Fine mapping and identification of candidate genes for the peach powdery mildew resistance gene Vr3

Abstract

Powdery mildew is one of the major diseases of peach (Prunus persica), caused by the ascomycete Podosphaera pannosa. Currently, it is controlled through calendar-based fungicide treatments starting at petal fall, but an alternative is to develop resistant peach varieties. Previous studies mapped a resistance gene (Vr3) in interspecific populations between almond (‘Texas’) and peach (‘Earlygold’). To obtain molecular markers highly linked to Vr3 and to reduce the number of candidate genes, we fine-mapped Vr3 to a genomic region of 270 kb with 27 annotated genes. To find evidence supporting one of these positional candidate genes as being responsible of Vr3, we analyzed the polymorphisms of the resequences of both parents and used near-isogenic lines (NILs) for expression analysis of the positional candidate genes in symptomatic or asymptomatic leaves. Genes differentially expressed between resistant and susceptible individuals were annotated as a Disease Resistance Protein RGA2 (Prupe2G111700) or an Eceriferum 1 protein involved in epicuticular wax biosynthesis (Prupe2G112800). Only Prupe2G111700 contained a variant predicted to have a disruptive effect on the encoded protein, and was overexpressed in both heterozygous and homozygous individuals containing the Vr3 almond allele, compared with susceptible individuals. This information was also useful to identify and validate molecular markers tightly linked and flanking Vr3. In addition, the NILs used in this work will facilitate the introgression of this gene into peach elite materials, alone or pyramided with other known resistance genes such as peach powdery mildew resistance gene Vr2.

References

[1] Arús, P., Verde, I., Sosinski, B., Zhebentyayeva, T. & Abbott, A. G. The peach genome. Tree Genet. Genomes 8, 531-547 (2012).

[2] Verde, I. et al. The high-quality draft genome of peach (Prunus persica) identifies unique patterns of genetic diversity, domestication and genome evolution. Nat. Genet. 45, 487-494 (2013).

[3] Peace, C. DNA-informed breeding of rosaceous crops: promises, progress and prospects. Hortic. Res. 4, 17006 (2017).

[4] FAOSTAT. FAO Corporate statistical database. Food and Agriculture Organization of the United Nations. http://www.fao.org/faostat/en/#data/QC (2018).

[5] Pascal, T., Pfeiffer, F. & Kervella, J. Powdery mildew resistance in the peach cultivar Pamirskij 5 is genetically linked with the Gr gene for leaf color. HortScience 45, 150-152 (2010).

[6] Pascal, T. et al. Mapping of a new resistance (Vr2, Rm1) and ornamental (Di2, pl) Mendelian trait loci in peach. Euphytica 213, 132 (2017).

[7] Dirlewanger, E., Pascal, T., Zuger, C. & Kervella, J. Analysis of molecular markers associated with powdery mildew resistance genes in peach (Prunus persica (L.) Batsch) Prunus davidiana hybrids. Theor. Appl. Genet. 93, 909-919 (1996).

[8] Foulongne, M., Pascal, T., Pfeiffer, F. & Kervella, J. QTLs for powdery mildew resistance in peach x Prunus davidiana crosses: consistency across generations and environments. Mol. Breed. 12, 33-50 (2003).

[9] Grove, G. G. In Compendium of stone fruit diseases (eds Ogawa, J. M. et al. ) Powdery Mildew (APS Press, 1995).

[10] Marimon, N., Eduardo, I., Martínez-Minaya, J., Vicent, A. & Luque, J. A decision support system based on degree-days to initiate fungicide spray programs for peach powdery mildew in Catalonia, Spain. Plant Dis. https://doi.org/10.1094/PDIS-10-19-2130-RE (2020).

[11] Aranzana, M. J. et al. Prunus genetics and applications after de novo genome sequencing: achievements and prospects. Hortic. Res. 6, 58 (2019).

[12] Donoso, J. M. et al. Exploring almond genetic variability useful for peach improvement: mapping major genes and QTLs in two interspecific almond x peach populations. Mol. Breed. 36, 16 (2016).

[13] Pacheco-Cruz, I., Eduardo, I., Rossini, L., Vecchietti, A., Bassi, D. QTL mapping for peach (Prunus persica L. Batsch) resistance to powdery mildew and brown rot. In Proc 53rd Italian Society of Agricultural Genetics Annual Congress (2009).

[14] Dabov, S. Inheritance of powdery mildew resistance in the peach. IV. Data supporting the hypothesis about the main role of 2 loci controlling the reaction to the pathogen. Genet. Sel. 16, 349-355 (1983).

[15] Saunier, R. Contribution to the study of relationships between certain characteristics of simple genetic determination in the peach tree and susceptibility of peach cultivars to oidium, Sphaerotheca pannosa (Wallr.) Lev. Ann. Amelior. Plant. 23, 235-243 (1973).

[16] Verde, I. et al. The Peach v2.0. release: high-resolution linkage mapping and deep resequencing improve chromosome-scale assembly and contiguity. BMC Genom. 18, 225 (2017).

[17] Jung, S. et al. 15 years of GDR: New data and functionality in the Genome Database for Rosaceae. Nucleic Acids Res. 47, D1137-D1145 (2019).

[18] Cingolani, P. et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff. Fly 6, 80-92 (2012).

[19] Chen, X. R., Brurberg, M. B., Elameen, A., Klemsdal, S. S. & Martinussen, I. Expression of resistance gene analogs in woodland strawberry (Fragaria vesca) during infection with Phytophthora cactorum. Mol. Genet. Genom. 291, 1967-1978 (2016).

[20] Calenge, F. & Durel, C. E. Both stable and unstable QTLs for resistance to powdery mildew are detected in apple after four years of field assessments. Mol. Breed. 17, 329-339 (2006).

[21] Kim, J. et al. A genome-wide comparison of NB-LRR type of resistance gene analogs (RGA) in the plant kingdom. Mol. Cells 33, 385-392 (2012).

[22] Lalli, D. A. et al. Identification and mapping of resistance gene analogs (RGAs) in Prunus: a resistance map of Prunus. Theor. Appl. Genet. 111, 1504-1513 (2005).

[23] Dhanyalakshmi, K. H., Soolanayakanahally, R. Y., Rahman, T., Tanino, K. K. & Nataraja, K. N. In Abiotic and Biotic Stress in Plants (eds. Bosco de Oliveira, A.) Leaf cuticular wax, a trait for multiple stress resistance in crop plants (IntechOpen, 2019).

[24] Zong, X., Denler, B. J., Danial, G. H., Chang, Y. & Song, G. Adventitious shoot regeneration and Agrobacterium tumefaciens-mediated transient transformation of almond × peach hybrid rootstock ‘Hansen 536’. Hort. Sci. 54, 936-940 (2019).

[25] Petri, C., Scorza, R., Srinivasan, C. In Transgenic Plants, Vol. 847 (eds. Dunwell J., Wetten A.) Highly efficient transformation protocol for plum (Prunus domestica L.) methods and protocols (Humana Press, 2012).

[26] He et al. Pm21, encoding a typical CC-NBS-LRR protein, confers broad-spectrum resistance to wheat powdery mildew disease. Mol. Plant. 11, 879-882 (2018).

[27] Serra, O. et al. Marker-assisted introgression (MAI) of almond genes into the peach background: a fast method to mine and integrate novel variation from exotic sources in long intergeneration species. Tree Genet. Genomes 12, 96 (2016).

[28] Doyle, J. J. & Doyle, J. L. Isolation of plant DNA from fresh tissue. Focus 12, 13-15 (1990).

[29] Serra, O. Towards increasing genetic variability and improving fruit quality in peach using genomic and bioinformatic tools (Doctoral dissertation, Universitat Autònoma de Barcelona, Spain). www.tdx.cat/handle/10803/460882 (2017).

[30] Lindgreen, S. AdapterRemoval: Easy cleaning of Next Generation Sequencing reads. BMC Res. Notes 5, 337 (2012).

[31] Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler Transform. Bioinformatics 25, 1754-1760 (2009).

[32] Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078-2079 (2009).

[33] Robinson, J. T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24-26 (2011).

[34] Untergasser, A. et al. Primer 3-new capabilities and interfaces. Nucleic Acids Res. 40, e115 (2012).

[35] Hayden, M. J., Nguyen, T. M., Waterman, A. & Chalmers, K. J. Multiplex-ready PCR: a new method for multiplexed SSR and SNP genotyping. BMC Genom. 9, 80-85 (2008).

[36] Román, B., Gómez, P., Picó, B., López, C. & Janssen, D. Candidate gene analysis of tomato leaf curl New Delhi virus resistance in Cucumis melo. Sci. Hortic. 243, 12-20 (2019).

[37] Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403-410 (1990).

[38] Zuker, M . Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406-3415 (2003).

[39] Rubio, M. et al. Analysis of gene expression changes in peach leaves in response to Plum pox virus infection using RNA-Seq. Mol. Plant Pathol. 16, 164-176 (2015).

[40] Zúñiga, E., Luque, J. & Martos, S. Lignin biosynthesis as a key mechanism to repress Polystigma amygdalinum, the causal agent of red leaf blotch disease in almond. J. Plant Physiol. 236, 96-104 (2019).

[41] Tong, Z., Gao, Z., Wang, F., Zhou, J. & Zang, Z. Selection of reliable reference genes for gene expression studies in peach using real-time PCR. BMC Mol. Biol. 10, 71 (2009).

[42] Rancurel, C., van Tran, T., Elie, C. & Hilliou, F. SATQPCR: Website for statistical analysis of real-time quantitative PCR data. Mol. Cell. Probe 46, 101418 (2019).

[43] Vandesompele, J. et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 18, 7 (2002).

[44] Bustin, S. A. et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55, 611-622 (2009).

[45] R Core Team . R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, Vienna, Austria, 2020).