Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Genetic diversity of ten black walnut (Juglans nigra L.) cultivars and construction of a mapping population

  • 188 Accesses


Black walnut (Juglans nigra L.) nut production stands on the brink of potentially great market expansion that can only be realized with significant genetic and horticultural improvement, such that commercial production becomes feasible. The goal of this study was to incorporate microsatellite/simple sequence repeat (SSR) markers to genotype 11 cultivars and selections, and establish a mapping population from a cross of two cultivars, “Football” × “Sparrow.” Morphology of these two cultivars coupled with phylogenetic analysis supports the notion that a cross between them may yield desirable hybrids. Of the 51 SSR markers amplified, 23 were polymorphic and useful for genotyping. A selection mislabeled as “Mintyle” was definitively identified as “Mintle”; therefore, the number of cultivars was reduced to 10. Furthermore, seven of the 23 markers were used to identify 91 intraspecific hybrids (“Football” × “Sparrow”) resulting from open pollination, thereby establishing the first mapping population of black walnut using SSR markers.

This is a preview of subscription content, log in to check access.

Fig. 1


  1. Adhikari P, Chen L-L, Chen X, Sapkota SD, Hwang C-F (2014) Interspecific hybrid identification of Vitis aestivalis-derived ‘Norton’-based populations using microsatellite markers. Sci Horti 179:363–366

  2. Aradhya MK, Potter D, Gao F, Simon CJ (2006) Molecular phylogeny of Juglans (Juglandaceae): a biogeographic perspective. Tree Genet Genomes 3:363–378

  3. Beedanagari SR, Dove SK, Wood BW, Conner PJ (2005) A first linkage map of pecan cultivarsbased on RAPD and AFLP markers. Theor Appl Genet 110:1127–1137

  4. Bentley NB, Grauke LJ, Klein P (2019) Genotyping by sequencing (GBS) and SNP marker analysis of diverse accessions of pecan (Carya illinoinensis). Tree Genet Genomes 15:1

  5. Bernard A, Lheureux F, Dirlewanger E (2018) Walnut: past and future of genetic improvement. Tree Genet Genomes 14:1

  6. Brawner SA, Warmund MR (2008) Husk softening and kernel characteristics of three black walnut cultivars at successive harvest dates. HortSci 43:691–695

  7. Camara CR, Schlegel V (2016) A review on the potential human health benefits of the black walnut: a comparison with the English walnuts and other tree nuts. Int J Food Prop 19:2175–2189

  8. Coggeshall MV (2011) Black walnut: a crop for the Midwestern United States. HortScience 46:340–342

  9. Coggeshall MV, Woeste KE (2010) The use of microsatellite and phenological descriptors to identify a collection of eastern black walnut cultivars in Missouri, USA. International symposium on molecular markers in horticultural species. Acta Hortic 859:93–98

  10. Dangl GS, Mendum ML, Prins BH, Walker MA, Meredith CP, Simon CJ (2001) Simple sequence repeat analysis of a clonally propogated species: a tool for managing a grape germplasm collection. Genome 44:432–438

  11. Dangl GS, Woeste K, Aradhya MK, Koehmstedt A, Simon C, Potter D, Leslie CA, McGranahan G (2005) Characterization of 14 microsatellite markers for genetic analysis and cultivar identification of walnut. J Am Soc Hortic Sci 130:348–354

  12. Dogan Y, Kafkas S, Sutyemez M, Akca Y, Turemis N (2014) Assessment and characterization of genetic relationships of walnut (Juglans regia L.) genotypes by three types of molecular markers. Sci Hortic 168:81–87

  13. Flores P, Poggi D, Garcia SM, Catraro M, Gariglio N (2017) Effects of pre-stratification storage conditions on black walnut seed post-stratification germination capacity. Int J Fruit Sci 17:29–40

  14. Gold M, Cernusca MM, Godsey L (2004) Consumer preferences for chestnuts, black walnuts, and pecans. HortTechnology 14:583–589

  15. Goonetilleke SN, March TJ, Wirthensohn MG, Arus P, Walker AR (2018) Genotyping by sequencing in almond: SNP discovery, linkage mapping, and marker design. G3·Gen Gen Gen 8:161–172

  16. Hammers M, Sapkota S, Chen L-L, Hwang C-F (2017) Constructing a genetic linkage map of Vitis aestivalis-derived ‘Norton’ and its use in comparing Norton and Cynthiana. Mol Breed 37:64–78

  17. Hammons Products Company (2016) Purchasing guidelines for black walnuts—improved varieties

  18. Hokanson SC, Szewc-McFadden AK, Lamboy WF, McFerson JR (1998) Microsatellite (SSR) markers reveal genetic identities, genetic diversity and relationships in a Malus × domestica Borkh. Core subset collection. Theor Appl Genet 97:671–683

  19. Karimi R, Ahmad E, Vahdati K, Woeste K (2010) Molecular characterization of Persian walnut populations in Iran with microsatellite markers. HortScience 45:1403–1406

  20. Michler C, Woeste K, Pijut P (2007) Black walnut, in genome mapping and molecular breeding in plants v7. In: Kole C (ed) Forest Trees. Springer-Verlag, Heidelberg, pp p189–p198

  21. Moriguchi Y, Ujino-Ihara T, Uchiyama K, Futamura N, Saito M, Ueno S, Matsumoto A, Tani N, Taira H, Shinohara K, Tsumura Y (2012) The construction of a high-density linkage map for identifying SNP markers that are tightly linked to a nuclear-recessive major gene for male sterility in Cryptomeria japonica D. Don. BMC Genet 13:95

  22. Muranty H, Jorge V, Bastien C, Lepoittevin C, Bouffier L, Sanchez L (2014) Potential for marker-assisted selection for forest tree breeding: lessons from 20 years of MAS in crops. Tree Genet Genomes 10:1491–1510

  23. National Agricultural Statistics Service (NASS) - USDA (2016) 2016 California Walnut Objective Measurement Report

  24. Peakall R, Smouse PE (2006) GenAlEx 6: genetic analysis in excel. Population genetic software for teaching and research. Mol Ecol Notes 6:288–295

  25. Peakall R, Smouse PE (2012) GenAlEx 6.5: genetic analysis in excel. Population genetic software for teaching and research—an update. Bioinf 28:2537–2539

  26. Perrier X, Jacquemoud-Collet JP (2006) DARwin software. http://darwin.cirad.fr/

  27. Perrier X, Flori A, Bonnot F (2003) Data analysis methods. In: Hammon P, Seguin M, Perrier X, Glaszmann J (eds) Genetic diversity of cultivated tropical plants. Enfield Science Publishers, Montpellier, pp 43–76

  28. Pollegioni P, Woeste K, Major A, Mugnozza GS, Malvolti ME (2009a) Characterization of Juglans nigra (L.), Juglans regia (L.) and Juglans × intermedia (Carr.) by SSR markers: a case study in Italy. Silv Gen 58:68–78

  29. Pollegioni P, Woeste K, Mugnozza GS, Malvolti ME (2009b) Retrospective identification of hybridogenic walnut plants by SSR fingerprinting and parentage analysis. Mol Breed 24:321–335

  30. Pollegioni P, Van der Linden G, Belisario A, Gras M, Anselmi N, Olimpieri I, Luongo L, Santini A, Turco E, Mugnozza GS, Malvolti ME (2012) Mechanisms governing the responses to anthracnose pathogen in Juglans spp. J Biotechnol 159:251–264

  31. Reid W (1990) Eastern black walnut: potential for commercial nut producing cultivars. In: Janick J, Simon JE (eds) Advances in new crops. Timber Press, Portland, OR, pp 327–331

  32. Reid W (1997) Evaluation and management of black walnut for nut production. In: Knowledge for the future of black walnut. Proceedings of the fifth black walnut symposium. USDA Forest Service, North Central Forest Experiment Station, General Technical Report NC-191. p 211–216

  33. Reid W, Coggeshall MV, Hunt KL (2004) Cultivar evaluation and development for black walnut orchards. In: Michler CH, Pijut PM, Van Sambeek JW, Coggeshall MV, Seifert J, Woeste K, Overton R, Ponder Jr F (eds) Proceedings of the 6th walnut council research symposium; gen. Tech. Rep. NC-243. St. Paul, MN: U.S. Department of Agriculture, Forest Service, North Central Research Station, pp 18–24

  34. Reid W, Coggeshall M, Garrett HE, Van Sambeek J (2009) Growing black walnut for nut production. AF1011–2009 agroforestry in action. University of Missouri Center for Agroforestry, pp 1–16

  35. Riaz S, Tenscher AC, Graziani R, Krivanek AF, Ramming DW, Walker MA (2009) Using marker-assisted selection to breed Pierce’s disease-resistant grapes. Am J Enol Vitic 60:199–207

  36. Robichaud RL, Glaubitz JC, Rhodes OE Jr, Woeste K (2006) A robust set of black walnut microsatellites for parentage and clonal identification. New For 32:179–196

  37. Ruiz-Garcia L, Lopez-Ortega G, Fuentes Denia A, Frutos Tomas D (2011) Identification of a walnut (Juglans regia L.) germplasm collection and evaluation of their genetic variability by microsatellite markers. Span J Agric Res 9:179–192

  38. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Bio Eviron 4:406–425

  39. Sapkota S, Chen L-L, Yang S, Hyma K, Cadle-Davidsion, Hwang C-F (2019) Construction of a high-density linkage map and QTL detection of downy mildew resistance in Vitis aestivalis-derived ‘Norton’. Theor Appl Genet 132:137–147

  40. Thomas AL, Reid WR (2006) Hardiness of black walnut and pecan cultivars in response to an early hard freeze. J Am Pomol Soc 60:90–94

  41. Thomas AL, Reid WR (2017) Productivity and characteristics of six nut-producing black walnut cultivars in Missouri. Proceedings of the 15th North American Agroforestry Conference 15:156

  42. Vahdati K, Karimi R, Ershadi A (2014) Genetic structure of some wild walnut populations in Iran. Acta Hortic 1074:125–128

  43. Victory ER, Glaubitz JC, Rhodes OE Jr, Woeste K (2006) Genetic homogeneity in Juglans nigra (Juglandaceae) at nuclear microsatellites. Am J Bot 93:118–126

  44. Wang A, Aldwinckle H, Forsline P, Main D, Fazio G, Brown S, Xu K (2012) EST contig-based SSR linkage maps for Malus × domestica cv Royal Gala and an apple scab resistant accession of M. sieversii, the progenitor species of domestic apple. Mol Breed 29:379–397

  45. Williams RD (1990) Juglans nigra L. black walnut. In: Burns RM, Honkala BH (eds) Silvics of North America, Vol. 2: Hardwoods. Agricultural Handbook 654 Of Forest Service, United States Department of Agriculture, Washington DC, pp 391–399

  46. Woeste KE, Beineke WF (2001) An efficient method for evaluating black walnut for resistance to walnut anthracnose in field plots and the identification of resistant genotypes. Pl Breed 120:454–456

  47. Woeste K, Burns R, Rhodes O, Michler C (2002) Thirty polymorphic nuclear microsatellite loci from black walnut. J Hered 93:58–60

  48. Xu K, Wang A, Brown S (2012) Genetic characterization of the Ma locus with pH and titratable acidity in apple. Mol Breed 30:899–912

  49. Zhao P, Zhou H, Coggeshall MV, Reid B, Woeste K (2017) Discrimination and assessment of black walnut (Juglans nigra L.) nut cultivars using phenology and microsatellite markers (SSR). Can J Plant Sci 98:616–627. https://doi.org/10.1139/CJPS-2017-0214

  50. Zhu Y, Yin Y, Yang K, Li J, Sang Y, Huang L, Fan S (2015) Construction of a high-density genetic map using specific length amplified fragment markers and identification of a quantitative trait locus for anthracnose resistance in walnut (Juglans regia L.). BMC Genomics 16:614–626

Download references


The authors thank Marilyn Odneal for valuable discussions and constructive comments on the manuscript. The authors also thank Brigette Williams for technical assistance.


This work is supported by Capacity Building Grants for Non-Land Grant College of Agriculture no. 2018-70001-27832 from the USDA National Institute of Food and Agriculture.

This research contains no data that requires submission to a public database. The names and allele sizes of the 23 SSR loci used in this study are listed in Table 3. Primer sequences for the SSR markers can be found in the original publications, which are referenced within the materials and methods of the manuscript.

Author information

Correspondence to Chin-Feng Hwang.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Communicated by A. M. Dandekar

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Schneider, S.J., Hwang, A.Y., Land, S.D. et al. Genetic diversity of ten black walnut (Juglans nigra L.) cultivars and construction of a mapping population. Tree Genetics & Genomes 15, 62 (2019). https://doi.org/10.1007/s11295-019-1369-y

Download citation


  • Black walnut breeding
  • Microsatellite markers
  • SSR
  • Mapping population