Historical Introgression of the Downy Mildew Resistance Gene <i>Rpv12</i> from the Asian Species <i>Vitis amurensis</i> into Grapevine Varieties

<div><p>The Amur grape (<i>Vitis amurensis</i> Rupr.) thrives naturally in cool climates of Northeast Asia. Resistance against the introduced pathogen <i>Plasmopara viticola</i> is common among wild ecotypes that were propagated from Manchuria into Chinese vineyards or collected by Soviet botanists in Siberia, and used for the introgression of resistance into wine grapes (<i>Vitis vinifera</i> L.). A QTL analysis revealed a dominant gene <i>Rpv12</i> that explained 79% of the phenotypic variance for downy mildew resistance and was inherited independently of other resistance genes. A Mendelian component of resistance–a hypersensitive response in leaves challenged with <i>P. viticola</i>–was mapped in an interval of 0.2 cM containing an array of coiled-coil NB-LRR genes on chromosome 14. We sequenced 10-kb genic regions in the <i>Rpv12⁺</i> haplotype and identified polymorphisms in 12 varieties of <i>V. vinifera</i> using next-generation sequencing. The combination of two SNPs in single-copy genes flanking the NB-LRR cluster distinguished the resistant haplotype from all others found in 200 accessions of <i>V. vinifera</i>, <i>V. amurensis</i>, and <i>V. amurensis</i> x <i>V. vinifera</i> crosses. The <i>Rpv12⁺</i> haplotype is shared by 15 varieties, the most ancestral of which are the century-old ‘Zarja severa’ and ‘Michurinets’. Before this knowledge, the chromosome segment around <i>Rpv12⁺</i> became introgressed, shortened, and pyramided with another downy mildew resistance gene from North American grapevines (<i>Rpv3</i>) only by phenotypic selection. <i>Rpv12⁺</i> has an additive effect with <i>Rpv3⁺</i> to protect vines against natural infections, and confers foliar resistance to strains that are virulent on <i>Rpv3⁺</i> plants.</p> </div

Phenotypic distribution of downy mildew resistance.

<p>Two families segregating for <i>Rpv12⁺</i> (panel <b>A</b>) and for the combination of <i>Rpv12⁺</i> and <i>Rpv3⁺</i> (panel <b>B</b>) were analysed. Resistance scores in panel <b>A</b> are based on the OIV452 parameter (1 = most sensitive, 9 = most resistant) scored on field-grown seedlings under natural infection. Resistance scores in panel <b>B</b> are based on the cumulative OIV452 parameter (∑OIV452 = sum of daily OIV452 scores from 3 to 8 dpi) in artificially inoculated leaf discs. The average phenotypic value in the upper left corner of the panels <b>A</b>–<b>B</b> refers to individuals grouped by their allelic status at the <i>Rpv12</i> and <i>Rpv3</i> genes, which was estimated based on the flanking markers UDV014/UDV370 for <i>Rpv12</i>, and on UDV305/UDV737 for <i>Rpv3 </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061228#pone.0061228-DiGaspero1" target="_blank">[8]</a>. Recombinants in those intervals were excluded from this estimate. QTL plots that explain the phenotypic variance shown in panel <b>B</b> are given in panel <b>C</b>.</p

Host–pathogen interaction observed between host and pathogen genotypes.

<p>Leaf discs of four host genotypes including (panels <b>A</b>, <b>E</b>) a double homozygous recessive grapevine (<i>Rpv12⁻</i> and <i>Rpv3⁻</i>), (panels <b>B</b>, <b>F</b>) a grapevine carrying <i>Rpv3⁺</i> in the absence of <i>Rpv12⁺</i>, (panels <b>C</b>, <b>G</b>) a grapevine carrying <i>Rpv12⁺</i> in the absence of <i>Rpv3⁺</i>, and (panels <b>D</b>, <b>H</b>) a double heterozygous grapevine for both <i>Rpv12⁺</i> and <i>Rpv3⁺</i> were inoculated with two isolates of <i>P. viticola</i>, (panels <b>A</b>–<b>D</b>) <i>Rude</i> (<i>avrRpv3⁺</i>/<i>avrRpv12⁺</i>) and (panels <b>E</b>–<b>H</b>) <i>Pv127</i> (<i>avrRpv3⁻</i>/<i>avrRpv12⁺</i>). Pictures were taken at 6 dpi.</p

Additional file 3: of A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants

Comparison of predicted paired end distance to genome.Heatmaps of alignment distance scores for the alignment of the read pairs from the 9Kb long-insert mate-paired-end (LIMP) library to each of the 29 chromosomes within the Red5 whole genome assembly and. Individual chromosome plots were prepared using hagfish_blockplot from the software program ‘hagfish’ ( https://github.com/mfiers/hagfish/ ). Individual images were cropped for height (not length) then cut and pasted into a table format for easier viewing. Each image depicted the entire length of the chromosome but all images are of standard length irrespective of chromosome length. Green regions indicate mate pairs aligning to the whole genome sequence within the expected distance of the library. Black indicates regions without mate pair alignment. Pinkish-red indicates regions where the distance between mated paired end reads is shorter (assembly compression relative to physical genome) or longer (assembly expansion relative to physical genome). (PPTX 432 kb

Additional file 8: of A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants

Sequenced cDNA’s used to verify the gene models.Fasta formatted predicted protein sequences of 550 bidirectionally sequenced expressed sequence tag clones from A. chinensis var. chinensis used in evaluating manually annotated gene models of Red5. (FASTA 220 kb

Additional file 4: of A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants

BLASTP comparison of manually edited gene models to the revised ‘Hongyang’ gene models. List of best reciprocal BLASTp matches between the revised Actinidia chinensis ‘Hongyang’ genes [18]and the Red5 gene set (XLSX 436 kb

Additional file 7: of A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants

Details of sequenced cDNA’s generated. Fasta formatted sequences of 812 bidirectionally sequenced expressed sequence tag clones from A. chinensis var. chinensis used in evaluating manually annotated gene models of Red5. (FASTA 1204 kb

Additional file 6: of A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants

Revised ‘Hongyang’ genes omitted from the manually edited gene set. Average RNA-Seq read coverage of the 1069 KIR V2 models perfectly aligned to the Red5 genome without a protein match in the Red5 gene set. (XLSX 114 kb

Additional file 1: of A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants

Map back rates to the Red5 genome sequence.Summary of the numbers of input reads reads that align to the RED5 genome construction (XLSX 10 kb

Additional file 10: of A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants

The manual annotation process.Flow diagram of manual annotation process. A. Timeline showing the manual annotation process. *see materials and methods. B. Annotation followed a 5 step process. The annotator training was completed in the form of both workshops and YouTube training videos. ** https://www.youtube.com/playlist?list=PLcBe8nhQVgUg1zqOsdeRuVq9QVsLfj_Y9 . (PPTX 47 kb