Abstract
We identified haplotype-tagging insertion/deletions (InDels) for downy mildew resistance (Rpv3-1) in grapevine and converted them into InDel markers. InDel-25,941 and InDel-26,032 were validated by fragment analysis via capillary electrophoresis in 174 varieties of Vitis vinifera, 50 resistant varieties of the ‘Seibel 4614’ lineage that share Rpv3-1 by descent, and in 83 Vitis accessions. Amplicon sequencing of ancestral and derived alleles revealed that both mutations were caused by deletions. The 25,941-deletion is most likely recent. The derived allele is present only in resistant varieties obtained from ‘Seibel 4614’ and has originated in North American populations through two successive deletions within a predicted multiple stem-loop ssDNA structure, consisting of three nearby short inverted repeats, which shortened the ancestral DNA stepwise. The 26,032-deletion is more ancient. The derived allele is always present in resistant varieties of the ‘Seibel 4614’ lineage, completely absent from V. vinifera, not found in other North American accessions, and rarely present in Asian species. It may have originated in a common ancestral population before the continental disjunction, followed by incomplete lineage sorting, or in either lineage followed by introgression via secondary contacts. Genotyping with these markers does not require special instruments or chemistry for routine screening in breeding practice. Differences in amplicon size between grapes that carry or do not carry Rpv3-1 are detectable via standard agarose gel electrophoresis, or classical melting curve analysis using nonsaturating fluorescent dyes. The recombination rate between each marker and the trait locus is 0.118% for InDel-25,941 and 0.071% for InDel-26,032.
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Acknowledgements
Grape accessions were provided by the following germplasm repositories: Consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, Italy; INRA Unité Expérimentale du Domaine de Vassal, France; Julius Kuhn Institute, Germany; Kmetijsko Gozdarski Zavod Nova Gorica, Slovenia; University of Udine, Italy; and Vivai Cooperativi Rauscedo, Italy (VCR). Chin-Feng Hwang, Dániel Pap, and Laszlo Kovács (Missouri State University) kindly provided DNA samples of V. aestivalis ‘Cynthiana’ and V. rupestris natural populations. We thank Irena Jurman, Federica Cattonaro, Nicola Ietri, and Giacomo Prete for DNA sequencing.
Funding
This research was partially funded by the EU-FP7 Innovine project (grant agreement no. 311775) and partially by the FP7-IDEAS-ERC Novabreed grant (agreement no. 294780).
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ESM 1
Genotypes of 307 grape accessions at InDel-25,941 and InDel-26,032, obtained by separation in capillary electrophoresis and fragment analysis with GeneMapper using default settings. (XLSX 22 kb)
ESM 2
Vitis accessions from species typical of the Midwestern US: V. rupestris, V. lincecumii, V. champinii, V. arizonica, V. aestivalis, V. longii, V. slavinii, V. doaniana, V. girdiana. (PDF 545 kb)
ESM 3
Genotypes of Vitis rupestris accessions from natural populations in Oklahoma and Missouri. (XLSX 11 kb)
ESM 4
Detailed information on InDel primers and PCR amplicons. (XLSX 12 kb)
ESM 5
Size distribution of InDels identified in the ‘PN40024’ chromosomal interval chr18:25,876,014..25,963,847 in comparison with the collinear region of BAC clone 10G13 from ‘Bianca’. Panel A shows small InDels predicted by GATK UnifiedGenotyper. Panel B shows other InDels predicted by DELLY2. (JPG 968 kb)
ESM 6
Size distribution of InDels identified in the ‘PN40024’ chromosomal intervals chr18:26,032,019..26,032,971; chr18:26,033,569..26,033,898; and chr18:26,034,098..26,034,354 in comparison with amplicons obtained from genomic DNA of ‘UD-21,076’. (JPEG 683 kb)
ESM 7
Fragment analysis chromatograms for InDel-25,941 in a set of grape accessions representative of all genotypes found in the genus Vitis. Peak height is expressed in Relative Fluorescence Units (RFU). From top to bottom: Rpv3-1homozygous, KL1270/2/14, Seyve Villard 12–358, Pinot Noir, V. rupestris du Lot. (JPG 1378 kb)
ESM 8
Fragment analysis chromatograms for InDel-26,032 in a set of grape accessions representative of the major genotypes found in the genus Vitis. Peak height is expressed in Relative Fluorescence Units (RFU). From top to bottom: Rpv3–1homozygous, Seyve Villard 12–358, Teréz, Marandi Shemakhinskii, V. acerifolia, Sauvignon Blanc, Negroamaro, Pinot Noir. (JPG 1747 kb)
ESM 9
Melting peaks of InDel-25,941 (panel A) and InDel-26,032 (panel B) amplicons. (PDF 549 kb)
ESM 10
Alignment of InDel-25,941 amplicon sequences in homozygous individuals carrying the 248-bp, 247-bp, 228-bp, and 196-bp electromorphs. (PDF 400 kb)
ESM 11
Alignment of InDel-26,032 amplicon sequences in homozygous individuals carrying the 262-bp, 244-bp, 242-bp, and 178-bp electromorphs. (PDF 398 kb)
ESM 12
Predicted ssDNA secondary structures of InDel-26,032 alleles. Highlighted in cyan are nucleotides involved in insertion/deletion mutations. (JPG 1519 kb)
ESM 13
Diagram of recombinant chromosomes between InDel-25,941 or InDel-26,032 and the Rpv3–1 phenotype in eight individuals from 4221 segregant genotypes. (PDF 453 kb)
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Foria, S., Magris, G., Copetti, D. et al. InDel markers for monitoring the introgression of downy mildew resistance from wild relatives into grape varieties. Mol Breeding 38, 124 (2018). https://doi.org/10.1007/s11032-018-0880-4
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DOI: https://doi.org/10.1007/s11032-018-0880-4