Plant Molecular Biology Reporter

, Volume 33, Issue 2, pp 239–252

QTL Analysis Coupled with PTR-ToF-MS and Candidate Gene-Based Association Mapping Validate the Role of Md-AAT1 as a Major Gene in the Control of Flavor in Apple Fruit

  • Luca Cappellin
  • Brian Farneti
  • Mario Di Guardo
  • Nicola Busatto
  • Iuliia Khomenko
  • Andrea Romano
  • Riccardo Velasco
  • Guglielmo Costa
  • Franco Biasioli
  • Fabrizio Costa
Original Paper


Volatile organic compounds (VOCs) are fundamental elements of flavor, one of the most important fruit-quality traits. Despite its importance, this aspect is still poorly considered in assisted breeding programs, due to the lack of suitable and fast detection systems as well as validated functional markers. In this work, a full-sib parental mapping population (‘Fuji × Delearly’) was initially employed to perform a comprehensive quantitative trait locus (QTL) survey, to assess the VOC segregation detected by a novel proton-transfer-reaction time-of-flight mass spectrometry (PTR-ToF-MS) on fruit collected after a 2-month period of postharvest storage. Among this set of genomic regions, on chromosome 2 was also verified the coincident location between a group of QTLs, mainly associated to esters and alcohols, with a functional marker designed for Md-AAT1, a gene involved in the last step of the ester biosynthetic pathway. The allelic effect of this marker (here named Md-AAT1SSR) was further validated by candidate gene association mapping approach in a collection of 124 apple accessions. In this case, the volatile profiling was performed on peeled fruit flesh, as an important fraction of the aromatic blend of apple is released only after cutting. This work proposed a new and fast method for aroma phenotyping as well as a novel marker for an easy and widely applicable apple fruit quality advanced selection.


Fruit quality Volatile organic compounds Apple flavor QTL analysis Candidate gene-based association mapping 

Supplementary material

11105_2014_744_MOESM1_ESM.xlsx (15 kb)
Table S1List of VOC related genes annotated within each QTL interval (XLSX 15 kb)
11105_2014_744_MOESM2_ESM.xlsx (12 kb)
Table S2List of the 124 apple accessions as reported on Fig. 5 (XLSX 12 kb)
11105_2014_744_MOESM3_ESM.xlsx (10 kb)
Table S3List of VOCs as reported on Fig. 5 (XLSX 10 kb)
11105_2014_744_MOESM4_ESM.docx (134 kb)
Table S4Pearson correlation analysis performed among seven VOCs over the 124 accessions of the apple collection (DOCX 133 kb)
11105_2014_744_MOESM5_ESM.doc (66 kb)
Fig. S1Sequence of the contig (MDC018196.110) on which the AAT gene (MDP0000214714) sequence is highlighted in gray, while the microsatellite motif on which the Md-AAT1SSR marker was designed in visualized using bold text (DOC 65 kb)
11105_2014_744_MOESM6_ESM.pptx (220 kb)
Fig. S2Population structure of the apple collection. a The most probable number of groups as defined by the Evanno calculation. b The population structure based on the STRUCTURE analysis, with K = 4 (PPTX 219 kb)
11105_2014_744_MOESM7_ESM.ppt (20 kb)
Fig. S3Variation in ester content between the two classes defined according to the absence/presence of the allele Md-AAT1SSR_201. The change is depicted as log2 of the rate of change between the two groups (PPT 20 kb)


  1. Aharoni A, Keizer LCP, Bouwmeester HJ, Sun Z, Alvarez- Huerta M, Harrie A, Verhoeven HA, Blaas J, van Houwelingen AMML, De Vos RCH, van der Voet H, Jansen RC, Guis M, Mol J, Davis RW, Schena M, van Tunen AJ, O’Connell AP (2000) Identification of the SAAT gene involved in strawberry flavor biogenesis by use of DNA microarrays. Plant Cell 12:647–661CrossRefPubMedCentralPubMedGoogle Scholar
  2. Arvisenet G, Billy L, Poinot P, Vigneau E, Bertrand D, Prost C (2008) Effect of apple particle state on the release of volatile compounds in a new artificial mouth device. J Agric Food Chem 56:3245–3253CrossRefPubMedGoogle Scholar
  3. Beekwilder J, Alvarez-Huerta M, Neef E, Verstappen FWA, Bouwmeester HJ, Aharoni A (2004) Functional characterization of enzymes forming volatile esters from strawberry and banana. Plant Physiol 135:1865–1878CrossRefPubMedCentralPubMedGoogle Scholar
  4. Berger RG (1991) In volatile compounds in food and beverages, H. Maarse (ed.). Marcel Dekker: New York, pp. 283–297Google Scholar
  5. Berger RG (2007) Flavours and Fragrances—chemistry, bioprocessing and suistainability. Springer-Verlag, Berlin, GermanyCrossRefGoogle Scholar
  6. Beyesr T, Perry G (1992) Dietary carotenes, vitamin C, and vitamin E as protective antioxidants in human cancers. Annu Rev Nutr 12:139–159CrossRefGoogle Scholar
  7. Bourne M (2002) Food Texture and Viscosity: Concept and measurement, 2nd edn. Academic Press, San DiegoGoogle Scholar
  8. Bradbury PJ, Zhang Z, Kroon DE, Casstevens TM, Ramdoss Y, Buckler ES (2007) TASSEL: software for association mapping of complex traits in diverse samples. Bioinformatics 23:2633–2635CrossRefPubMedGoogle Scholar
  9. Cappellin L, Biasioli F, Schuhfried E, Soukoulis C, Märk TD, Gasperi F (2011a) Extending the dynamic range of proton transfer reaction time-of-flight mass spectrometers by a novel dead time correction. Rapid Commun Mass Spectrom RCM 25:179–183CrossRefGoogle Scholar
  10. Cappellin L, Biasioli F, Granitto PM, Schuhfried E, Soukoulis C, Costa F, Märk TD, Gasperi F (2011b) On data analysis in PTR-TOF-MS: From raw spectra to data mining. Sens. Actuators B Chem 155:183–190CrossRefGoogle Scholar
  11. Cappellin L, Soukoulis C, Aprea E, Granitto P, Dallabetta N, Costa F, Viola R, Märk TD, Gasperi F, Biasioli F (2012a) PTR-ToF-MS and data mining methods: a new tool for fruit metabolomics. Metabolomics 8:761–770CrossRefGoogle Scholar
  12. Cappellin L, Karl T, Probst M, Ismailova O, Winkler PM, Soukoulis C, Aprea E, Märk TD, Gasperi F, Biasioli F (2012b) On quantitative determination of volatile organic compound concentrations using proton transfer reaction time-of-flight mass spectrometry. Environ Sci Technol 46:2283–2290CrossRefPubMedGoogle Scholar
  13. Cevik V, Ryder CD, Popovich A, Manning K, King GJ, Seymour GB (2009) A FRUITFULL-like gene is associated with genetic variation for fruit flesh firmness in apple (Malus domestica Borkh.). Tree Genet Genome 6:271–279CrossRefGoogle Scholar
  14. Contreras C, Beaudry R (2013) Lipoxygenase-associated apple volatiles and their relationship with aroma perception during ripening. Postharvest Biol Technol 82:28–38CrossRefGoogle Scholar
  15. Costa F, Stella S, Van de Weg WE, Guerra W, Cecchinel M, Dallavia J, Koller B, Sansavini S (2005) Role of the genes Md-ACO1 and Md-ACS1 in ethylene production and shelf life of apple (Malus domestica Borkh). Euphytica 141:181–190CrossRefGoogle Scholar
  16. Costa F, Van de Weg WE, Stella S, Dondini L, Pratesi D, Musacchi S, Sansavini S (2008) Map position and functional allelic diversity of Md-Exp7, a new putative expansin gene associated with fruit softening in apple (Malus 9 domestica Borkh.) and pear (Pyrus communis). Tree Genet Genome 4:575–586CrossRefGoogle Scholar
  17. Costa F, Peace CP, Stella S, Serra S, Musacchi S, Bazzani M, Sansavini S, Van de Weg WE (2010) QTL dynamics for fruit firmness and softening around an ethylene-dependent polygalacturonase gene in apple (Malus 9 domestica Borkh.). J Exp Bot 11:2029–3039Google Scholar
  18. Costa F, Cappellin L, Longhi S, Guerra W, Magnano P, Porro D, Soukoulis C, Salvi S, Velasco R, Biasioli F, Gasperi F (2011) Assessment of apple (Malus×domestica Borkh.) fruit texture by a combined acoustic-mechanical profiling strategy. Postharvest Biol Technol 6:21–28CrossRefGoogle Scholar
  19. Costa F, Cappellin L, Fontanari M, Longhi S, Guerra W, Magnago P, Gasperi F, Biasioli F (2012) Texture dynamics during postharvest cold storage ripening in apple (Malus x domestica Borkh.). Postharvest Biol Technol 69:54–63CrossRefGoogle Scholar
  20. Costa F, Cappellin L, Zini E, Patocchi A, Kellerhals M, Komjanc M, Gessler C, Biasioli F (2013) QTL validation and stability for volatile organic compounds (VOCs) in apple. Plant Sci 211:1–7CrossRefPubMedGoogle Scholar
  21. De Roos KB (2003) Effect of texture and microstructure on flavour retention and release. Int Dairy J 13:593–605CrossRefGoogle Scholar
  22. DellaPenna D (1999) Nutritional genomics: manipulating plant micronutrients to improve human health. Science 285:375CrossRefPubMedGoogle Scholar
  23. Dixon J, Hewett EW (2000) Factors affecting apple aroma/flavor volatile concentration: a review. NZJ Crop Hortic Sci 28:155–173CrossRefGoogle Scholar
  24. Dudareva N, Klempien A (2013) Biosynthesis, function and metabolic engineering of plant volatile organic compounds. New Phytol 198:16–32CrossRefPubMedGoogle Scholar
  25. Dunemann F, Ulrich D, Boudichevskaia A, Grafe C, Weber WE (2009) QTL mapping of aroma compounds analysed by headspace solid-phase microextraction gas chromatography in the apple progeny ‘Discovery’ × ‘Prima’. Mol Breeding 23:501–521CrossRefGoogle Scholar
  26. Dunemann F, Ulrich D, Malysheva-Otto L, Weber WE, Longhi S, Velasco R, Costa F (2012) Functional allelic diversity of the apple alcohol acyl-transferase gene MdAAT1 associated with fruit ester volatile contents in apple cultivars. Mol Breeding 29:609–625CrossRefGoogle Scholar
  27. Earl D, vonHoldt BM (2012) STRUCTURE HARVESTER: a Website and program for visualizing STRUCTURE output and implementing the Evanno method. Conserv Genet Resour 4(2):359–361. doi:10.1007/s12686-011-9548-7 CrossRefGoogle Scholar
  28. El-Sharkawy I, Manriques D, Flores FB, Regad F, Bouzayen M, Latche A, Pech JC (2005) Functional characterization of a melon alcohol acyl-transferase gene family involved in the biosynthesis of ester volatiles. Identification of the crucial role of a threonine residue for enzyme activity. Plant Mol Biol 59:345–362CrossRefPubMedGoogle Scholar
  29. Evanno G, Regnaut S, Goudet J (2005) Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol Ecol 14:2611–2620CrossRefPubMedGoogle Scholar
  30. Excoffier L, Laval G, Schneider S (2005) Arlequin ver. 3.0: An integrated software package for population genetics data analysis. Evol Bioinformatics Online 1:47–50Google Scholar
  31. Fellman JK, Rudell DR, Mattinson DS, Mattheis JP (2003) Relationship of harvest maturity to flavor regeneration after CA storage of “Delicious” apples. Postharvest Biol Technol 27:39–51CrossRefGoogle Scholar
  32. Fuhrmann E, Grosch W (2002) Character impact odorants of the apple cultivars Elstar and Cox Orange. Nahrung/Food 46(3):187–193CrossRefGoogle Scholar
  33. Gapper NE, McQuinn RP, Giovannoni JJ (2013) Molecular and genetic regulation of fruit ripening. Plant Mol Biol 82:575–591CrossRefPubMedGoogle Scholar
  34. Giovannoni JJ (2001) Molecular biology of fruit maturation and ripening. Annu Rev Plant Physiol Plant Mol Biol 52:725–749CrossRefPubMedGoogle Scholar
  35. Glaubitz JC (2004) CONVERT: A user-friendly program to reformat diploid genotypic data for commonly used population genetic software packages. Mol Ecol Notes 4:309–310CrossRefGoogle Scholar
  36. Harker FR, Maindonald J, Murray SH, Gunson FA, Hallett IC, Walker SB (2002) Sensory interpretation of instrumental measurements 1: texture of apple fruit. Postharvest Biol Technol 24:225–239CrossRefGoogle Scholar
  37. Harker FR, White A, Gunson FA, Hallett IC, De Silva HN (2006) Instrumental measurement of apple texture: a comparison of the single-edge notched test and the penetrometer. Postharvest Biol Technol 39:185–192CrossRefGoogle Scholar
  38. Holland D, Larkov O, Bar-Yaákov I, Bar E, Zax A, Brandeis E (2005) Developmental and varietal differences in volatiles ester formation and acetyl-CoA:alcohol acetyl transferase activities in apple (Malus domestica Borkh.) fruit. J Agric Food Chem 53:7198–7203CrossRefPubMedGoogle Scholar
  39. Jordan A, Haidacher S, Hanel G, Hartungen E, Mark L, Seehauser H, Schottkowsky R, Sulzer P, Mark T (2009) A high resolution and high sensitivity proton-transfer-reaction time-of-flight mass spectrometer (PTR-TOF-MS). Int J Mass Spectrom 286:122–128CrossRefGoogle Scholar
  40. Klee HJ (2010) Improving the flavor of fresh fruits: genomics, biochemistry, and biotechnology. New Phytol 187:44–56CrossRefPubMedGoogle Scholar
  41. Klee HJ, Giovannoni JJ (2011) Genetics and control of tomato fruit ripening and quality attributes. Annu Rev Genet 45:41–59CrossRefPubMedGoogle Scholar
  42. Lindinger W, Hansel A, Jordan A (1998) On-line monitoring of volatile organic compounds at pptv levels by means of proton-transfer-reaction mass spectrometry (PTR-MS)—medical applications, food control and environmental research. Int J Mass Spectrom Ion Phys 173:191–241CrossRefGoogle Scholar
  43. Longhi S, Moretto M, Viola R, Velasco R, Costa F (2012) Comprehensive QTL mapping survey dissects the complex fruit texture physiology in apple (Malus x domestica Borkh.). J Exp Bot 63(3):1107–1121CrossRefPubMedGoogle Scholar
  44. Longhi S, Cappellin L, Guerra W, Costa F (2013) Validation of a functional molecular marker suitable for marker-assisted breeding for fruit texture in apple (Malus 3 domestica Borkh.). Mol Breeding 32:841–852CrossRefGoogle Scholar
  45. Newcomb RD, Crowhurst RN, Gleave AP, Rikkerink EHA, Allan AC, Beuning LL, Bowen JH, Gera E, Jamieson KR, Janssen BJ, Laing WA, McArtney S, Nain B, Ross GS, Snowden KC, Souleyre EJF, Walton EF, Yauk YK (2006) Analyses of expressed sequence tags from apple. Plant Physiol 141:147–166CrossRefPubMedCentralPubMedGoogle Scholar
  46. Nijssen LM, van Ingen-Visscher CA, Donders JJH (2011) VCF Volatile Compounds in Food: database (version 13.1). Zeist (The Netherlands)Google Scholar
  47. Oraguzie NC, Iwanami H, Soejima J, Harada T, Hall A (2004) Inheritance of the Md-ACS1 gene and its relationship to fruit softening in apple (Malus × domestica Borkh.). Theor Appl Genet 108:1526–1533CrossRefPubMedGoogle Scholar
  48. Oraguzie NC, Volz RK, Whitworth CJ, Bassett HCM, Hall AJ, Gardiner SE (2007) Influence of Md-ACS1 allelotype and harvest season within an apple germplesm collection on fruit softening during cold air storage. Postharvest Biol Technol 44:212–219CrossRefGoogle Scholar
  49. Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155:945–959PubMedCentralPubMedGoogle Scholar
  50. Schaffer RJ, Friel EN, Souleyre EJF, Bolitho K, Thodey K, Ledger S, Bowen JH, Ma JH, Nain B, Cohen D, Gleave AP, Crowhurst RN, Janssen BJ, Yao JL, Newcomb RD (2007) A genomics approach reveals that aroma production in apple is controlled by ethylene predominantly at the final step in each biosynthetic pathway. Plant Physiol 144:1899–1912CrossRefPubMedCentralPubMedGoogle Scholar
  51. Singh N, Choudhury DR, Singh AK, Kumar S, Srinivasan K, Tyagi RK, Singh NK, Singh R (2013) Comparison of SSR and SNP markers in estimation of genetic diversity and population structure of Indian rice varieties. Plos ONE 8(12):e84136CrossRefPubMedCentralPubMedGoogle Scholar
  52. Song J, Forney CF (2008) Flavour volatile production and regulation in fruit. Can J Plant Sci 88:537–550CrossRefGoogle Scholar
  53. Soukoulis C, Cappellin L, Aprea E, Costa F, Viola R, Märk TD, Gasperi F, Biasioli F (2013) PTR-ToF-MS, a novel, rapid, high sensitivity and non-invasive tool to monitor volatile compound release during fruit post-harvest storage: the case study of apple ripening. Food Bioproc Technol 6:2831–2843CrossRefGoogle Scholar
  54. Stich B, Möhring J, Piepho HP, Heckenberger M, Buckler ES, Melchinger AE (2008) Comparison of mixed-model approaches for association mapping. Genetics 178:1745–1754CrossRefPubMedCentralPubMedGoogle Scholar
  55. Ting VJL, Soukoulis C, Silcock P, Cappellin L, Romano A, Aprea E, Bremer PJ, Märk T, Gasperi F, Biasioli F (2012) In vitro and in vivo flavour release from intact and freshcut apple in relation with genetic, textural, and physicochemical parameters. J Food Sci 77:1226–1233CrossRefGoogle Scholar
  56. Ulrich D, Dunemann F (2012) Towards the development of molecular markers for apple volatiles. Flavour Fragr J 27:286–289CrossRefGoogle Scholar
  57. Van Ooijen JW (2006) Joinmap 4®, software for the calculation of genetic linkage maps in experimental populations. Kyazma BV, Wageningen, NetherlandsGoogle Scholar
  58. Van Ooijen JW (2009) MAPQTL 6®, Software for the mapping of quantitative trait loci in experimental populations of diploid species. Kyazma BV, Wageningen, NetherlandsGoogle Scholar
  59. Velasco R, Zharkikh A, Affourtit J, Dhingra A, Cestaro A et al (2010) The genome of the domesticated apple (Malus x domestica Borkh.). Nat Genet 42(10):833–839. doi:10.1038/ng.654 CrossRefPubMedGoogle Scholar
  60. Vorrips RE (2002) MapChart: software for the graphical presentation of linkage maps and QTLs. J Hered 93:77–78CrossRefGoogle Scholar
  61. Welch RM, Graham RD (2004) Breeding for micronutrients in staple food crops from a human nutrition perspective. J Exp Bot 55(396):353–364CrossRefPubMedGoogle Scholar
  62. Yang X, Xu Y, Shah T, Li H, Han Z, Li J, Yan J (2011) Comparison of SSR and SNPs in assessment of genetic relatedness in maize. Genetica 139:1045–1054CrossRefPubMedGoogle Scholar
  63. Yu J, Buckler ES (2006) Genetic association mapping and genome organization of maize. Curr Opin Biotechnol 17:155–160CrossRefPubMedGoogle Scholar
  64. Zhu YM, Barritt BH (2008) Md-ACS1 and Md-ACO1 geno- typing of apple (Malus 9 domestica Borkh.) breeding parents and suitability for marker-assisted selection. Tree Genet Genome 4:555–562CrossRefGoogle Scholar
  65. Zini E, Biasioli F, Gasperi F, Mott D, Aprea E, Mark TD, Patocchi A, Gessler C, Komjanc M (2005) QTL mapping of volatile compounds in ripe apples detected by proton transfer reaction-mass spectrometry. Euphytica 145:269–279CrossRefGoogle Scholar
  66. Ziosi V, Noferini M, Fiori G, Tadiello A, Trainotti L, Casadoro G, Costa G (2008) A new index based on Vis spectroscopy to characterize the progression of ripening in peach fruit. Postharvest Biol Technol 49:319–329CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Luca Cappellin
    • 1
  • Brian Farneti
    • 2
  • Mario Di Guardo
    • 1
  • Nicola Busatto
    • 2
  • Iuliia Khomenko
    • 1
  • Andrea Romano
    • 1
  • Riccardo Velasco
    • 1
  • Guglielmo Costa
    • 2
  • Franco Biasioli
    • 1
  • Fabrizio Costa
    • 1
  1. 1.Research and Innovation CentreFondazione Edmund MachSan Michele all’Adige (Trento)Italy
  2. 2.Department of Agricultural SciencesBologna UniversityBolognaItaly

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