Fire blight QTL analysis in a multi-family apple population identifies a reduced-susceptibility allele in ‘Honeycrisp’

Abstract

Breeding apple cultivars with resistance offers a potential solution to fire blight, a damaging bacterial disease caused by Erwinia amylovora. Most resistance alleles at quantitative trait loci (QTLs) were previously characterized in diverse Malus germplasm with poor fruit quality, which reduces breeding utility. This study utilized a pedigree-based QTL analysis approach to elucidate the genetic basis of resistance/susceptibility to fire blight from multiple genetic sources in germplasm relevant to U.S. apple breeding programs. Twenty-seven important breeding parents (IBPs) were represented by 314 offspring from 32 full-sib families, with ‘Honeycrisp’ being the most highly represented IBP. Analyzing resistance/susceptibility data from a two-year replicated field inoculation study and previously curated genome-wide single nucleotide polymorphism data, QTLs were consistently mapped on chromosomes (Chrs.) 6, 7, and 15. These QTLs together explained ~28% of phenotypic variation. The Chr. 6 and Chr. 15 QTLs colocalized with previously reported QTLs, while the Chr. 7 QTL is possibly novel. ‘Honeycrisp’ inherited a rare reduced-susceptibility allele at the Chr. 6 QTL from its grandparent ‘Frostbite’. The highly resistant IBP ‘Enterprise’ had at least one putative reduced-susceptibility allele at all three QTLs. In general, lower susceptibility was observed for individuals with higher numbers of reduced-susceptibility alleles across QTLs. This study highlighted QTL mapping and allele characterization of resistance/susceptibility to fire blight in complex pedigree-connected apple breeding germplasm. Knowledge gained will enable more informed parental selection and development of trait-predictive DNA tests for pyramiding favorable alleles and selection of superior apple cultivars with resistance to fire blight.

References

[1] Norelli, J. L., Jones, A. L. & Aldwinckle, H. S. Fire blight management in the twenty-first century using new technologies to enhance host resistance in apple. Plant Dis. 87, 756-765 (2003).

[2] Kellerhals, M., Schütz, S. & Patocchi, A. Breeding for host resistance to fire blight. J. Plant Pathol. 99, 37-43 (2017).

[3] Peil, A., Emeriewen, O. F., Khan, A., Kostick, S., & Malnoy, M. Status of fire blight resistance breeding in Malus. J. Plant Pathol. https://doi.org/10.1007/s42161-020-00581-8 (2020).

[4] Brown, S. in Fruit Breeding, Handbook of Plant Breeding (eds Badenes M. L., & Byrne, D. H.) Ch. 10 (Springer-Verlag, 2012).

[5] Lee, S. A. et al. Virulence characteristics accounting for fire blight disease severity in apple trees and seedlings. Phytopathology 100, 539-550 (2010).

[6] van der Zwet, T., Orolaza-Halbrendt, N., & Zeller, W. Fire Blight: History, Biology, and Management (APS Press, 2012).

[7] Kostick, S. A., Norelli, J. L., Teh, S. & Evans, K. M. Quantitative variation and heritability estimates of fire blight resistance in a pedigree-connected apple germplasm set. J. Plant Pathol. https://doi.org/10.1007/s42161-020-00543-0 (2020).

[8] Emeriewen, O. F., Wöhner, T., Flachowsky, H. & Peil, A. Malus hosts-Erwinia amylovora interactions: strain pathogenicity and resistance mechanisms. Front. Plant Sci. 10, 551 (2019).

[9] Calenge, F. et al. Identification of major QTL together with several minor additive or epistatic QTLs for resistance to fire blight in apple in two related progenies. Theor. Appl. Genet. 111, 128-135 (2005).

[10] Desnoues, E. et al. Identification of novel strain-specific and environment-dependent minor QTLs linked to fire blight resistance in apple. Plant. Mol. Biol. Rep. 36, 247-256 (2018).

[11] Durel, C.-E., Denancé, C. & Brisset, M. N. Two distinct major QTL for resistance to fire blight co-localize on linkage group 12 in apple genotypes ‘Evereste’ and Malus floribunda clone 821. Genome 52, 139-147 (2009).

[12] Emeriewen, O. F. et al. Evidence of a major QTL for fire blight resistance in apple wild species Malus fusca. Acta Hortic. 1056, 289-294 (2014).

[13] Emeriewen, O. F. et al. Fire blight resistance of Malus × arnoldiana is controlled by a quantitative trait locus located at the distal end of linkage group 12. Eur. J. Plant Pathol. 148, 1011-1018 (2017).

[14] Gardiner, S. E. et al. Putative resistance gene markers associated with quantitative trait loci for fire blight resistance in Malus ‘Robusta 5’ accessions. BMC Genet. 13, 25 (2012).

[15] Khan, M. A., Duffy, B., Gessler, C. & Patocchi, A. QTL mapping of fire blight resistance in apple. Mol. Breed. 17, 299-306 (2006).

[16] Khan, M. A., Zhao, Y. & Korban, S. S. Identification of genetic loci associated with fire blight resistance in Malus through combined use of QTL and association mapping. Physiol. Plant 148, 344-353 (2013).

[17] Le Roux, P.-M. F. et al. Mapping of quantitative trait loci for fire blight resistance in the apple cultivars ‘Florina’ and ‘Nova Easygro.’. Genome 53, 710-722 (2010).

[18] Peil, A. et al. Strong evidence for a fire blight resistance gene of Malus robusta located on linkage group 3. Plant Breed. 126, 470-475 (2007).

[19] Peil, A. et al. Mapping of fire blight resistance in Malus ×robusta 5 flowers following artificial inoculation. BMC Plant Biol. 19, 532 (2019).

[20] van de Weg, E. et al. Epistatic fire blight resistance QTL alleles in the apple cultivar ‘Enterprise’ and selection X-6398 discovered and characterized through pedigree-informed analysis. Mol. Breed. 38, 5 (2018).

[21] Daccord, N. et al. High-quality de novo assembly of the apple genome and methylome dynamics of early fruit development. Nat. Genet. 49, 1099-1108 (2017).

[22] Luo, F., Evans, K., Norelli, J. L., Zhang, Z. & Peace, C. Prospects for achieving durable disease resistance with elite fruit quality in apple breeding. Tree Genet. Genomes 16, 21 (2020).

[23] Kostick, S. A., Norelli, J. L. & Evans, K. M. Novel metrics to classify fire blight resistance of 94 apple cultivars. Plant Pathol. 68, 985-996 (2019).

[24] Evans, K. & Peace, C. in Achieving Sustainable Cultivation of Apples (ed. Evans, K. ) (Burleigh Dodds Science Publishing, 2017).

[25] Vanderzande, S. et al. Crossing the finish line: How to develop diagnostic DNA tests as breeding tools after QTL discovery. J. Hortic. 5, 228 (2018).

[26] Bink, M. C. A. M. et al. Bayesian QTL analyses using pedigreed families of an outcrossing species, with application to fruit firmness in apple. Theor. Appl. Genet. 127, 1073-1090 (2014).

[27] van de Weg, W. E. et al. Pedigree genotyping: a new pedigree-based approach of QTL identification and allele mining. Acta Hortic. 663, 45-50 (2004).

[28] Peace, C. P., Luby, J. J., van de Weg, W. E., Bink, M. C. A. M. & Iezzoni, A. F. A strategy for developing representative germplasm sets for systematic QTL validation, demonstrated for apple, peach, and sweet cherry. Tree Genet. Genomes 10, 1679-1694 (2014).

[29] Allard, A. et al. Detecting QTLs and putative candidate genes involved in budbreak and flowering time in an apple multiparental population. J. Exp. Bot. 67, 2875-2888 (2016).

[30] Di Guardo, M. et al. Deciphering the genetic control of fruit texture in apple by multiple family-based analysis and genome-wide association. J. Exp. Bot. 68, 1451-1466 (2017).

[31] Guan, Y., Peace, C., Rudell, D., Verma, S. & Evans, K. QTLs detected for individual sugars and soluble solids content in apple. Mol. Breed. 35, 334 (2015).

[32] Howard, N. P. et al. Two QTL characterized for soft scald and soggy breakdown in apple (Malus × domestica) through pedigree-based analysis of a large population of interconnected families. Tree Genet. Genomes 14, 2 (2018).

[33] Howard, N. P., Tillman, J., Vanderzande, S. & Luby, J. J. Genetics of zonal leaf chlorosis and genetic linkage to a major gene regulating skin anthocyanin production (MdMYB1) in the apple (Malus × domestica) cultivar Honeycrisp. PLoS ONE 14, e0210611 (2019).

[34] Verma, S. et al. Two large-effect QTLs, Ma and Ma3, determine genetic potential for acidity in apple fruit: breeding insights from a multi-family study. Tree Genet. Genomes 15, 18 (2019).

[35] Chagné, D. et al. Genome-wide SNP detection, validation, and development of an 8 K SNP array for apple. PLoS ONE 7, e31745 (2012).

[36] Vanderzande, S. et al. High-quality, genome-wide SNP genotypic data for pedigreed germplasm of the diploid outbreeding species apple, peach, and sweet cherry through a common workflow. PLoS ONE 14, e0210928 (2019).

[37] Bink, M. C. A. M., Uimari, P., Sillanpää, M., Janss, L. & Jansen, R. Multiple QTL mapping in related plant populations via a pedigree-analysis approach. Theor. Appl. Genet. 104, 751-762 (2002).

[38] Bink, M. C. A. M. et al. Bayesian analysis of complex traits in pedigreed plant populations. Euphytica 161, 85-96 (2008).

[39] Bink, M. C. A. M. et al. QTL linkage analysis of connected populations using ancestral marker and pedigree information. Theor. Appl. Genet. 124, 1097-1113 (2012).

[40] Luby, J. J. & Bedford, D. S. Honeycrisp Apple. Minnesota Report 225-1992 (University of Minnesota: Minnesota Agricultural Experiment Station, 1992). https://conservancy.umn.edu/bitstream/handle/11299/141481/MR225.pdf?sequence=1&isAllowed=y. Accessed 19 June 2020.

[41] Evans, K. M., Barritt, B. H., Konishi, B. S., Brutcher, L. J. & Ross, C. F. ‘WA 38’ Apple. HortScience 48, 1177-1179 (2012).

[42] Norelli, J. L. & Aldwinckle, H. S. Differential susceptibility of Malus spp. Cultivars Robusta 5, Novole, and Ottawa 523 to Erwinia amylovora. Plant Dis. 70, 1017-1019 (1986).

[43] Norelli, J. L., Holleran, H. T., Johnson, W. C., Robinson, T. L. & Aldwinckle, H. S. Resistance of Geneva and other apple rootstocks to Erwinia amylovora. Plant Dis. 87, 26-32 (2003).

[44] Flachowsky, H., Hanke, M.-V., Peil, A., Strauss, S. H. & Fladung, M. A review of transgenic approaches to accelerate breeding of woody plants. Plant Breed. 128, 217-226 (2009).

[45] Flachowsky, H. et al. Application of a high-seed breeding technology to apple (Malus × domestica) based on transgenic early flowering plants and marker-assisted selection. N. Phytol. 192, 364-377 (2011).

[46] Weigl, K., Wenzel, S., Flachowsky, H., Peil, A. & Hanke, M.-V. Integration of BpMADS4 on various linkage groups improves the utilization of the rapid cycle breeding system in apple. Plant Biotechnol. J. 13, 246-258 (2015).

[47] Johnson, K. B. et al. Effect of antagonistic bacteria on establishment of honey bee-dispersed Erwinia amylovora in pear blossoms and on fire blight control. Phytopathol 83, 995-1002 (1993).

[48] Stockwell, V. O., Johnson, K. B. & Loper, J. E. Compatibility of bacterial antagonists of Erwinia amylovora with antibiotics used to control fire blight. Phytopathol 86, 834-840 (1996).

[49] Bates, D., Mächler, M., Bolker, B. & Walker, S. Fitting linear mixed-effects models using lme4. J. Stat. Softw. 67, 1-48 (2015).

[50] Amyotte, B., Bowen, A. J., Banks, T., Rajcan, I. & Somers, D. J. Mapping the sensory perception of apple using descriptive sensory evaluation in a genome wide association study. PLoS ONE 12, e0171710 (2017).

[51] Iezzoni, A. et al. RosBREED: enabling marker-assisted breeding in Rosaceae. Acta Hortic. 859, 389-394 (2010).

[52] Iezzoni, A. et al. RosBREED2: progress and future plans to enable DNA-informed breeding in the Rosaceae. Acta Hortic. 1172, 115-118 (2017).

[53] Iezzoni, A. et al. RosBREED: Bridging the chasm between discovery and application to enable DNA-informed breeding in rosaceous crops. Hortic. Res. 7, 177 (2020).

[54] Kass, R. E. & Raftery, A. E. Bayes factors. J. Am. Stat. Assoc. 90, 773-795 (1995).

[55] Buels, R. et al. JBrowse: a dynamic web platform for genome visualization and analysis. Genome Biol. 17, 66 (2016).

[56] 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).

[57] Voorrips, R. E., Bink, M. C. A. M., Kruisselbrink, J. W., Koehorst-van Putten, H. J. J. & van de Weg, W. E. Pedihaplotyper: software for consistent assignment of marker haplotypes in pedigrees. Mol. Breed. 36, 119 (2016).

[58] Voorrips, R. E., Bink, M. C. A. M. & van de Weg, W. E. Pedimap: software for the visualization of genetic and phenotypic data in pedigrees. J. Hered. 103, 903-907 (2012).

[59] Kassambara, A. ggpubr: ‘ggplot2’ based publication ready plots. https://rpkgs.datanovia.com/ggpubr/.

[60] de Mendiburu, F. agricolae: statistical procedures for agricultural research. http://tarwi.lamolina.edu.pe/~fmendiburu.