Percent sequence variation within and between <i>Capsella spp</i>.

<p>Neutral variation within and between <i>Capsella</i> populations. Percent sequence differences at synonymous sites averaged across pairs of individuals within and between <i>C. rubella</i> and <i>C. grandiflora</i>. This matrix is symmetric and comparisons between partially overlapping sets (e.g.<i>C. rubella</i> x Greek <i>C. rubella</i>) are noted as ‘NA’. Redundant cells above the main diagonal are intentionally left blank.</p

Variation within and among <i>C. rubella</i>'s founding haplotypes.

<p><i>A)</i> Pairwise nucleotide diversity () within and among <i>C. rubella</i>'s founding haplotypes at synonymous sites (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003754#pgen.1003754.s013" target="_blank">Table S3</a> for values). <i>B)</i> Ratio of nucleotide diversity at non-synonymous relative to synonymous sites () within and among <i>C. rubella</i>'s founding haplotypes. Error bars mark the upper and lower 2.5% tails and are generated by resampling blocks assigned to different (left hand side) or same (right hand side) founding haplotypes. In the top panel (A and B), orange, green, and blue horizontal lines are drawn for reference to interspecific comparisons, comparisons within <i>C. grandiflora</i>, and genome-wide <i>C. rubella</i> comparisons, respectively (taken from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003754#pgen-1003754-t001" target="_blank">Table 1</a>). <i>C)</i> Neighbor joining trees in <i>Capsella</i>, using all comparisons (<i>C.1</i>), comparisons within (<i>C.2</i>), or among (<i>C.3</i>) founding haplotypes to generate entries in the pairwise distance matrix for comparisons within <i>C. rubella</i>. All distances are generated from nucleotide diversity at synonymous sites.</p

A summary of our coalescent model of the history of <i>C. rubella.</i>

<p><i> A)</i> The relative composite log-likelihood surface as function of and . <i>B)</i> The probability that all individuals coalesce to the same founding haplotype () as a function of and three estimates of values (the MLE, lower and upper confidence intervals). The dotted red line indicates the value of () directly estimated from the data. <i>C)</i> A summary of simulation results (assuming ). <i>C1)</i> The frequency of singletons, doubletons, and tripletons observed in simulation (full lines), and estimated from our data (dashed lines) conditional on all four samples deriving from the same founding haplotype. <i>C2)</i> The frequency of one, two, three or four lineages surviving to the founding event. When is large, is the probability that all samples coalesce to the same founding haplotype. The dotted black line portrays the estimated frequency of all four samples residing on one founding haplotype . <i>D)</i> The estimated current effective number of chromosomes in <i>C. rubella</i> () as a function of the number of founding chromosomes (). We plot this for three different values of (the MLE, as well as the lower and upper confidence intervals). These results are robust to haplotype labeling criteria in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003754#pgen-1003754-g006" target="_blank">Figure 6</a> (see <i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003754#pgen.1003754.s014" target="_blank">Text S1</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003754#pgen.1003754.s006" target="_blank">Figure S6</a></i>).</p

The founding of <i>C. rubella</i> and the identification of its founding haplotypes.

<p><i>A)</i> A cartoon coalescent model of <i>C. rubella</i>'s origin. At time, , a population ancestral to <i>C. rubella</i> is formed by sampling chromosomes (i.e. haplotypes, haps) from a large outcrossing population ancestral to both species, and this selfing population quickly recovers to size, . Because some of the lineages are lost to drift, we can identify the founding haplotypes surviving to the present, which we color in red and blue. While recombination scrambles ancestral chromosomes in <i>C. grandiflora</i>, the low effective recombination rate in <i>C. rubella</i> ensures that large chunks of founding haplotypes remain intact. <i>B)</i> We aim to identify these founding haplotypes by using patterns of sequence variation (see text and <i>METHODS</i> for details of our algorithm). Here, we present an example of founding haplotype identification in a typical genomic region. To aid visualization, we label the major allele in <i>C. rubella</i> as ‘0’, and the allele that is rare or absent <i>C. rubella</i> as ‘1’, and only display genotypes at sites with common variants in <i>C. grandiflora</i>. In the left hand side of <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003754#pgen-1003754-g001" target="_blank">Figure 1B</a>, there are clearly two distinct founding haplotypes on the basis of patterns of variation at sites polymorphic in both species. On the right hand side, all <i>C. rubella</i> individuals are identical at sites polymorphic in <i>C. grandiflora</i>, so we infer a single founding haplotype.</p

Diversity across chromosome seven in <i>C. rubella</i>.

<p>Mean pairwise synonymous diversity (purple, upward pointing lines) and major founding haplotype frequency (orange, downward pointing lines) across chromosome seven. Red points mark regions putatively containing more than two extant founding haplotypes. Values of and major founding haplotype frequency are averaged across overlapping sliding windows (ten kb windows with a two kb slide), here only windows with data for sites of pairwise comparisons are evaluated. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003754#pgen.1003754.s007" target="_blank">Figure S7</a>, for plots of all chromosomes.</p

The allele frequency spectrum within <i>C. rubella</i>'s founding haplotypes.

<p>The proportion of polymorphic derived alleles within a founding haplotype observed as singletons or doubletons, split by geography and synonymy. Light and dark blue represent comparisons within Greek and Out-of-Greece samples, respectively. Filled and hatched bars represent synonymous and non-synonymous sites, respectively. Error bars represent the upper and lower 2.5% tails of the allele frequency spectrum when founding haplotypes are resampled with replacement. Grey lines represent expectations of a model for neutral mutations at mutation-drift equilibrium.</p

Linked neutral diversity and divergence as a function of distance from fixed substitutions across the <i>C. grandiflora</i> genome.

<p>A) Diversity at 4-fold degenerate sites, B) Divergence at 4-fold degenerate sites, and C) Diversity/divergence at 4-fold degenerate sites. In all figures, black lines represent measures surrounding fixed replacement substitutions and gray shading represents 95% confidence intervals, from bootstrapping, surrounding silent substitutions.</p

Estimates of negative and positive selection on coding and noncoding sites in <i>C. grandiflora</i>.

<p>A) The proportion of sites found in each bin of purifying selection strength, separated by site type, B) The proportion of divergent sites fixed by positive selection, and C) the rate of adaptive substitution relative to neutral divergence. Error bars represent 95% bootstrap confidence intervals.</p

Linked neutral diversity/divergence surrounding conserved noncoding sequences (CNSs).

<p>A) Diversity/divergence at 4-fold degenerate sites as a function of distance from fixed substitutions in CNSs (black lines) and fixed substitutions in non-conserved intergenic sequence (gray shading, 95% confidence interval). B) Diversity/divergence at 4-fold degenerate sites as a function of distance from CNSs containing fixed substitutions (black line) and CNSs without any fixed substitutions (gray shading, 95% confidence interval).</p

Image3.JPEG

<p>Sodium (Na⁺) accumulation in the cytosol will result in ion homeostasis imbalance and toxicity of transpiring leaves. Studies of salinity tolerance in the diploid wheat ancestor Triticum monococcum showed that HKT1;5-like gene was a major gene in the QTL for salt tolerance, named Nax2. In the present study, we were interested in investigating the molecular mechanisms underpinning the role of the HKT1;5 gene in salt tolerance in barley (Hordeum vulgare). A USDA mini-core collection of 2,671 barley lines, part of a field trial was screened for salinity tolerance, and a Genome Wide Association Study (GWAS) was performed. Our results showed important SNPs that are correlated with salt tolerance that mapped to a region where HKT1;5 ion transporter located on chromosome four. Furthermore, sodium (Na⁺) and potassium (K⁺) content analysis revealed that tolerant lines accumulate more sodium in roots and leaf sheaths, than in the sensitive ones. In contrast, sodium concentration was reduced in leaf blades of the tolerant lines under salt stress. In the absence of NaCl, the concentration of Na⁺ and K⁺ were the same in the roots, leaf sheaths and leaf blades between the tolerant and the sensitive lines. In order to study the molecular mechanism behind that, alleles of the HKT1;5 gene from five tolerant and five sensitive barley lines were cloned and sequenced. Sequence analysis did not show the presence of any polymorphism that distinguishes between the tolerant and sensitive alleles. Our real-time RT-PCR experiments, showed that the expression of HKT1;5 gene in roots of the tolerant line was significantly induced after challenging the plants with salt stress. In contrast, in leaf sheaths the expression was decreased after salt treatment. In sensitive lines, there was no difference in the expression of HKT1;5 gene in leaf sheath under control and saline conditions, while a slight increase in the expression was observed in roots after salt treatment. These results provide stronger evidence that HKT1;5 gene in barley play a key role in withdrawing Na⁺ from the xylem and therefore reducing its transport to leaves. Given all that, these data support the hypothesis that HKT1;5 gene is responsible for Na⁺ unloading to the xylem and controlling its distribution in the shoots, which provide new insight into the understanding of this QTL for salinity tolerance in barley.</p