QTL analysis for ascorbic acid content in strawberry fruit reveals a complex genetic architecture and association with GDP-L-galactose phosphorylase

Abstract

Strawberry (Fragaria × ananassa) fruits are an excellent source of L-ascorbic acid (AsA), a powerful antioxidant for plants and humans. Identifying the genetic components underlying AsA accumulation is crucial for enhancing strawberry nutritional quality. Here, we unravel the genetic architecture of AsA accumulation using an F1 population derived from parental lines ‘Candonga’ and ‘Senga Sengana’, adapted to distinct Southern and Northern European areas. To account for environmental effects, the F1 and parental lines were grown and phenotyped in five locations across Europe (France, Germany, Italy, Poland and Spain). Fruit AsA content displayed normal distribution typical of quantitative traits and ranged five-fold, with significant differences among genotypes and environments. AsA content in each country and the average in all of them was used in combination with 6,974 markers for quantitative trait locus (QTL) analysis. Environmentally stable QTLs for AsA content were detected in linkage group (LG) 3A, LG 5A, LG 5B, LG 6B and LG 7C. Candidate genes were identified within stable QTL intervals and expression analysis in lines with contrasting AsA content suggested that GDP-L-Galactose Phosphorylase FaGGP(3A), and the chloroplast-located AsA transporter gene FaPHT4;4(7C) might be the underlying genetic factors for QTLs on LG 3A and 7C, respectively. We show that recessive alleles of FaGGP(3A) inherited from both parental lines increase fruit AsA content. Furthermore, expression of FaGGP(3A) was two-fold higher in lines with high AsA. Markers here identified represent a useful resource for efficient selection of new strawberry cultivars with increased AsA content.

Acknowledgements

This research was supported by the European Union’s Horizon 2020 research and innovation program (GoodBerry; grant agreement number 679303), Agencia Estatal de Investigación (PID2019-111496RR-I00 / AEI / 10.13039/501100011033) and PR.AVA.AVA2019.034 (IFAPA, FEDER funds). P. Muñoz acknowledged a PhD FPI-INIA contract co-financed by FSE. We are grateful to Francisco J. Durán for his excellent care of strawberry plants and Javier Roldan, Nicolas Oiza, Lidia Jiménez, Rocío Torreblanca and Elsa Martinez-Ferri for their technical assistance.

Author Contributions

P.M. performed the experiments, analyzed data, and wrote the paper; C.C. performed experimentation, analyzed data, and critically edited and reviewed the paper; L.M. and J.A.G. cultivated plants and collected samples at Spain; S.L. and K.O. generated and propagated the Can×SS population, cultivated plants and collected samples at Germany; A.P and P. C. cultivated plants, processed samples and evaluated DM content at France; A. H., J.T., and W.S. processed samples from Germany and analyzed data; A.M. and E.Z. processed samples and evaluated DM content at Poland; F.M.R.Z and B.U. provided RNAseq data from F₁ lines and detected SNPs in FaGGP(3A); L.M. and B.M. cultivated plants, processed samples and evaluated DM content at Italy; S.O. provided RNAseq data and obtained funding; J.S.S. analyzed RNAseq gene expression data and mapped SNP markers to the diploid and octoploid genomes. I.A. conceived the study, analyzed the data, obtained funding and wrote the paper. All authors reviewed and approved the final paper.

Data availability

All the data used in this study is provided in the Supplementary data.

Conflict of interest

The authors declare no competing interest.

References

[1] Davey MW, Montagu M, van, Inzé D et al. Plant L-ascorbic acid: chemistry, function, metabolism, bioavailability and effects of processing. J Sci Food Agric. 2000; 80: 825-60.

[2] Mellidou I, Kanellis AK . Genetic control of ascorbic acid biosynthesis and recycling in horticultural crops. Front Chem. 2017; 5: 177-8.

[3] Fenech M, Amaya I, Valpuesta V et al. Vitamin C content in fruits: biosynthesis and regulation. Front Plant Sci. 2019; 9: 2006.

[4] Nishikimi M, Fukuyama R, Minoshima S et al. Cloning and chromosomal mapping of the human nonfunctional gene for L-gulono-gamma-lactone oxidase, the enzyme for L-ascorbic acid biosynthesis missing in man. J Biol Chem. 1994; 269: 13685-8.

[5] Carr AC, Maggini S . Vitamin C and immune function. Nutrients. 2017; 9: 1211.

[6] Lester GE . Environmental regulation of human health nutrients (ascorbic acid, β-carotene, and folic acid) in fruits and vegetables. HortScience. 2006; 41: 59-64.

[7] Wheeler G, Ishikawa T, Pornsaksit V et al. Evolution of alternative biosynthetic pathways for vitamin C following plastid acquisition in photosynthetic eukaryotes. elife. 2015; 4: e06369.

[8] Wheeler GL, Jones MA, Smirnoff N . The biosynthetic pathway of vitamin C in higher plants. Nature. 1998; 393: 365-9.

[9] Dowdle J, Ishikawa T, Gatzek S et al. Two genes in Arabidopsis thaliana encoding GDP-L-galactose phosphorylase are required for ascorbate biosynthesis and seedling viability. Plant J. 2007; 52: 673-89.

[10] Laing WA, Martínez-Sánchez M, Wright MA et al. An upstream open reading frame is essential for feedback regulation of ascorbate biosynthesis in Arabidopsis. Plant Cell. 2015; 27: 772-86.

[11] Bulley S, Laing W . The regulation of ascorbate biosynthesis. Curr Opin Plant Biol. 2016; 33: 15-22.

[12] Fenech M, Amorim-Silva V, Valle AED et al. The role of GDP-L-galactose phosphorylase in the control of ascorbate biosynthesis. Plant Physiol. 2021; 185: 1574-94.

[13] Zhang H, Si X, Ji X et al. Genome editing of upstream open reading frames enables translational control in plants. Nat Biotechnol. 2018; 36: 894-8.

[14] Mellidou I, Chagné D, Laing WA et al. Allelic variation in paralogs of GDP-L-galactose phosphorylase is a major determinant of vitamin C concentrations in apple fruit. Plant Physiol. 2012; 160: 1613-29.

[15] Li T, Yang X, Yu Y et al. Domestication of wild tomato is accelerated by genome editing. Nat Biotechnol. 2018; 36: 1160-3.

[16] Mellidou I, Keulemans J, Kanellis AK et al. Regulation of fruit ascorbic acid concentrations during ripening in high and low vitamin C tomato cultivars. BMC Plant Biol. 2012; 12: 239.

[17] Bulley S, Wright M, Rommens C et al. Enhancing ascorbate in fruits and tubers through over-expression of the L-galactose pathway gene GDP-L-galactose phosphorylase. Plant Biotechnol J. 2012; 10: 390-7.

[18] Mellidou I, Koukounaras A, Kostas S et al. Regulation of vitamin C accumulation for improved tomato fruit quality and alleviation of abiotic stress. Genes. 2021; 12: 694.

[19] Wolucka BA, Montagu MV . GDP-mannose 3′,5′-epimerase forms GDP-L-gulose, a putative intermediate for the de novo biosynthesis of vitamin C in plants. J Biol Chem. 2003; 278: 47483-90.

[20] Badejo A, Wada K, Gao Y et al. Translocation and the alternative D-galacturonate pathway contribute to increasing the ascorbate level in ripening tomato fruits together with the D-mannose/L-galactose pathway. J Exp Bot. 2012; 63: 229-39.

[21] Agius F, González-Lamothe R, Caballero JL et al. Engineering increased vitamin C levels in plants by overexpression of a D-galacturonic acid reductase. Nat Biotechnol. 2003; 21: 177-81.

[22] Lorence A, Chevone BI, Mendes P et al. Myo-inositol oxygenase offers a possible entry point into plant ascorbate biosynthesis. Plant Physiol. 2004; 134: 1200-5.

[23] Kanter U, Usadel B, Guerineau F et al. The inositol oxygenase gene family of Arabidopsis is involved in the biosynthesis of nucleotide sugar precursors for cell-wall matrix polysaccharides. Planta. 2005; 221: 243-54.

[24] Kavkova EI, Blöchl C, Tenhaken R . The Myo-inositol pathway does not contribute to ascorbic acid synthesis. Plant Biol. 2018; 21: 95-102.

[25] Foyer CH, Halliwell B . The presence of glutathione and glutathione reductase in chloroplasts: a proposed role in ascorbic acid metabolism. Planta. 1976; 133: 21-5.

[26] Miyaji T, Kuromori T, Takeuchi Y et al. AtPHT4;4 is a chloroplast-localized ascorbate transporter in Arabidopsis. Nature. 2015; 6: 5928-11.

[27] Sechet J, Htwe S, Urbanowicz B et al. Suppression of Arabidopsis GGLT1 affects growth by reducing the L-galactose content and borate cross-linking of rhamnogalacturonan-II. Plant J. 2018; 96: 1036-50.

[28] Darrow GM . The strawberry. In: History, Breeding and Physiology. Holt, Rinehart and Winston: New York, NY, USA, 1966.

[29] Hancock JF . Strawberries. CAB International; 1999.

[30] Edger PP, Poorten TJ, VanBuren R et al. Origin and evolution of the octoploid strawberry genome. Nat Genet. 2019; 51: 541-7.

[31] Edger PP, Mckain MR, Yocca AE et al. Reply to: revisiting the origin of octoploid strawberry. Nat Genet. 2020; 52: 5-7.

[32] Liston A, Wei N, Tennessen JA et al. Revisiting the origin of octoploid strawberry. Nat Genet. 2020; 52: 2-4.

[33] Tennessen JA, Govindarajulu R, Ashman T-L et al. Evolutionary origins and dynamics of octoploid strawberry subgenomes revealed by dense targeted capture linkage maps. Genome Biol Evolution. 2014; 6: 3295-313.

[34] Giampieri F, Forbes-Hernandez TY, Gasparrini M et al. Strawberry as a health promoter: an evidence based review. Food Funct. 2015; 6: 1386-98.

[35] Cruz-Rus E, Amaya I, Sánchez-Sevilla JF et al. Regulation of L-ascorbic acid content in strawberry fruits. J Exp Bot. 2011; 62: 4191-201.

[36] Mezzetti B, Balducci F, Capocasa F et al. Breeding strawberry for higher phytochemicals content and claim it: is it possible? J Hortic Sci. 2016; 16: 194-206.

[37] Mazzoni L, Vittori LD, Balducci F et al. Sensorial and nutritional quality of inter and intra-specific strawberry genotypes selected in resilient conditions. Sci Hortic. 2020; 261: 108945.

[38] Zorrilla-Fontanesi Y, Cabeza A, Domínguez P et al. Quantitative trait loci and underlying candidate genes controlling agronomical and fruit quality traits in octoploid strawberry (Fragaria × ananassa). Theor Appl Genet. 2011; 123: 755-78.

[39] Whitaker VM, Lee S, Osorio LF et al. Advances in strawberry breeding at the University of Florida. Acta Hortic. 2017; 1156: 1-6.

[40] Whitaker VM, Knapp SJ, Hardigan MA et al. A roadmap for research in octoploid strawberry. Hortic Res 2020; 7: 33.

[41] Senger E, Osorio S, Olbricht K et al. Towards smart and sustainable development of modern berry cultivars in Europe. Plant J. 2022; 111: 1238-51.

[42] Stevens R, Buret M, Duffé P et al. Candidate genes and quantitative trait loci affecting fruit ascorbic acid content in three tomato populations. Plant Physiol. 2007; 143: 1943-53.

[43] Stevens R, Page D, Gouble B et al. Tomato fruit ascorbic acid content is linked with monodehydroascorbate reductase activity and tolerance to chilling stress. Plant Cell Environ. 2008; 31: 1086-96.

[44] Davey MW, Kenis K, Keulemans J . Genetic control of fruit vitamin C contents. Plant Physiol. 2006; 142: 343-51.

[45] Vallarino JG, Pott DM, Cruz-Rus E et al. Identification of quantitative trait loci and candidate genes for primary metabolite content in strawberry fruit. Hortic Res. 2019; 6: 4.

[46] Sánchez-Sevilla JF, Horvath A, Botella MA et al. Diversity arrays technology (DArT) marker platforms for diversity analysis and linkage mapping in a complex crop, the octoploid cultivated strawberry (Fragaria × ananassa). PLoS One. 2015; 10: e0144960.

[47] Hardigan MA, Feldmann MJ, Lorant A et al. Genome synteny has been conserved among the octoploid progenitors of cultivated strawberry over millions of years of evolution. Front Plant Sci. 2020; 10: 1789.

[48] van Ooijen JW . In: Kyazma BV, ed. JoinMap® 5, Software for the Calculation of Genetic Linkage Maps in Experimental Populations of Diploid Species. Wageningen, Netherlands, 2018.

[49] Hardigan MA, Feldmann MJ, Pincot DDA et al. Blueprint for phasing and assembling the genomes of heterozygous Polyploids: application to the Octoploid genome of strawberry. Biorxiv. 2021: 2021.11.03.467115.

[50] Liu X, Wu R, Bulley SM et al. Kiwifruit MYBS1-like and GBF3 transcription factors influence l-ascorbic acid biosynthesis by activating transcription of GDP-L-galactose phosphorylase 3. New Phytol. 2022; 234: 1782-800.

[51] Liao G, Chen L, He Y et al. Three metabolic pathways are responsible for the accumulation and maintenance of high AsA content in kiwifruit (Actinidia eriantha). BMC Genomics. 2021; 22: 13.

[52] Lerceteau-Köhler E, Moing A, Guerin G et al. Genetic dissection of fruit quality traits in the octoploid cultivated strawberry highlights the role of homoeo-QTL in their control. Theor Appl Genet. 2012; 124: 1059-77.

[53] Sánchez-Sevilla JF, Vallarino JG, Osorio S et al. Gene expression atlas of fruit ripening and transcriptome assembly from RNA-seq data in octoploid strawberry (Fragaria × ananassa). Sci Rep. 2017; 7: 13737.

[54] Liu H, Wei L, Ni Y et al. Genome-wide analysis of ascorbic acid metabolism related genes in Fragaria × ananassa and its expression pattern analysis in strawberry fruits. Front Plant Sci. 2022; 13: 954505.

[55] Li D, Li L, Luo Z et al. Comparative transcriptome analysis reveals the influence of abscisic acid on the metabolism of pigments, ascorbic acid and folic acid during strawberry fruit ripening. PLoS One. 2015; 10: e0130037.

[56] Schauer N, Semel Y, Balbo I et al. Mode of inheritance of primary metabolic traits in tomato. Plant Cell. 2008; 20: 509-23.

[57] Pott DM, Vallarino JG, Cruz-Rus E et al. Genetic analysis of phenylpropanoids and antioxidant capacity in strawberry fruit reveals mQTL hotspots and candidate genes. Sci Rep. 2020; 10: 20197-15.

[58] Toor RK, Savage GP, Lister CE . Seasonal variations in the antioxidant composition of greenhouse grown tomatoes. J Food Compos Anal. 2006; 19: 1-10.

[59] Dumas Y, Dadomo M, Lucca GD et al. Effects of environmental factors and agricultural techniques on antioxidant content of tomatoes. J Sci Food Agr. 2003; 83: 369-82.

[60] Tulipani S, Mezzetti B, Capocasa F et al. Antioxidants, phenolic compounds, and nutritional quality of different strawberry genotypes. J Agric Food Chem. 2008; 56: 696-704.

[61] Ye J, Li W, Ai G et al. Genome-wide association analysis identifies a natural variation in basic helix-loop-helix transcription factor regulating ascorbate biosynthesis via D-mannose/L-galactose pathway in tomato. PLoS Genet. 2019; 15: e1008149.

[62] Cheng CH, Seal AG, Boldingh HL et al. Inheritance of taste characters and fruit size and number in a diploid Actinidia chinensis (kiwifruit) population. Euphytica. 2004; 138: 185-95.

[63] Sargent DJ, Buti M, Surbanovski N et al. Identification of QTLs for powdery mildew (Podosphaera aphanis; syn. Sphaerotheca macularis f. sp. fragariae) susceptibility in cultivated strawberry (Fragaria × ananassa). PLoS One. 2019; 14: e0222829-17.

[64] Capel C, Carmen AF d, Alba JM et al. Wide-genome QTL mapping of fruit quality traits in a tomato RIL population derived from the wild-relative species Solanum pimpinellifolium L. Theor Appl Genet. 2015; 128: 2019-35.

[65] Roth C, Menzel G, Petétot JM-C et al. Characterization of a protein of the plastid inner envelope having homology to animal inorganic phosphate, chloride and organic-anion transporters. Planta. 2004; 218: 406-16.

[66] Semagn K, Babu R, Hearne S et al. Single nucleotide polymorphism genotyping using Kompetitive allele specific PCR (KASP): overview of the technology and its application in crop improvement. Mol Breed. 2014; 33: 1-14.

[67] de Meaux J, de. Cis-regulatory variation in plant genomes and the impact of natural selection. Am J Bot. 2018; 105: 1788-91.

[68] Davey MW, Dekempeneer E, Keulemans J . Rocket-powered high-performance liquid chromatographic analysis of plant ascorbate and glutathione. Anal Biochem. 2003; 316: 74-81.

[69] Harapanhalli RS, Howell RW, Rao DV . Testicular and plasma ascorbic acid levels in mice following dietary intake: a high-performance liquid chromatographic analysis. J Chromatogr B Biomed Sci Appl. 1993; 614: 233-43.

[70] Alseekh S, Tohge T, Wendenberg R et al. Identification and mode of inheritance of quantitative trait loci for secondary metabolite abundance in tomato. Plant Cell. 2015; 27: 485-512.

[71] Doyle JJ, Doyle JL . Isolation of plant DNA from fresh tissue. Focus. 1990; 12: 13-5.

[72] Zorrilla-Fontanesi Y, Cabeza A, Torres A et al. Development and bin mapping of strawberry genic-SSRs in diploid Fragaria and their transferability across the Rosoideae subfamily. Mol Breed. 2011; 27: 137-56.

[73] Sánchez-Sevilla JF, Cruz-Rus E, Valpuesta V et al. Deciphering gamma-decalactone biosynthesis in strawberry fruit using a combination of genetic mapping, RNA-Seq and eQTL analyses. Genomics. 2014; 15: 218.

[74] Cruz-Rus E, Sesmero R, Ángel-Pérez JA et al. Validation of a PCR test to predict the presence of flavor volatiles mesifurane and γ-decalactone in fruits of cultivated strawberry (Fragaria × ananassa). Mol Breed. 2017; 37: 131.

[75] Edger PP, VanBuren R, Colle M et al. Single-molecule sequencing and optical mapping yields an improved genome of woodland strawberry (Fragaria vesca) with chromosome-scale contiguity. GigaScience. 2018; 7: 1-7.

[76] van Ooijen JW . In: Kyazma BV, ed. MapQTL 6®: Software for the Mapping of Quantitative Trait Loci in Experimental Populations of Diploid Species. Wageningen, The Netherlands, 2009.

[77] Gambino G, Perrone I, Gribaudo I . A rapid and effective method for RNA extraction from different tissues of grapevine and other woody plants. Phytochem Anal. 2008; 19: 520-5.