High-density linkage maps and loci for berry color and flower sex in muscadine grape (Vitis rotundifolia)

  • Jennifer Lewter
  • Margaret L. WorthingtonEmail author
  • John R. Clark
  • Aruna V. Varanasi
  • Lacy Nelson
  • Christopher L. Owens
  • Patrick Conner
  • Gunawati Gunawan
Original Article


Key message

Linkage maps of muscadine grape generated using genotyping-by-sequencing (GBS) provide insight into genome collinearity between Muscadinia and Euvitis subgenera and genetic control of flower sex and berry color.


The muscadine grape, Vitis rotundifolia, is a specialty crop native to the southeastern USA. Muscadine vines can be male, female, or perfect-flowered, and berry color ranges from bronze to black. Genetic linkage maps were constructed using genotyping-by-sequencing in two F1 populations segregating for flower sex and berry color. The linkage maps consisted of 1244 and 2069 markers assigned to 20 linkage groups (LG) for the ‘Black Beauty’ × ‘Nesbitt’ and ‘Supreme’ × ‘Nesbitt’ populations, respectively. Data from both populations were used to generate a consensus map with 2346 markers across 20 LGs. A high degree of collinearity was observed between the genetic maps and the Vitis vinifera physical map. The higher chromosome number in muscadine (2n = 40) compared to V. vinifera (2n = 38) was accounted for by the behavior of V. vinifera chromosome 7 as two independently segregating LGs in muscadine. The muscadine sex locus mapped to an interval that aligned to 4.64–5.09 Mb on V. vinifera chromosome 2, a region which includes the previously described V. vinifera subsp. sylvestris sex locus. While the MYB transcription factor genes controlling fruit color in V. vinifera are located on chromosome 2, the muscadine berry color locus mapped to an interval aligning to 11.09–11.88 Mb on V. vinifera chromosome 4, suggesting that a mutation in a different gene in the anthocyanin biosynthesis pathway determines berry color in muscadine. These linkage maps lay the groundwork for marker-assisted breeding in muscadine and provide insight into the evolution of Vitis species.



We thank the staff of the University of Arkansas System Division of Agriculture Fruit Research Station for planting and maintaining the mapping populations. The authors also acknowledge the USDA-ARS Grape Genetics Research Unit, the Cornell University Biotechnology Resource Center, and the Institute of Genomic Diversity, Cornell University, for their contributions to data generation.


This research was supported by a grant from the Southern Region Small Fruit Consortium, SRSFC Project # 2012-02.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

122_2019_3302_MOESM1_ESM.jpg (2.5 mb)
Supplementary Fig. 1 Pedigrees of ‘Black Beauty,’ ‘Supreme,’ and ‘Nesbitt’ based on breeders’ records as documented in Clark (1997), Conner (2013), and Goldy and Nesbitt (1985). Lines connecting female and male parents to offspring are colored red and blue, respectively. Bronze- and black-fruited cultivars and breeding selections are colored yellow and purple, respectively. Breeding selections with unknown berry color are indicated in white (JPEG 2522 kb)
122_2019_3302_MOESM2_ESM.pdf (254 kb)
Supplementary Fig. 2 High-density genetic linkage map of the ‘Black Beauty’ × ‘Nesbitt’ mapping population. Markers segregating in only the female parent (lm × ll), the male parent (nn × np), and both parents (hk × hk) are colored red, blue, and black, respectively. The VR006 and VR009 muscadine sex markers developed by Conner et al. (2017) are colored orange, the sex locus is colored green, and the color locus is colored pink. Marker positions are expressed in centimorgans (PDF 253 kb)
122_2019_3302_MOESM3_ESM.pdf (308 kb)
Supplementary Fig. 3 High-density genetic linkage map of the ‘Supreme’ × ‘Nesbitt’ mapping population. Markers segregating in only the female parent (lm × ll), the male parent (nn × np), and both parents (hk × hk) are colored red, blue, and black, respectively. The VR006 and VR009 muscadine sex markers developed by Conner et al. (2017) are colored orange, the sex locus is colored green, and the color locus is colored pink. Marker positions are expressed in centimorgans (PDF 308 kb)
122_2019_3302_MOESM4_ESM.pdf (329 kb)
Supplementary Fig. 4 High-density consensus linkage map of muscadine grape created from ‘Black Beauty’ × ‘Nesbitt’ (BB × N) and ‘Supreme’ × ‘Nesbitt’ (S × N) F1 populations. Markers present in only the BB × N linkage map and segregating in only the female parent (lm × ll), the male parent (nn × np), and both parents (hk × hk) are colored orange, aqua, and forest green, respectively. Markers present in only the S × N linkage map and segregating in only the female parent (lm × ll), the male parent (nn × np), and both parents (hk × hk) are colored red, blue, and neon green, respectively. Markers present in both linkage maps and segregating in only the female parents (lm × ll), the male parent (nn × np), and all three parents (hk × hk) are colored pink, dark blue, and black, respectively. The VR006 and VR009 muscadine sex markers developed by Conner et al. (2017), sex locus, and color locus are all colored yellow. Marker positions are expressed in centimorgans (PDF 328 kb)
122_2019_3302_MOESM5_ESM.xlsx (894 kb)
Supplementary Table 1 Marker positions in the ‘Black Beauty’ × ‘Nesbitt’ linkage map, physical positions on the V. vinifera reference genome, deviations from the expected segregation ratios, and genotype scores in the progeny (XLSX 894 kb)
122_2019_3302_MOESM6_ESM.xlsx (1.5 mb)
Supplementary Table 2 Marker positions in the ‘Supreme’ × ‘Nesbitt’ linkage map, physical positions on the V. vinifera reference genome, deviations from the expected segregation ratios, and genotype scores in the progeny. (XLSX 1504 kb)
122_2019_3302_MOESM7_ESM.xlsx (196 kb)
Supplementary Table 3 Marker positions in the muscadine consensus map, physical positions on the V. vinifera reference genome, and marker positions and classes in the ‘Supreme’ × ‘Nesbitt’ and ‘Black Beauty’ × ‘Nesbitt’ F1 linkage maps. (XLSX 195 kb)


  1. Agurto M, Schlechter RO, Armijo G, Solano E, Serrano C, Contreras RA, Zúñiga GE, Arce-Johnson P (2017) RUN1 and REN1 pyramiding in grapevine (Vitis vinifera cv. Crimson Seedless) displays an improved defense response leading to enhanced resistance to powdery mildew (Erysiphe necator). Front Plant Sci 8:1–15. CrossRefGoogle Scholar
  2. Ahmedullah M, Himelrick DG (1990) Grape management. In: Galletta G, Himelrick DG (eds) Small fruit crop management. Prentice Hall, Englewood Cliffs, pp 383–471Google Scholar
  3. Antcliff AJ (1980) Inheritance of sex in Vitis. Ann l’Amelioration des Plantes 30:113–122Google Scholar
  4. Argout X, Salse J, Aury JM, Guiltinan MJ, Droc G, Gouzy J, Allegre M, Chaparro C, Legavre T, Maximova SN, Abrouk M, Murat F, Fouet O, Poulain J, Ruiz M, Roguet Y, Rodier-Goud M, Barbosa-Neto JF et al (2011) The genome of Theobroma cacao. Nat Genet 43:101–108. CrossRefGoogle Scholar
  5. Barker CL, Donald T, Pauquet J, Ratnaparkhe MB, Bouquet A, Adam-Blondon A-F, Thomas MR, Dry I (2005) Genetic and physical mapping of the grapevine powdery mildew resistance gene, Run1, using a bacterial artificial chromosome library. Theor Appl Genet 111:370–377. CrossRefGoogle Scholar
  6. Battilana J, Lorenzi S, Moreira FM, Moreno-Sanz P, Failla O, Emanuelli F, Grando MS (2013) Linkage mapping and molecular diversity at the flower sex locus in wild and cultivated grapevine reveal a prominent SSR haplotype in hermaphrodite plants. Mol Biotechnol 54:1031–1037. CrossRefGoogle Scholar
  7. Biasi LA, Conner PJ (2016) Reproductive traits of hermaphroditic muscadine cultivars. HortScience 51:255–261CrossRefGoogle Scholar
  8. Blanc S, Wiedemann-Merdinoglu S, Dumas V, Mestre P, Merdinoglu D (2012) A reference genetic map of Muscadinia rotundifolia and identification of Ren5, a new major locus for resistance to grapevine powdery mildew. Theor Appl Genet 125:1663–1675. CrossRefGoogle Scholar
  9. Brizicky GK (1965) The genera of Vitaceae in the southeastern United States. J Arnold Arbor 46:48–67Google Scholar
  10. Carbonneau A (1983) Sterilites male et femelle dans le genre Vitis, 1: modelisation de leur heredite. Agronomie 3:635–644CrossRefGoogle Scholar
  11. Clark JR (1997) Grapes. In: Brooks RM, Olmo HP (eds) Register of fruit and nut varieties, 3rd edn. ASHS Press, Alexandria, pp 248–299Google Scholar
  12. Coito JL, Ramos MJN, Cunha J, Silva HG, Amâncio S, Costa MMR, Rocheta M (2017) VviAPRT3 and VviFSEX: two genes involved in sex specification able to distinguish different flower types in Vitis. Front Plant Sci 8:1–11. CrossRefGoogle Scholar
  13. Conner PJ (2009) A century of muscadine grape (Vitis rotundifolia Michx.) breeding at the University of Georgia. Acta Hortic 827:481–484CrossRefGoogle Scholar
  14. Conner PJ (2013) ‘Lane’: an early-season self-fertile black muscadine grape. HortScience 48:128–129CrossRefGoogle Scholar
  15. Conner PJ, MacLean D (2013) Fruit anthocyanin profile and berry color of muscadine grape cultivars and Muscadinia germplasm. HortScience 48:1235–1240CrossRefGoogle Scholar
  16. Conner PJ, Conner J, Catotti P, Lewter J, Clark JR, Biasi LA (2017) Development and characterization of molecular markers associated with female plants in muscadine grape. J Am Soc Hortic Sci 142:143–150. CrossRefGoogle Scholar
  17. Dalbó MA, Ye GN, Weeden NF, Steinkellner H, Sefc KM, Reisch BI (2000) A gene controlling sex in grapevines placed on a molecular marker-based genetic map. Genome 43:333–340CrossRefGoogle Scholar
  18. Dearing C (1948) New muscadine grapes, vol 769. US Department of Agriculture CircularGoogle Scholar
  19. Detjen LR (1917) Inheritance of sex in Vitis rotundifolia, vol 17. North Carolina Agricultural Experiment Station Technical BulletinGoogle Scholar
  20. Detjen LR (1919) The limits in hybridization of Vitis rotundifolia with related species and genera, vol 17. North Carolina Agricultural Experiment Station Technical Bulletin, pp 1–25Google Scholar
  21. Doligez A, Adam-Blondon A-F, Cipriani G, Di Gaspero G, Laucou V, Merdinoglu D, Meredith CP, Riaz S, Roux C, This P (2006) An integrated SSR map of grapevine based on five mapping populations. Theor Appl Genet 113:369–382CrossRefGoogle Scholar
  22. Dunstan RT (1964) Hybridization of Euvitis × V. rotundifolia: backcrosses to muscadine. J Am Soc Hortic Sci 84:238–242Google Scholar
  23. Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES, Mitchell SE (2011) A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. PLoS ONE 6:1–10. CrossRefGoogle Scholar
  24. Fechter I, Hausmann L, Daum M, Rosleff Sörensen T, Viehöver P, Weisshaar B, Töpfer R (2012) Candidate genes within a 143 kb region of the flower sex locus in Vitis. Mol Genet Genomics 287:247–259. CrossRefGoogle Scholar
  25. Feechan A, Anderson C, Torregrosa L, Jermakow A, Mestre P, Wiedemann-Merdinoglu S, Merdinoglu D, Walker AR, Cadle-Davidson L, Reisch B, Aubourg S, Bentahar N, Shrestha B, Bouquet A, Adam-Blondon AF, Thomas MR, Dry IB (2013) Genetic dissection of a TIR-NB-LRR locus from the wild North American grapevine species Muscadinia rotundifolia identifies paralogous genes conferring resistance to major fungal and oomycete pathogens in cultivated grapevine. Plant J 76:661–674. CrossRefGoogle Scholar
  26. Feechan A, Kocsis M, Riaz S, Zhang W, Gadoury DM, Walker MA, Dry IB, Reisch B, Cadle-davidson L (2015) Strategies for RUN1 deployment using RUN2 and REN2 to manage grapevine powdery mildew informed by studies of race specificity. Phytopathology 105:1104–1113CrossRefGoogle Scholar
  27. Firoozabady E, Olmo HP (1982) Resistance to grape phylloxera in Vitis vinifera × V. rotundifolia grape hybrids. Vitis 21:1–4Google Scholar
  28. Fournier-Level A, Le Cunff L, Gomez C, Doligez A, Ageorges A, Roux C, Bertrand Y, Souquet J-M, Cheynier V, This P (2009) Quantitative genetic bases of anthocyanin variation in grape (Vitis vinifera L. ssp. sativa) berry: a quantitative trait locus to quantitative trait nucleotide integrated study. Genetics 183:1127–1139CrossRefGoogle Scholar
  29. Glaubitz JC, Casstevens TM, Lu F, Harriman J, Elshire RJ, Sun Q, Buckler ES (2014) TASSEL-GBS: a high capacity genotyping by sequencing analysis pipeline. PLoS ONE. Google Scholar
  30. Goldy RG, Nesbitt WB (1985) “Nesbitt” muscadine grape. HortScience 20:777Google Scholar
  31. Haldane JBS (1919) The combination of linkage values and the calculation of distances between the loci of linked factors. J Genet 8:299–309CrossRefGoogle Scholar
  32. Himelrick DG (2001) Vineyard site selection, establishment, and floor management. In: Basiouny FM, Himelrick DG (eds) Muscadine grapes. ASHS Press, Alexandria, pp 133–152Google Scholar
  33. Jaillon O, Aury J-M, Noel B, Policriti A, Clepet C, Casagrande A, Choisne N, Aubourg S, Vitulo N, Jubin C (2007) The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature 449:463CrossRefGoogle Scholar
  34. Karkamkar SP, Patil SG, Misra SC (2010) Cyto-morphological studies and their significance in evolution of family Vitaceae. Nucleus 53:37–43. CrossRefGoogle Scholar
  35. Kobayashi S, Goto-Yamamoto N, Hirochika H (2004) Retrotransposon-induced mutations in grape skin color. Science 304:982. CrossRefGoogle Scholar
  36. Lane R (1997) Breeding muscadine and southern bunch grapes. Fruit Var J 51:144–148Google Scholar
  37. Levadoux L (1946) Étude de la fleur et de la sexualité chez la vigne. Ann l’École Natl d’agriculture Montpellier 27:1–89Google Scholar
  38. Liu XQ, Ickert-Bond SM, Nie ZL, Zhou Z, Chen LQ, Wen J (2016) Phylogeny of the Ampelocissus–Vitis clade in Vitaceae supports the New World origin of the grape genus. Mol Phylogenet Evol 95:217–228. CrossRefGoogle Scholar
  39. Loomis NH, Williams CF, Murphy MM (1954) Inheritance of flower types in muscadine grapes. Proc Am Soc Hortic Sci 64:279–283Google Scholar
  40. Marguerit E, Boury C, Manicki A, Donnart M, Butterlin G, Némorin A, Wiedemann-Merdinoglu S, Merdinoglu D, Ollat N, Decroocq S (2009) Genetic dissection of sex determinism, inflorescence morphology and downy mildew resistance in grapevine. Theor Appl Genet 118:1261–1278. CrossRefGoogle Scholar
  41. Merdinoglu D, Wiedeman-Merdinoglu S, Coste P, Dumas V, Haetty S, Butterlin G, Greif C (2003) Genetic analysis of downy mildew resistance derived from Muscadinia rotundifolia. Acta Hortic 603:451–456CrossRefGoogle Scholar
  42. Myles S, Boyko AR, Owens CL, Brown PJ, Grassi F, Aradhya MK, Prins B, Reynolds A, Chia J-M, Ware D, Bustamante C, Buckler ES (2011) Genetic structure and domestication history of the grape. Proc Natl Acad Sci 108:3530–3535. CrossRefGoogle Scholar
  43. Olien WC (1990) The muscadine grape: botany, viticulture, history, and current industry. HortScience 25:732–739CrossRefGoogle Scholar
  44. Olmo HP (1971) Vinifera rotundifolia hybrids as wine grapes. Am J Enol Vitic 22:87–91Google Scholar
  45. Olmo HP (1986) The potential role of (vinifera × rotundifolia) hybrids in grape variety improvement. Experientia 42:921–926CrossRefGoogle Scholar
  46. Patel GI, Olmo HP (1955) Cytogenetics of Vitis: I. The hybrid V. vinifera × V. rotundifolia. Am J Bot 42:141–159CrossRefGoogle Scholar
  47. Pauquet J, Bouquet A, This P, Adam-Blondon AF (2001) Establishment of a local map of AFLP markers around the powdery mildew resistance gene Run1 in grapevine and assessment of their usefulness for marker assisted selection. Theor Appl Genet 103:1201–1210. CrossRefGoogle Scholar
  48. Picq S, Santoni S, Lacombe T, Latreille M, Weber A, Ardisson M, Ivorra S, Maghradze D, Arroyo-Garcia R, Chatelet P (2014) A small XY chromosomal region explains sex determination in wild dioecious V. vinifera and the reversal to hermaphroditism in domesticated grapevines. BMC Plant Biol 14:229CrossRefGoogle Scholar
  49. Ramos MJN, Coito JL, Fino J, Cunha J, Silva H, de Almeida PG, Costa MMR, Amâncio S, Paulo OS, Rocheta M (2017) Deep analysis of wild Vitis flower transcriptome reveals unexplored genome regions associated with sex specification. Plant Mol Biol 93:151–170. CrossRefGoogle Scholar
  50. Riaz S, Krivanek AF, Xu K, Walker MA (2006) Refined mapping of the Pierce’s disease resistance locus, PdR1, and Sex on an extended genetic map of Vitis rupestris × V. arizonica. Theor Appl Genet 113:1317–1329. CrossRefGoogle Scholar
  51. Riaz S, Tenscher AC, Smith BP, Ng DA, Walker MA (2008) Use of SSR markers to assess identity, pedigree, and diversity of cultivated muscadine grapes. J Am Soc Hortic Sci 133:559–568CrossRefGoogle Scholar
  52. Riaz S, Tenscher AC, Ramming DW, Walker MA (2011) Using a limited mapping strategy to identify major QTLs for resistance to grapevine powdery mildew (Erysiphe necator) and their use in marker-assisted breeding. Theor Appl Genet 122:1059–1073. CrossRefGoogle Scholar
  53. Riaz S, Hu R, Walker MA (2012) A framework genetic map of Muscadinia rotundifolia. Theor Appl Genet 125:1195–1210. CrossRefGoogle Scholar
  54. Sandhu AK, Gu L (2010) Antioxidant capacity, phenolic content, and profiling of phenolic compounds in the seeds, skin, and pulp of Vitis rotundifolia (muscadine grapes) as determined by HPLC-DAD-ESI-MS. J Agric Food Chem 58:4681–4692. CrossRefGoogle Scholar
  55. Small JK (1913) Flora of the southeastern United States: being descriptions of the seed-plants, ferns and fern-allies growing naturally in North Carolina, South Carolina, Georgia, Florida, Tennessee, Alabama, Mississippi, Arkansas, Louisiana and in Oklahoma and Texas eas. J.K. Small, New YorkGoogle Scholar
  56. Staudt G (1997) Evaluation of resistance to grapevine powdery mildew (Uncinula necator [Scuw.] Burr., anamorph Oidium tuckeri Berk.) in accessions of Vitis species. Vitis 36:151–154Google Scholar
  57. Striegler RK, Morris JR, Carter PM, Clark JR, Threlfall RT, Howard LR (2005) Yield, quality, and nutraceutical potential of selected muscadine cultivars grown in southwestern Arkansas. Horttechnology 15:276–284CrossRefGoogle Scholar
  58. Stucky HP (1919) Work with Vitis rotundifolia, a species of muscadine grapes, vol 133. Georgien Experiment Station BulletinGoogle Scholar
  59. This P, Lacombe T, Owens MCCL (2007) Wine grape (Vitis vinifera L.) color associates with allelic variation in the domestication gene VvmybA1. Theor Appl Genet 114:723–730. CrossRefGoogle Scholar
  60. Van Ooijen JW (2006) JoinMap 4, software for the calculation of genetic linkage maps in experimental populations. Kyazma BV, WageningenGoogle Scholar
  61. Voorrips RE (2002) MapChart: software for the graphical presentation of linkage maps and QTLs. J Hered 93:77–78. CrossRefGoogle Scholar
  62. Wan Y, Schwaninger HR, Baldo AM, Labate JA, Zhong GY, Simon CJ (2013) A phylogenetic analysis of the grape genus (Vitis L.) reveals broad reticulation and concurrent diversification during neogene and quaternary climate change. BMC Evol Biol 13:141. CrossRefGoogle Scholar
  63. Wen J (2007) Vitaceae. In: Kubitzki K (ed) The families and genera of vascular plants, vol 9. Springer, Berlin, pp 466–478Google Scholar
  64. Wen J, Nie Z, Soejima A, Meng Y (2007) Phylogeny of Vitaceae based on the nuclear GAI1 gene sequences. Can J Bot 85:731–745. CrossRefGoogle Scholar
  65. Wickham H (2016) ggplot2: elegant graphics for data analysis. Springer, New YorkCrossRefGoogle Scholar
  66. Winsor J (ed) (2016) Narrative and critical history of America, vol 3 (of 8) English explorations and settlements in North America, pp 1497–1689Google Scholar
  67. Zecca G, Abbott JR, Sun WB, Spada A, Sala F, Grassi F (2012) The timing and the mode of evolution of wild grapes (Vitis). Mol Phylogenet Evol 62:736–747. CrossRefGoogle Scholar
  68. Zheng C, Chen E, Albert VA, Lyons E, Sankoff D (2013) Ancient eudicot hexaploidy meets ancestral eurosid gene order. BMC Genom 14:S3. CrossRefGoogle Scholar
  69. Zhou Y, Massonnet M, Sanjak JS, Cantu D, Gaut BS (2017) Evolutionary genomics of grape (Vitis vinifera ssp. vinifera) domestication. Proc Natl Acad Sci 114:11715–11720. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of HorticultureUniversity of ArkansasFayettevilleUSA
  2. 2.USDA-ARS Grape Genetics Research UnitCornell UniversityGenevaUSA
  3. 3.Department of HorticultureUniversity of GeorgiaTiftonUSA
  4. 4.IFGBakersfieldUSA

Personalised recommendations