Molecular characterization of intergeneric hybrids between Malus and Pyrus

Abstract

Apple (Malus) and pear (Pyrus) are economically important fruit crops well known for their unique textures, flavours, and nutritional qualities. Both genera are characterised by a distinct pattern of secondary metabolites, which directly affect not only resistance to certain diseases, but also have significant impacts on the flavour and nutritional value of the fruit. The identical chromosome numbers, similar genome size, and their recent divergence date, together with DNA markers have shown that apple and pear genomes are highly co-linear. This study utilized comparative genomic approaches, including simple sequence repeats, high resolution single nucleotide polymorphism melting analysis, and single nucleotide polymorphism chip analysis to identify genetic differences among hybrids of Malus and Pyrus, and F2 offspring. This research has demonstrated and validated that these three marker types, along with metabolomics analysis are very powerful tools to detect and confirm hybridity of progeny derived from crosses between apple and pear in both cross directions. Furthermore, this work analysed the genus-specific metabolite patterns and the resistance to fire blight (Erwinia amylovora) in progeny. The findings of this work will enhance and accelerate the breeding of novel tree fruit crops that benefit producers and consumers, by enabling marker assisted selection of desired traits introgressed between pear and apple.

Acknowledgements

This work was funded by The Autonomous Province of Trento, Italy (ADP) and a PhD fellowship to GM co-funded by FEM and PFR. We are grateful to Dr Lester Brewer and Richard Volz for performing intergeneric crosses in the field. We thank Dr Diego Micheletti (Fondazione Edmund Mach, Italy) for his assistance with the analysis of the SNP dataset from the array.

Author Contributions

MAM, SEG, SM, VGMB and DC conceived and designed the study. CW raised the PFR hybrid progenies. GP conducted the experiments and analysed the data, with input from CW, LP, VGMB, SM and DC. GP and SEG wrote the manuscript with input from VGMB, LP, DC and SM. All authors read and approved the final manuscript.

Data availability

All data were included in the paper and its Supplementary Materials published online.

Conflicts of interest statement

The authors have no competing interests to declare.

References

[1] Espley R, Martens S . Health Properties of Apple and Pear. West Sussex: Wiley; 2013: 81-100.

[2] Cornille A, Antolín F, Garcia E et al. A multifaceted overview of apple tree domestication. Trends Plant Sci. 2019; 24: 770-82.

[3] Awad MA, de Jager A, van Westing LM . Flavonoid and chlorogenic acid levels in apple fruit: characterisation of variation. Sci Hortic. 2000; 83: 249-63.

[4] Petkou D, Diamantidis G, Vasilakakis M . Arbutin oxidation by pear (Pyrus communis L.) peroxidases . Plant Sci. 2002; 162: 115-9.

[5] Velasco R, Zharkikh A, Affourtit J et al. The genome of the domesticated apple (Malus × domestica Borkh.). Nat Genet. 2010; 42: 833-9.

[6] Chagné D, Crowhurst RN, Pindo M et al. The draft genome sequence of European pear (Pyrus communis L ‘Bartlett’). PLoS One. 2014; 9: 1-12, 92644.

[7] Wu J, Wang Z, Shi Z et al. The genome of the pear (Pyrus bretschneideri Rehd.). Genome Res. 2013; 23: 396-408.

[8] Linsmith G, Rombauts S, Montanari S et al. Pseudo-chromosome-length genome assembly of a double haploid ‘Bartlett’ pear (Pyrus communis L.). Gigascience. 2019; 8: 1-17.

[9] Van Tuyl JM, De Jeu MJ . Methods for overcoming interspecific crossing barriers. In: Sawhney VK, Shivanna KR, eds. Pollen Biotechnol Crop Prod Improv. 1997, 273-92.

[10] Kamiri M, Stift M, Costantino G et al. Preferential homologous chromosome pairing in a tetraploid intergeneric somatic hybrid (Citrus reticulata + Poncirus trifoliata) revealed by molecular marker inheritance . Front Plant Sci. 2018; 9: 327.

[11] Brewer L, Aldsworth M, Bus V et al. Breeding for fire blight resistance in an interspecific pear breeding programme. Acta Hortic. 2021; 1303: 49-54.

[12] Fischer TC, Malnoy M, Hofmann T et al. F1 hybrid of cultivated apple (Malus x domestica) and European pear (Pyrus communis) with fertile F2 offspring . Mol Breed. 2014; 34: 817-28.

[13] Crane MB, Marks E . Pear-apple hybrids. Nature. 1952; 170: 1017-7.

[14] Rudenko IS, Rotaru GI . Morphological and anatomical characteristics of an intergeneric apple x pear hybrid. Strukt Osob Sochni Myasnsi Plodov. 1970; 5: 40-51.

[15] Inoue E, Sakuma F, Kasumi M et al. Effect of high-temperature on suppression of the lethality exhibited in the intergeneric hybrid between Japanese pear (Pyrus pyrifolia Nakai) and apple (Malus × domestica Borkh.). Sci Hortic. 2003; 98: 385-96.

[16] Gonai T, Manabe T, Inoue E et al. Overcoming hybrid lethality in a cross between Japanese pear and apple using gamma irradiation and confirmation of hybrid status using flow cytometry and SSR markers. Sci Hortic. 2006; 109: 43-7.

[17] Gunen Y, Misirli A, Gulcan R . Leaf phenolic content of pear cultivars resistant or susceptible to fire blight. Sci Hortic. 2005; 105: 213-21.

[18] Distefano G, Caruso M, La Malfa S et al. High resolution melting analysis is a more sensitive and effective alternative to gel-based platforms in analysis of SSR - an example in citrus. PLoS One. 2012; 7: 1-11, e44202.

[19] Wittwer CT . High-resolution DNA melting analysis: advancements and limitations. Hum Mutat. 2009; 30: 857-9.

[20] Duan N, Bai Y, Sun H et al. Genome re-sequencing reveals the history of apple and supports a two-stage model for fruit enlargement. Nat Commun. 2017; 8: 1-16, 249.

[21] Montanari S, Perchepied L, Renault D et al. A QTL detected in an interspecific pear population confers stable fire blight resistance across different environments and genetic backgrounds. Mol Breed. 2016; 36: 47.

[22] Verde I, Bassil N, Scalabrin S et al. Development and evaluation of a 9K SNP Array for peach by internationally coordinated SNP detection and validation in breeding germplasm. PLoS One. 2012; 7: 1-13, e35668.

[23] García C, Guichoux E, Hampe A . A comparative analysis between SNPs and SSRs to investigate genetic variation in a juniper species (Juniperus phoenicea ssp. turbinata). Tree Genet Genomes. 2018; 14: 1-9.

[24] Singh N, Choudhury DR, Singh AK et al. Comparison of SSR and SNP markers in estimation of genetic diversity and population structure of Indian rice varieties. PLoS One. 2013; 8: 1-14, e84136.

[25] Zurn JD, Nyberg A, Montanari S et al. A new SSR fingerprinting set and its comparison to existing SSR-and SNP-based genotyping platforms to manage Pyrus germplasm resources. Tree Genet Genomes. 2020; 16: 1-10.

[26] Montanari S, Postman J, Bassil NV et al. Reconstruction of the largest pedigree network for pear cultivars and evaluation of the genetic diversity of the USDA-ARS national Pyrus collection . G3 (Bethesda). 2020; 10: 3285-97.

[27] Heo S, Kim C, Chung YS . High-resolution melting analysis for identification of apple cultivars using simple sequence repeat markers. Plant Biotechnol Rep. 2019; 13: 337-44.

[28] Van Dijk T, Pagliarani G, Pikunova A et al. Genomic rearrangements and signatures of breeding in the Allo-octoploid strawberry as revealed through an allele dose based SSR linkage map. BMC Plant Biol. 2014; 14: 1-16.

[29] Tiwari VK, Wang S, Sehgal S et al. SNP discovery for mapping alien introgressions in wheat. BMC Genomics. 2014; 15: 1-11.

[30] Rodionov AV, Amosova AV, Belyakov EA et al. Genetic consequences of interspecific hybridization, its role in speciation and phenotypic diversity of plants. Russ J Genet. 2019; 55: 278-94.

[31] Du XZ, Ge XH, Zhao ZG et al. Chromosome elimination and fragment introgression and recombination producing intertribal partial hybrids from Brassica napus x Lesquerella fendleri crosses . Plant Cell Rep. 2008; 27: 261-71.

[32] Faure N, Serieys H, Bervillé A et al. Occurrence of partial hybrids in wide crosses between sunflower (Helianthus annuus) and perennial species H. mollis and H. orgyalis. Theor Appl Genet. 2002; 104: 652-60.

[33] Rathod V, Behera T, Munshi A et al. Developing partial interspecific hybrids of Momordica charantia × Momordica balsamina and their advance generations . Sci Hortic. 2021; 281: 1-11, 109985.

[34] Polgári D, Mihók E, Sági L . Composition and random elimination of paternal chromosomes in a large population of wheat × barley (Triticum aestivum L. × Hordeum vulgare L.) hybrids . Plant Cell Rep. 2019; 38: 767-75.

[35] Chen HF, Wang H, Li ZY . Production and genetic analysis of partial hybrids in intertribal crosses between brassica species (B. rapa, B. napus) and Capsella bursa-pastoris. Plant Cell Rep. 2007; 26: 1791-800.

[36] Tu Y, Sun J, Ge X et al. Production and genetic analysis of partial hybrids from intertribal sexual crosses between Brassica napus and Isatis indigotica and progenies . Genome. 2010; 53: 146-56.

[37] Gutierrez BL, Zhong GY, Brown SK . Increased phloridzin content associated with russeting in apple (Malus domestica (Suckow) Borkh) fruit. Genet Resour Crop Evol. 2018; 65: 2135-49.

[38] Le Roux P-M, Flachowsky H, Hanke M-V et al. Use of a transgenic early flowering approach in apple (Malus domestica Borkh.) to introgress fire blight resistance from cultivar Evereste . Mol Breed. 2012; 30: 857-74.

[39] Myburg AA, Vogl C, Griffin AR et al. Genetics of postzygotic isolation in eucalyptus: whole-genome analysis of barriers to introgression in a wide interspecific cross of Eucalyptus grandis and E. globulus. Genetics. 2004; 166: 1405-18.

[40] Doyle J, Doyle J . A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull. 1987; 19: 11-5.

[41] Knäbel M, Friend AP, Palmer JW et al. Genetic control of pear rootstock-induced dwarfing and precocity is linked to a chromosomal region syntenic to the apple Dw1 loci . BMC Plant Biol. 2015; 15: 1-16.

[42] Kloosterman AD, Budowle B, Daselaar P . PCR-amplification and detection of the human D1S80 VNTR locus - amplification conditions, population genetics and application in forensic analysis. Int J Legal Med. 1993; 105: 257-64.

[43] Peakall R, Smouse PE . GenALEx 6.5: genetic analysis in excel. Population genetic software for teaching and research-an update. Bioinformatics. 2012; 28: 2537-9.

[44] Langmead B, Salzberg SL . Fast gapped-read alignment with bowtie 2. Nat Methods. 2012; 9: 357-9.

[45] Paetkau D, Slade R, Burden M et al. Genetic assignment methods for the direct, real-time estimation of migration rate: a simulation-based exploration of accuracy and power. Mol Ecol. 2004; 13: 55-65.

[46] Paetkau D, Calvert W, Stirling I et al. Microsatellite analysis of population structure in Canadian polar bears. Mol Ecol. 1995; 4: 347-54.

[47] Chagné D, Crowhurst RN, Troggio M et al. Genome-wide SNP detection, validation, and development of an 8K SNP Array for apple. PLoS One. 2012; 7: 1-12, e31745.

[48] Montanari S, Saeed M, Knäbel M et al. Identification of Pyrus single nucleotide polymorphisms (SNPs) and evaluation for genetic mapping in European pear and interspecific Pyrus hybrids . PLoS One. 2013; 8: 1-11, e77022.

[49] Vrhovsek U, Masuero D, Gasperotti M et al. A versatile targeted metabolomics method for the rapid quantification of multiple classes of phenolics in fruits and beverages. J Agric Food Chem. 2012; 60: 8831-40.

[50] Maas Geesteranus H, Heyting J . The value of topleaf inoculation to demonstrate genetic resistance in Pomoideae species to Erwinia amylovora (Burr.) Winslow . Acta Hortic. 1981; 117: 75-82.

[51] Jeger MJ, Viljanen-Rollinson SLH . The use of the area under the disease-progress curve (AUDPC) to assess quantitative disease resistance in crop cultivars. Theor Appl Genet. 2001; 102: 32-40.

[52] Le LM, Paulin J . Shoot susceptibility to fireblight of some apple cultivars. Acta Hortic. 1984; 151: 277-81.