<p>(A) Sequence diversity in the genomic region centered on <i>RKS1</i>. Red, blue and orange bars indicate non-synonymous mutations, silent mutations and indels, respectively. ‘35/46’ indicates the location of the 35 SNPs in complete linkage disequilibrium. The red cross indicates the position of the stop codon at the fourth amino-acid. Arrows indicate the orientation of the genes along the genome. (B) Observed nucleotide diversity between the two intergenic haplotypes. (C) Maximum Likelihood circular tree based on nucleotide variation of <i>RKS1</i> and the intergenic region between <i>RKS1</i> and <i>At3g57720</i>. (D) Scan for association with the relative gene expression of <i>RKS1-L</i> in the sequenced region centered on <i>RKS1</i>. Red points indicate the 35 SNPs in complete LD. The <i>y</i>-axis indicates the –log¹⁰<i>p</i>-values using the EMMAX method. (E) Tajima's <i>D</i> values along the 5 kb genomic region centered on <i>RKS1</i>. The dashed line denotes the 1% significance level of an empirical distribution based on 876 short fragments obtained for the same set of accessions <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003766#pgen.1003766-Nordborg1" target="_blank">[63]</a>.</p

<i>RKS1</i> confers resistance to multiple strains and races and pathovars of <i>Xcc</i>.

<p>Time course evaluation of disease index in lines differing only by the presence of <i>RKS1</i> gene (<i>rks1-1</i> mutant (blue), complemented <i>rks1-1</i> mutant (purple) and the parental lines Col-0 (green) and Kas-1 (red) after inoculation with different strains of (A) <i>Xcc</i> belonging to races as defined by Vicente <i>et al.. </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003766#pgen.1003766-Vicente2" target="_blank">[49]</a> and Fargier <i>et al.. </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003766#pgen.1003766-Fargier2" target="_blank">[50]</a>, and of (B) <i>Xc</i> pathovars <i>raphani</i> (<i>Xcr</i>), <i>armoriaceae</i> (<i>Xca</i>), <i>vesicatoria</i> (<i>Xcv</i>) and <i>incanae</i> (<i>Xci</i>).</p

Phenotypic analysis of insertional mutants corresponding to genes of the <i>QRX3</i> locus.

<p>(A) Structure of the kinase cluster contained within the <i>QRX3</i> locus and positions of the insertional mutations are indicated with vertical lines. (B) Disease symptoms were observed on leaves of mutant and wild-type plants, 10 days post-inoculation with a bacterial suspension adjusted to 2×10⁸ cfu/mL. (C) Time course evaluation of disease index after inoculation with <i>Xcc568</i> under the same conditions. Means and standard errors were calculated from 3–8 plants.</p

The genetics of <i>Xcc568</i> quantitative resistance at the species level identified by nested GWA mapping.

<p>(A) Violin plots (i.e. box-and-whisker plot overlaid with a kernel density plot) of phenotypic variation of our disease index. Whole-genome scan of 214,051 SNPs for association with disease index at 10 dpi across (B) 381 accessions, (C) within the allelic group SNP-3-21386192-T and (D) within the allelic group SNP-3-21386192-C.</p

Genetic evidence that <i>RKS1</i> is causal for <i>QRX3</i> QTL.

<p>Disease symptoms were observed on leaves of wild-type plants, mutants, HIF lines or lines complemented with the <i>RKS1</i> gene, at 7 (C) or 10 (A and B) days post-inoculation. Time course evaluation of our disease index was performed after inoculation with <i>Xcc568</i> under the same conditions. (A) Mutant complementation (lines #9, #E9, #F9). (B) amiRNA silencing (lines #23 and #24). (C) HIF line complementation (lines #105 and #106 for the susceptible HIF (HIF685), lines #107 and #110 for the resistant HIF (HIF1011)). Means and standard errors were calculated for 16–60 plants (4–9 independent experiments). (D) Bacterial growth measurement (colony forming unit (CFU)/cm² expressed in a log10 scale) in leaves of lines differing only by the presence of <i>RKS1</i> gene (wild type (Col-0), <i>rks1-1</i> mutant, and the complemented mutant line (#9)). The susceptible accession Kas-1 has been included as a positive control. Bacterial growth has been measured 0 (grey bars) and 7 (black bars) days after inoculation with <i>Xcc</i> strain 568. Data were collected from two independent experiments, each timepoint corresponds to 6 independent measurements, each on 3–5 individual plants (four leaves/plant).</p

Identification and mapping of the major QTL, <i>QRX3</i>, for resistance to <i>Xcc</i>.

<p>(A and B) Phenotype of susceptible (Kas-1) and resistant (Col-0) accessions (Col-5 and Col-0 (used here) show similar phenotypes) : (A) symptoms 7 days post-inoculation (dpi) and (B) bacterial invasion of leaf tissue using an <i>Xcc568</i> reporter strain that carries the <i>Photorhabdus luminescens</i> lux operon. (C) QTL maps of resistance to <i>Xcc</i> in the Col-5 x Kas-1 recombinant inbred line population at four inoculation times: yellow, 3 dpi; orange, 5 dpi; red, 7 dpi and purple, 10 dpi. The horizontal dotted line represents the significance threshold for the LOD score (average = 2.50). (D to F) Map-based isolation of the <i>QRX3</i> locus. (D) Genetic map of chromosome III is shown between markers <i>T04109</i> and <i>MS005</i> with the defined target interval for <i>QRX3</i> (in red). (E) A number of additional markers and recombinant lines were used to reduce the <i>QRX3</i> locus to a 44.9 kb region between the markers <i>3-57670</i> and <i>3-57810</i>. (F) The corresponding physical interval contains 17 open reading frames (ORFs). Genes are represented by arrows. The black arrows correspond to a cluster of putative kinase genes, the red arrow corresponds to <i>RKS1</i>.</p

<i>RKS1</i> allelic forms and expression in susceptible and resistant accessions of <i>Arabidopsis thaliana</i>.

<p>(A) Schematic representation of resistant (Col-0) and susceptible (Kas-1) <i>RKS1</i> allele polymorphisms. Sequence changes in both alleles are indicated. (B) Schematic representation of the most frequent 5′ and 3′ ends of <i>RKS1</i> transcripts found by 5′ and 3′ RACE experiments in resistant (Col-0, green) and susceptible (Kas-1, red) accessions. (C) <i>RKS1</i> gene expression evaluated by Q-RT-PCR in germinating seeds and in leaves, healthy or inoculated with <i>Xcc568</i>, from the resistant accession (Col-0, green) and the susceptible accession (Kas-1, red). Different primers are used to evaluate long (L) and long+short (L+S) transcripts (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003766#pgen.1003766.s018" target="_blank">Table S2</a>) A.U.: arbitrary units. (D) Correlation between <i>RKS1</i> gene expression after infection with <i>Xcc568</i> and resistance phenotype. The dashed line indicates an exponentially decreasing function fitted on the median values of the 13 types of genetic line. Numbers in brackets indicate the number of representatives of each type of transgenic line.</p

Pairs of trait with a latitudinal gradient of co-variation.

<p>Significance was tested with a linear regression model, with latitude as a dependent variable (see methods). p-values associated to the interaction between the traits and the percentage of the latitudinal variation explained by the interaction are shown below and above the diagonal, respectively.</p

Pearson correlation coefficients between life history traits (112 genotypes).

<p>Coefficient of correlation (<i>r</i>) and the Bonferroni corrected p-values are shown below and above the diagonal, respectively. Secondary dormancy is not correlated with other traits (not shown).</p

Histograms showing natural genetic variation of life history traits using adjusted means.

<p>(a) Primary dormancy (days): measured by number of days required to reach 50% of germination (DSDS50). (b) Secondary dormancy (% of germination decrease per day): measured by the reduction in germination rate of fully after-ripened seed after a 6-week long exposure to 4°C in darkness. (c) Vegetative growth rate (cm²/day): measured by increase of leaf area during one week in the greenhouse. (d) Flowering time (days): number of days until opening of the first flower.</p