Stable QTL for malate levels in ripe fruit and their transferability across Vitis species

Abstract

Malate is a major contributor to the sourness of grape berries (Vitis spp.) and their products, such as wine. Excessive malate at maturity, commonly observed in wild Vitis grapes, is detrimental to grape and wine quality and complicates the introgression of valuable disease resistance and cold hardy genes through breeding. This study investigated an interspecific Vitis family that exhibited strong and stable variation in malate at ripeness for five years and tested the separate contribution of accumulation, degradation, and dilution to malate concentration in ripe fruit in the last year of study. Genotyping was performed using transferable rhAmpSeq haplotype markers, based on the Vitis collinear core genome. Three significant QTL for ripe fruit malate on chromosomes 1, 7, and 17, accounted for over two-fold and 6.9 g/L differences, and explained 40.6% of the phenotypic variation. QTL on chromosomes 7 and 17 were stable in all and in three out of five years, respectively. Variation in pre-veraison malate was the major contributor to variation in ripe fruit malate (39%), and based on two and five years of data, respectively, their associated QTL overlapped on chromosome 7, indicating a common genetic basis. However, use of transferable markers on a closely related Vitis family did not yield a common QTL across families. This suggests that diverse physiological mechanisms regulate the levels of this key metabolite in the Vitis genus, a conclusion supported by a review of over a dozen publications from the past decade, showing malate-associated genetic loci on all 19 chromosomes.

References

[1] Fernie AR, Martinoia EM . Jack of all trades or master of a few? Phytochemistry . 2009; 70: 828-32. https://doi.org/10.1016/j.phytochem.2009.04.023.

[2] Walker RP, Famiani F . Organic Acids in Fruits: metabolism, functions and contents. In: Warrington I, editor. Horticultural Reviews.Vol. 45. John Wiley & Sons, Inc.; 2018. p. 371-430.

[3] Neta ERD, Johanningsmeier SD, McFeeters RF . The chemistry and physiology of sour taste - a review. J Food Sci. 2007; 72: R33-8. https://doi.org/10.1111/j.1750-3841.2007.00282.x.

[4] Klee HJ . Improving the flavor of fresh fruits: genomics, biochemistry, and biotechnology. New Phytol. 2010; 187: 44-56. https://doi.org/10.1111/j.1469-8137.2010.03281.x.

[5] Cleary M et al. Advances in Wine Research. In: Advances in Wine Research . American Chemical Society; 2015. p. 365-78.

[6] Ruffner HP . Metabolism of tartaric and malic-acids in vitis - a review. B Vitis. 1982; 21: 346-58.

[7] Sweetman C, Deluc LG, Cramer GR et al. Regulation of malate metabolism in grape berry and other developing fruits. Phytochemistry. 2009; 70: 1329-44. https://doi.org/10.1016/j.phytochem.2009.08.006.

[8] Coombe BG . Distribution of solutes within the developing grape berry in relation to its morphology. Am J Enol Vitic. 1987; 38: 120-7.

[9] Famiani F, Farinelli D, Pallioti A et al. Is stored malate the quantitatively most important substrate utilised by respiration and ethanolic fermentation in grape berry pericarp during ripening? Plant Physiol Biochem. 2014; 76: 52-7. https://doi.org/10.1016/j.plaphy.2013.12.017.

[10] Terrier N, Romieu C . Molecular Biology & Biotechnology of the Grapevine. In: Kalliopi A, Roubelakis-Angelakis, editors. Springer: Netherlands; 2001. p. 35-57.

[11] Etienne A, Genard M, Lobit P et al. What controls fleshy fruit acidity? A review of malate and citrate accumulation in fruit cells. J Exp Bot. 2013; 64: 1451-69. https://doi.org/10.1093/jxb/ert035.

[12] Burzynski-Chang EA, Brown EJ, Reshef N et al. Malate content in wild Vitis spp. demonstrates a range of Behaviors during berry maturation. Am J Enol Vitic. 2020; 71: 80-7. https://doi.org/10.5344/ajev.2019.19015.

[13] Kliewer WM . Concentration of tartrates, Malates, glucose and fructose in the fruits of the genus Vitis. Am J Enol Vitic. 1967; 18: 87-96.

[14] Reisch BI, Owens CL, Cousins PS . Grape Fruit Breeding. In: Badenes M, Byrne D, editors. Fruit Breeding.Springer; 2012.

[15] Cadle-Davidson L, Chicoine DR, Consolie NH . Variation within and among Vitis spp. for foliar resistance to the powdery mildew pathogen Erysiphe necator. Plant Dis. 2011; 95: 202-11. https://doi.org/10.1094/PDIS-02-10-0092.

[16] Staudt G, Kassemeyer HH . Evaluation of downy mildew resistance in various accessions of wild Vitis species. Vitis. 1995; 34: 225-8.

[17] Hyma KE, Barba P, Wang M et al. Heterozygous mapping strategy (HetMappS) for high resolution genotyping-by-sequencing markers: a case study in grapevine. PLoS One. 2015; 10. https://doi.org/10.1371/journal.pone.0134880.

[18] Yang SS, Fresnedo-Ramirez J, Wang M et al. A next-generation marker genotyping platform (AmpSeq) in heterozygous crops: a case study for marker-assisted selection in grapevine. Horticulture Research. 2016; 3. https://doi.org/10.1038/hortres.2016.2.

[19] Doligez A, Adam-Blondon AF, Cipriani G et al. An integrated SSR map of grapevine based on five mapping populations. Theor Appl Genet. 2006; 113: 369-82. https://doi.org/10.1007/s00122-006-0295-1.

[20] Zou C, Karn A, Reisch B et al. Haplotyping the Vitis collinear core genome with rhAmpSeq improves marker transferability in a diverse genus. Nat Commun. 2020; 11: 11. https://doi.org/10.1038/s41467-019-14280-1.

[21] Chen J, Wang N, Fang LC et al. Construction of a high-density genetic map and QTLs mapping for sugars and acids in grape berries. BMC Plant Biol. 2015; 15. https://doi.org/10.1186/s12870-015-0428-2.

[22] Houel C, Chatbanyong R, Doligez A et al. Identification of stable QTLs for vegetative and reproductive traits in the microvine (Vitis vinifera L.) using the 18 K Infinium chip . BMC Plant Biol. 2015; 15. https://doi.org/10.1186/s12870-015-0588-0.

[23] Duchene E, Dumas V, Jaegli N et al. Genetic variability of descriptors for grapevine berry acidity in Riesling, Gewurztraminer and their progeny. Aust J Grape Wine Res. 2014; 20: 91-9. https://doi.org/10.1111/ajgw.12051.

[24] Bayo-Canha A, Costantini L, Fernandez-Fernandez JI et al. QTLs related to berry acidity identified in a wine grapevine population grown in warm weather. Plant Mol Biol Rep. 2019; 37: 157-69. https://doi.org/10.1007/s11105-019-01145-6.

[25] Higginson EG . The genetic basis of acid composition in developing berries of the cultivated grapevine Vitis vinifera. Ph.D. thesis. University of Adelaide; 2016.

[26] Duchene E, Dumas V, Butterlin G et al. Genetic variations of acidity in grape berries are controlled by the interplay between organic acids and potassium. Theor Appl Genet. 2020; 133: 993-1008. https://doi.org/10.1007/s00122-019-03524-9.

[27] Kliewer WM . Berry composition of vitis-vinifera cultivars as influenced by photo-temperatures and nycto-temperatures during maturation. J Am Soc Hortic Sci. 1973; 98: 153-9.

[28] Ban Y, Mitani N, Sato A et al. Genetic dissection of quantitative trait loci for berry traits in interspecific hybrid grape (Vitis labruscana x Vitis vinifera). Euphytica. 2016; 211: 295-310. https://doi.org/10.1007/s10681-016-1737-8.

[29] Viana AP, Riaz S, Walker MA . Genetic dissection of agronomic traits within a segregating population of breeding table grapes. Genet Mol Res. 2013; 12: 951-64. https://doi.org/10.4238/2013.April.2.11.

[30] Yang SS, Fresnedo-Ramirez J, Sun Q et al. Next generation mapping of Enological traits in an F-2 interspecific grapevine hybrid family. PLoS One. 2016; 11. https://doi.org/10.1371/journal.pone.0149560.

[31] Waterhouse AL, Sacks GL, Jeffery DW . Understanding Wine Chemistry. John Wiley & Sons, Ltd; 2016.

[32] Bai Y, Dougherty L, Li M et al. A natural mutation-led truncation in one of the two aluminum-activated malate transporter-like genes at the ma locus is associated with low fruit acidity in apple. Mol Gen Genomics. 2012; 287: 663-78. https://doi.org/10.1007/s00438-012-0707-7.

[33] Regalado A, Leonardo C, Bitetto M et al. Characterization of mitochondrial dicarboxylate/tricarboxylate transporters from grape berries. Planta. 2013; 237: 693-703. https://doi.org/10.1007/s00425-012-1786-8.

[34] Homble F, Krammer EM, Prevost M . Plant VDAC: facts and speculations. Biochimica Et Biophysica Acta-Biomembranes. 2012; 1818: 1486-501. https://doi.org/10.1016/j.bbamem.2011.11.028.

[35] Li SJ, Yin XR, Xie XL et al. The citrus transcription factor, CitERF13, regulates citric acid accumulation via a protein-protein interaction with the vacuolar proton pump, CitVHA-c4. Sci Rep. 2016; 6. https://doi.org/10.1038/srep20151.

[36] Hu DG, Li YY, Zhang QY et al. The R2R3-MYB transcription factor MdMYB73 is involved in malate accumulation and vacuolar acidification in apple. Plant J. 2017; 91: 443-54. https://doi.org/10.1111/tpj.13579.

[37] Jia D, Shen F, Wang Y et al. Apple fruit acidity is genetically diversified by natural variations in three hierarchical epistatic genes: MdSAUR37, MdPP2CH and MdALMTII. Plant J. 2018; 95: 427-43. https://doi.org/10.1111/tpj.13957.

[38] Fortes AM, Agudelo-Romero P, Silva MS et al. Transcript and metabolite analysis in Trincadeira cultivar reveals novel information regarding the dynamics of grape ripening. BMC Plant Biol. 2011; 11: 149. https://doi.org/10.1186/1471-2229-11-149.

[39] Fasoli M, Richter CL, Zenoni S et al. Timing and order of the molecular events marking the onset of berry ripening in grapevine. Plant Physiol. 2018; 178: 1187-206. https://doi.org/10.1104/pp.18.00559.

[40] Terrier N, Glissant D, Grimplet J et al. Isogene specific oligo arrays reveal multifaceted changes in gene expression during grape berry (Vitis vinifera L.) development. Planta. 2005; 222: 832-47. https://doi.org/10.1007/s00425-005-0017-y.

[41] OR E, Baybik J, Sadka A et al. Isolation of mitochondrial malate dehydrogenase and phosphoenolpyruvate carboxylase cDNA clones from grape berries and analysis of their expression pattern throughout berry development. J Plant Physiol. 2000; 157: 527-34. https://doi.org/10.1016/S0176-1617(00)80108-X.

[42] Deluc LG, Grimplet J, Wheatley MD et al. Transcriptomic and metabolite analyses of cabernet sauvignon grape berry development. BMC Genomics. 2007; 8: 429. https://doi.org/10.1186/1471-2164-8-429.

[43] Ruffner HP, Hawker JS, Hale CR . Temperature and enzymic control of malate metabolism in berries of vitis-vinifera. Phytochemistry. 1976; 15: 1877-80. https://doi.org/10.1016/S0031-9422(00)88835-4.

[44] Duchene E, Butterlin G, Dumas V et al. Towards the adaptation of grapevine varieties to climate change: QTLs and candidate genes for developmental stages. Theor Appl Genet. 2012; 124: 623-35. https://doi.org/10.1007/s00122-011-1734-1.

[45] Kamal N, Schwandner A, Viehover P et al. Characterization of genes and alleles involved in the control of flowering time in grapevine. PLoS One. 2019; 14: e0214703. https://doi.org/10.1371/journal.pone.0214703.

[46] Kobayashi S, Goto-Yamamoto N, Hirochika H . Retrotransposon-induced mutations in grape skin color. Science. 2004; 304. https://doi.org/10.1126/science.1095011.

[47] Marrano A. Genome-Wide Patterns of Genetic Variation among Wild and Cultivated Grapevines (V. vinifera L.) . Ph.D. thesis. Università degli Studi di Padova; 2013.

[48] Schwander F, Zyprian E, Topfer R . Xi International Conference on Grapevine Breeding and Genetics. In: Li SH, Archbold D, London J, editors. Acta Horticulturae.Vol. 1082. 2015. p. 315-9.

[49] Reisch B, Robinson WB, Kimball K et al. Horizon grape. HortScience. 1983; 18: 108-9.

[50] Dalbo MA, Ye GN, Weeden NF et al. A gene controlling sex in grapevines placed on a molecular marker-based genetic map. Genome. 2000; 43: 333-40. https://doi.org/10.1139/gen-43-2-333.

[51] Bradbury PJ, Zhang Z, Kroon DE et al. TASSEL: software for association mapping of complex traits in diverse samples. Bioinformatics. 2007; 23: 2633-5. https://doi.org/10.1093/bioinformatics/btm308.

[52] Wickham H. ggplot2: Elegant Graphics for Data Analysis . Springer; 2009.

[53] Danecek P, Auton A, Abecasis G et al. The variant call format and VCFtools. Bioinformatics. 2011; 27: 2156-8. https://doi.org/10.1093/bioinformatics/btr330.

[54] Rastas P. Lep-MAP3: robust linkage mapping even for low-coverage whole genome sequencing data. Bioinformatics. 2017; 33: 3726-32. https://doi.org/10.1093/bioinformatics/btx494.

[55] Broman KW, Wu H, Sen S et al. R/QTL: QTL mapping in experimental crosses. Bioinformatics. 2003; 19: 889-90. https://doi.org/10.1093/bioinformatics/btg112.

[56] R Development Core Team . R: A language and environment for statistical computing. In: R Foundation for Statistical Computing.Vienna, Austria. URL https://www.R-project.org/; 2019.

[57] Castellari M, Versari A, Spinabelli U et al. An improved HPLC method for the analysis of organic acids, carbohydrates, and alcohols in grape musts and wines. J Liq Chromatogr Relat Technol. 2000; 23: 2047-56. https://doi.org/10.1081/jlc-100100472.

[58] Nally RM, Walsh CJ . Hierarchical partitioning public-domain software. Biodivers Conserv. 2004; 13: 659-60. https://doi.org/10.1023/B:BIOC.0000009515.11717.0b.

[59] Holland JB, Nyquist WE, Cervantes-Martínez CT . Plant Breeding Reviews . 2002: 9-112.

[60] Kawamura K, Hibrand-Saint Oyant L, Crespel L et al. Quantitative trait loci for flowering time and inflorescence architecture in rose. Theor Appl Genet. 2011; 122: 661-75. https://doi.org/10.1007/s00122-010-1476-5.

[61] Bates D, Maechler M, Bolker B et al. Fitting linear mixed-effects models using lme4. J Stat Softw. 2015; 67: 1-48. https://doi.org/10.18637/jss.v067.i01.

[62] Mendiburu F. agricolae: Statistical Procedures for Agricultural Research. R package version 1.2-4. https://CRAN.R-project.org/package=agricolae; 2016.

[63] Peterson BG, Carl P . PerformanceAnalytics: Econometric Tools for Performance and Risk Analysis. R package version 2.0.4. https://CRAN.R-project.org/package=PerformanceAnalytics; 2020.

[64] Voorrips RE . MapChart: software for the graphical presentation of linkage maps and QTLs. J Hered. 2002; 93: 77-8. https://doi.org/10.1093/jhered/93.1.77.

[65] Osier MV . VitisPathways: gene pathway analysis for V-vinifera. Vitis. 2016; 55: 129-33. https://doi.org/10.5073/vitis.2016.55.129-133.