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Development of a high-resolution melting approach for reliable and cost-effective genotyping of PPVres locus in apricot (P. armeniaca)

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Abstract

Sharka, caused by plum pox virus, is the most important viral disease of stone fruits. Important progresses have been recently achieved in apricot (Prunus armeniaca), identifying a major locus on chromosome 1 which explains most of the variability for plum pox virus (PPV) resistance trait. A set of molecular markers associated with the resistance has been developed and validated in different genetic backgrounds, endorsing their application for breeding purposes. Particularly for complex traits as the PPV resistance, requiring long and expensive phenotyping procedures, marker-assisted selection (MAS) bears a great potential to improve the efficiency of conventional breeding. In this work, novel HRM (high-resolution melting) assays were designed for the genotyping of resistant/susceptible alleles at PPV resistance (PPVres) locus. The assays were tested on 51 apricot cultivars and breeding selections already phenotyped for PPV resistance and cross-validated with standard short simple repeat marker data. We demonstrated that three HRM assays, PGS1.21_SNP, PGS1.24_SNP, and ZP002_DEL, represent a reliable, quick, and cost-effective genotyping approach, particularly suitable as high-throughput screening method for large-scale breeding programs.

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Acknowledgments

The authors wish to thank Stéphane Decroocq for providing SNP information and whole-genome sequencing data of 66 apricot accessions, funded by the France project ANR ABRIWG. The authors wish to thank Dr.ssa Lecchi Cristina (UNIMI, Milan, Italy) for technical assistance in HRM analysis and S. Foschi and M. Lama (CRPV, Cesena, Italy) for technical assistance in field operations.

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Authors and Affiliations

  1. Department of Agricultural and Environmental Sciences (DISAA), University of Milan, via Celoria 2, 20133, Milan, Italy

    Marco Passaro, Filippo Geuna, Daniele Bassi & Marco Cirilli

Authors
  1. Marco Passaro
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  2. Filippo Geuna
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  3. Daniele Bassi
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  4. Marco Cirilli
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Corresponding author

Correspondence to Marco Cirilli.

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Fundings

This work was supported by the MAS.PES (Italian project aimed at apricot and peach breeding, http://www.maspes.org/) and by FP7 project MARS (European project for Marker Assisted Resistance to Sharka, https://www6.inra.fr/mars/) (EC# 613654).

Electronic supplementary material

Supplemental Fig. 1

Derivative melting profiles of the four HRM assays (ZP002_DEL, PGS1.24_SNP, PGS1.21_SNP, and ZP002_SNP) as simulated in silico by using uMELT-HETS and uMELT-BATCH melting prediction tools. (JPEG 27 kb)

High resolution image (TIFF 395 kb)

Supplemental Fig. 2

Derivative fluorescence melting curve analyses for the marker ZP002_DEL on a 7300 Real Time PCR instrument (Thermo Scientific, USA) on two biological replicates of the samples ‘BO03615049’, ‘Lito’ and ‘Portici’ (left panel) and on a set of 48 among apricot accessions/breeding selections carrying on different dosage of the resistant alleles (right panel). Only a subset of the samples are depicted on the right panel to improve the visualization of the melting curves. (JPEG 13 kb)

High resolution image (TIFF 214 kb)

Supplemental Fig. 3

Amplicon size confirmation by 3% agarose-gel electrophoresis for the four tested markers ZP002_DEL, PGS1.24_SNP, PGS1.21_SNP and ZP002_SNP. (JPEG 7 kb)

High resolution image (TIFF 344 kb)

Supplemental Fig. 4

Derivative fluorescence and normalized fluorescence profiles of the HRM analysis for the markers ZP002_DEL (A, B), PGS1.24_SNP (C, D) and PGS1.21_SNP (E, F) on a set of 48 samples among apricot accessions/breeding selections carry on different dosage of the resistant alleles: homozygote resistant allele (brown lines), heterozygote (green lines) or homozygote for the susceptible allele (blue lines). (JPEG 45 kb)

High resolution image (TIFF 892 kb)

Supplemental Fig. 5

Frequencies of PPV resistant (no symptoms, RT-PCR negative), tolerant (no symptoms, RT-PCR positive) and susceptible individuals in an F1 progenies from the cross ‘Lito’ (resistant parents, heterozygous for the resistant allele at the three marker loci ZP002_DEL, PGS1.24_SNP, PGS1.21_SNP) x ‘BO81604311’ (susceptible parents). The presence/absence of the resistant/susceptible allele at all the three marker loci is indicate with R and S haplotype, respectively. (JPEG 13 kb)

High resolution image (TIFF 177 kb)

Supplemental Table 1

Name, origin, phenotype and genotype of the 51 among accessions and breeding selections sampled from CRPV (Centro Ricerche Produzioni Vegetali) experiment station (Faenza, Italy). 1Babini A.R., Phytosanitary Service, CRPV; 2Martínez-Gómez et al. (2003); 3Stylianidis et al. (2005); 4Rankovic et al. (1999); and 5Trandafirescu et al. (2013). (DOCX 19 kb)

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Passaro, M., Geuna, F., Bassi, D. et al. Development of a high-resolution melting approach for reliable and cost-effective genotyping of PPVres locus in apricot (P. armeniaca). Mol Breeding 37, 74 (2017). https://doi.org/10.1007/s11032-017-0666-0

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