Genome-wide Association Study Identifies New Loci for Resistance to Leptosphaeria maculans in Canola

Blackleg, caused by Leptosphaeria maculans, is a significant disease which affects the sustainable production of canola. This study reports a genome-wide association study based on 18,804 polymorphic SNPs to identify loci associated with qualitative and quantitative resistance to L. maculans. Genomic regions delimited with 503 significant SNP markers, that are associated with resistance evaluated using 12 single spore isolates and pathotypes from four canola stubble were identified. Several significant associations were detected at known disease resistance loci including in the vicinity of recently cloned Rlm2/LepR3 genes, and at new loci on chromosomes A01/C01, A02/C02, A03/C03, A05/C05, A06, A08, and A09. In addition, we validated statistically significant associations on A01, A07 and A10 in four genetic mapping populations, demonstrating that GWAS marker loci are indeed associated with resistance to L. maculans. One of the novel loci identified for the first time, Rlm12, conveys adult plant resistance and mapped within 13.2 kb from Arabidopsis R gene of TIR-NBS class. We showed that resistance loci are located in the vicinity of R genes of A. thaliana and B. napus on the sequenced genome of B. napus cv. Darmor-bzh. Significantly associated SNP markers provide a valuable tool to enrich germplasm for favorable alleles in order to improve the level of resistance to L. maculans in canola

Evaluation and Recommendations for Routine Genotyping Using Skim Whole Genome Re-sequencing in Canola

Whole genome sequencing offers genome wide, unbiased markers, and inexpensive library preparation. With the cost of sequencing decreasing rapidly, many plant genomes of modest size are amenable to skim whole genome resequencing (skim WGR). The use of skim WGR in diverse sample sets without the use of imputation was evaluated in silico in 149 canola samples representative of global diversity. Fastq files with an average of 10x coverage of the reference genome were used to generate skim samples representing 0.25x, 0.5x, 1x, 2x, 3x, 4x, and 5x sequencing coverage. Applying a pre-defined list of SNPs versus de novo SNP discovery was evaluated. As skim WGR is expected to result in some degree of insufficient allele sampling, all skim coverage levels were filtered at a range of minimum read depths from a relaxed minimum read depth of 2 to a stringent read depth of 5, resulting in 28 list-based SNP sets. As a broad recommendation, genotyping pre-defined SNPs between 1x and 2x coverage with relatively stringent depth filtering is appropriate for a diverse sample set of canola due to a balance between marker number, sufficient accuracy, and sequencing cost, but depends on the intended application. This was experimentally examined in two sample sets with different genetic backgrounds: 1x coverage of 1,590 individuals from 84 Australian spring type four-parent crosses aimed at maximizing diversity as well as one commercial F1 hybrid, and 2x coverage of 379 doubled haploids (DHs) derived from a subset of the four-parent crosses. To determine optimal coverage in a simpler genetic background, the DH sample sequence coverage was further down sampled in silico. The flexible and cost-effective nature of the protocol makes it highly applicable across a range of species and purposes

Permeabilization and cell viability of hyphae treated with 50 μg/ml pterostilbene.

<p>Uptake over time of 0.5 μM SYTOX green and 1μg/ml fluorescein diacetate (FDA) by hyphae cultures. Left axis: relative SYTOX green fluorescence (fold-change). Right axis: relative FDA fluorescence (%). Vertical bars represent standard deviation. All values were statistically significant at <i>p</i><0.01 in Student’s <i>t</i>-test with degrees of freedom = 8.</p

Inhibition of conidia germination of five <i>L</i>. <i>maculans</i> isolates by pterostilbene.

<p>Conidia (1 x 10⁷ spores/ml) germination after 10 days on 2% water agar amended with 50 μg/ml pterostilbene (PTE) or solvent (control, CTRL).</p

Conidia germination of <i>L</i>. <i>maculans</i> on 2% water agar amended with resveratrol at various concentrations.

<p>Colonies were counted after 10 days, and germination is expressed as percentage (%) of number of colonies in the treatment relative to the control. Statistical significance in Student’s <i>t</i>-test at <i>p</i><0.05 (*) and <i>p</i><0.01 (**) with degrees of freedom = 4. Vertical bars represent standard deviation.</p

SYTOX green (0.5 μM) uptake into hyphae treated with 50 μg/ml pterostilbene.

<p>Bright field and fluorescent images for <b>(a)</b> control, <b>(b)</b> hyphae treated for 2 hours and <b>(c)</b> 4 hours. Black arrowheads indicate coagulation of the cytosol within the cytoplasm. White arrowheads indicate SYTOX green staining in the nuclei. Scale bar = 20 μm.</p

Inhibition of mycelial growth of <i>L</i>. <i>maculans</i> by the stilbenes resveratrol and pterostilbene.

<p>Hyphal plugs were cultured for six days on half strength V8 agar amended with resveratrol or pterostilbene at various concentrations. Mycelial growth is expressed as the percentage (%) of growth in the treatment relative to the control. Vertical bars represent standard deviation. Chemical structures of resveratrol and pterostilbene are shown above graph.</p

Sporicidal activity of pterostilbene against <i>L</i>. <i>maculans</i> spores.

<p>Growth after 10 days on 2% water agar of <b>(a)</b> 1 x 10⁷ spores/ml and <b>(b)</b> 1 x 10² spores/ml cultures. Conidia (1 x 10⁷ spores/ml) were treated with 50 μg/ml of pterostilbene (PTE) or solvent (control, CTRL) and serially diluted to 1 x 10² spores/ml.</p