Rapid identification of candidate genes for resistance to tomato late blight disease using next-generation sequencing technologies

<div><p>Tomato late blight caused by <i>Phytophthora infestans</i> (Mont.) de Bary, also known as the Irish famine pathogen, is one of the most destructive plant diseases. Wild relatives of tomato possess useful resistance genes against this disease, and could therefore be used in breeding to improve cultivated varieties. In the genome of a wild relative of tomato, <i>Solanum habrochaites</i> accession LA1777, we identified a new quantitative trait locus for resistance against blight caused by an aggressive Egyptian isolate of <i>P</i>. <i>infestans</i>. Using double-digest restriction site–associated DNA sequencing (ddRAD-Seq) technology, we determined 6,514 genome-wide SNP genotypes of an F₂ population derived from an interspecific cross. Subsequent association analysis of genotypes and phenotypes of the mapping population revealed that a 6.8 Mb genome region on chromosome 6 was a candidate locus for disease resistance. Whole-genome resequencing analysis revealed that 298 genes in this region potentially had functional differences between the parental lines. Among of them, two genes with missense mutations, Solyc06g071810.1 and Solyc06g083640.3, were considered to be potential candidates for disease resistance. SNP and SSR markers linking to this region can be used in marker-assisted selection in future breeding programs for late blight disease, including introgression of new genetic loci from wild species. In addition, the approach developed in this study provides a model for identification of other genes for attractive agronomical traits.</p></div

Representation of high-confidence single nucleotide polymorphism (SNP) markers along chromosome 6 of tomato mapped on SL3.0 version of the tomato reference genome.

<p>Candidate genomic region tightly related to plant disease resistance was predicted on ch06 based on SnpEff annotation, (A) the double-digest restriction site–associated DNA sequencing (ddRAD-Seq), and (B) the whole-genome shotgun resequencing (WGRS) technologies. The remaining chromosomes ch00 –ch12 are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189951#pone.0189951.s001" target="_blank">S1 Fig</a>.</p

Genotyping of F₂ mapping population with five EST-SSR markers.

<p>Genotyping of F₂ mapping population with five EST-SSR markers.</p

Manhattan plots for genome-wide association studies of generalized linear model (GLM) analysis of late blight disease resistance using TASSEL software.

<p>The SNP markers were generated using NGS technology, double-digest restriction site–associated DNA sequencing (ddRAD-Seq).</p

Disease severity rating 0–6 of F₂ mapping population (n = 344) of the cross cv. Castlerock (<i>S</i>. <i>lycopersicum</i>) x <i>S</i>. <i>habrochaites</i> accession LA1777 to aggressive Egyptian isolate of <i>P</i>. <i>infestans</i>.

<p>Disease severity rating 0–6 of F₂ mapping population (n = 344) of the cross cv. Castlerock (<i>S</i>. <i>lycopersicum</i>) x <i>S</i>. <i>habrochaites</i> accession LA1777 to aggressive Egyptian isolate of <i>P</i>. <i>infestans</i>.</p

Screening the parental lines for resistance to <i>P</i>. <i>infestans</i> isolate EG_12 using whole-plant assay under controlled conditions.

<p>(A) Highly susceptible parent cv. Castlerock, (B) highly resistant parent <i>S</i>. <i>habrochaites</i> accession LA1777.</p