Comparison of Methods to Distinguish Diploid and Tetraploid Potato in Applied Diploid Breeding

  • Maher Alsahlany
  • Daniel Zarka
  • Joseph Coombs
  • David S. DouchesEmail author


Diploid (2n = 2x = 24) and tetraploid (2n = 4x = 48) germplasm are commonly used in potato breeding programs. Potato breeders need to efficiently and inexpensively differentiate between diploid and tetraploid progeny in dihaploid induction crosses as well as 2x-2x crosses where 2n gametes may occur in the parents. In this study, we compared the chloroplast count, genome-wide SNP genotyping and flow cytometry methods to determine ploidy. Twenty-eight clones were used as reference samples (14 diploid lines (2n = 2x = 24), 14 tetraploid (2n = 4x = 48) varieties and advanced breeding lines) to compare the three ploidy determination methods. The chloroplast count method was used to determine the ploidy level in the 28 reference samples, and 102 potato breeding lines derived from diploid (2x-2x) crosses. The Infinium 12 K V2 Potato Single Nucleotide Polymorphism (SNP) Array was used to examine the 28 reference samples and the 102 breeding lines. The results obtained from both chloroplasts counts and SNP genotyping techniques determined that there was a total of 84 diploid lines and 18 tetraploid lines. Flow cytometry was also used to determine ploidy level in a subset of 42 lines (28 reference lines set and 14 breeding lines). All three methods of ploidy determination (chloroplast counting, SNP genotyping and flow cytometry) agreed for all samples evaluated. These results demonstrate the usefulness of chloroplast counting as an efficient and inexpensive method for breeders to differentiate ploidy between diploid and tetraploid potato in applied breeding programs.


Potato ploidy level Chloroplast count Single nucleotide polymorphism (SNP) Flow cytometry Diploid potato (2n = 2x = 24) Tetraploid potato (2n = 4x = 48) 


El germoplasma diploide (2n = 2x = 24) y el tetraploide (2n = 4X = 48) se usan comúnmente en los programas de mejoramiento de la papa. Los fitomejoradores necesitan diferenciar eficiente y económicamente entre progenies diploide y tetraploide en cruzas de inducción de dihaploides así como en cruzas 2x-2x, donde los gamentos 2n pudieran presentarse en los progenitores. En este estudio comparamos el conteo de cloroplastos, la amplitud genómica de SNP y los métodos de citometría de flujo para determinar la ploidía. Se usaron 28 clones como muestras de referencia (14 líneas diploides (2n = 2x = 24), 14 variedades tetraploides (2n = 4x = 48) y clones avanzados) para comparar los tres métodos de determinación de ploidía. El método del conteo de cloroplastos se usó para determinar el nivel de ploidía en las 28 muestras de referencia, y 102 líneas de papa de mejoramiento derivadas de cruzas diploides (2x-2x). Se usó el arreglo de polimorfismo de nucleótido simple (SNP) de papa Infinium 12 K V2 para examinar las 28 muestras de referencia y los 102 clones avanzados. Los resultados que se obtuvieron de ambas técnicas, el conteo de cloroplastos y la determinación de genotipos por SNP, determinaron que hubo un total de 84 líneas diploides y 18 tetraploides. También se usó la citometría de flujo para determinar el nivel de ploidía en un subgrupo de 42 líneas (un juego de 28 líneas de referencia y 14 de mejoramiento). Los tres métodos para la determinación de ploidías (el conteo de cloroplastos, el genotipado por SNP, y la citometría de flujo) coincidieron para todas las muestras evaluadas. Estos resultados demuestran la utilidad del conteo de cloroplastos como un método eficiente y económico para los mejoradores para diferenciar ploidías entre papa diploide y tetraploide para aplicar en programas de mejoramiento.



This research was funded by USDA NIFA 2014-67013-22434 and the Higher Committee for Education Development in Iraq (HCED). The authors thank all Michigan State University Potato Breeding and Genetics Program members and especially Dr. Norma C. Manrique-Carpintero and Nick Garrity, who helped with chloroplast counting. The authors also thank the Kathiravetpillai Arumuganathan lab for processing leaf samples with flow cytometry to measure DNA content. The authors thank Dr. Linda Hanson (USDA/ARS Sugar Beet Pathology Lab) for the use of their microscope for chloroplast counts and Dr. Mitch McGrath (USDA/ARS) for reviewing the manuscript.


  1. Altmann, Thomas, Brigitte Damm, Wolf B. Frommer, Thomas Martin, Peter C. Morris, Dieter Schweizer, Lothar Willmitzer, and Renate Schmidt. 1994. Easy determination of ploidy level in Arabidopsis thaliana plants by means of pollen size measurement. Plant Cell Reports 13: 652–656.CrossRefGoogle Scholar
  2. Arumuganathan, K., and E.D. Earle. 1991. Estimation of nuclear DNA content of plants by flow cytometry. Plant Molecular Biology Reporter 9: 229–241. Scholar
  3. Bamberg, J.B., and R.E. Hanneman. 1991. Rapid ploidy screening of tuber-bearing Solanum (potato) species through pollen diameter measurement. American Potato Journal 68: 279–285. Scholar
  4. Chase, Sherret S. 1963. Analytical breeding in Solanum tuberosum L. – A scheme utilizing parthenotes and other diploid stocks. Canadian Journal of Genetics and Cytology 5: 359–363.CrossRefGoogle Scholar
  5. Doležel, J. 1991. Flow cytometric analysis of nuclear DNA content in higher plants*. Phytochemical Analysis 2: 143–154.CrossRefGoogle Scholar
  6. Doležel, J., P. Binarová, and S. Lucretti. 1989. Analysis of nuclear DNA content in plant cells by flow cytometry. Biologia Plantarum 31: 113–120. Scholar
  7. Douches, D., C.N. Hirsch, N.C. Manrique-Carpintero, A.N. Massa, J. Coombs, M. Hardigan, D. Bisognin, W. De Jong, and C.R. Buell. 2014. The contribution of the Solanaceae coordinated agricultural project to potato breeding. Potato Research 57: 215–224. Scholar
  8. Ellis, David, Oswaldo Chavez, Joseph J. Coombs, Julian V. Soto, Rene Gomez, David S. Douches, Ana Panta, Rocio Silvestre, and Noelle Lynette Anglin. 2018. Genetic identity in Genebanks: Application of the SolCAP 12K SNP Array in fingerprinting and diversity analysis in the global in trust potato collection. Genome 61: 523–537. Scholar
  9. Gebhardt, C., D. Bellin, H. Henselewski, W. Lehmann, J. Schwarzfischer, and J.P.T. Valkonen. 2006. Marker-assisted combination of major genes for pathogen resistance in potato. Theoretical and Applied Genetics 112: 1458–1464. Scholar
  10. Hao, Y.J., C.X. You, and X.X. Deng. 2002. Cell size as a morphological marker to calculate the mitotic index and ploidy level of citrus callus. Plant Cell Reports 20: 1123–1127. Scholar
  11. Hermsen, J.G.Th. 1969. Induction of haploids and aneuhaploids in colchicine-induced tetraploid Solanum chacoense bitt. Euphytica 18: 183–189. Google Scholar
  12. Hutten, R.C.B., M.G.M. Schippers, J.G.T.H. Hermsen, and E. Jacobsen. 1995. Comparative performance of diploid and tetraploid progenies from 2x. 2x crosses in potato. Euphytica 81: 187–192.CrossRefGoogle Scholar
  13. Jackson, M., J. Hawkes, and P. Rowe. 1980. An ethnobotanical field study of primitive potato varieties in Peru. Euphytica 29: 107–113.CrossRefGoogle Scholar
  14. Jacobsen, E. 1981. Polyploidization in leaf callus tissue and in regenerated plants of dihaploid potato. Plant Cell, Tissue and Organ Culture 1: 77–84. Scholar
  15. Jansky, Shelley H., and Stanley J. Peloquin. 2006. Advantages of wild diploid Solanum species over cultivated diploid relatives in potato breeding programs. Genetic Resources and Crop Evolution 53: 669–674. Scholar
  16. Jansky, Shelley H., Amy O. Charkowski, David S. Douches, Gabe Gusmini, Craig Richael, Paul C. Bethke, David M. Spooner, Richard G. Novy, Hielke de Jong, Walter S. de Jong, John B. Bamberg, A.L. Thompson, Benoit Bizimungu, David G. Holm, Chuck R. Brown, Kathleen G. Haynes, Vidyasagar R. Sathuvalli, Richard E. Veilleux, J. Creighton Miller, Jim M. Bradeen, and Jiming Jiang. 2016. Reinventing potato as a diploid inbred line-based crop. Crop Science 56: 1412–1422. Scholar
  17. Johnstone, Francis E. 1939. Chromosome doubling in potatoes induced by colchicine treatment. American Potato Journal 16: 288–304. Scholar
  18. Kessel, R., and P.R. Rowe. 1975. Production of intraspecific aneuploids in the genus Solanum 2. Triploids produced from tetraploid-diploid crosses in potato. Euphytica 24: 379–386. CrossRefGoogle Scholar
  19. Langton, F.A. 1974. A re-evaluation of the Dionne method of vegetatively doubling the chromosome number in potato. Potato Research 17: 296–306. Scholar
  20. Maris, B. 1990. Comparison of diploid and tetraploid potato families derived from Solanum phureja x dihaploid S. tuberosum hybrids and their vegetatively doubled counterparts. Euphytica 46: 15–33. Scholar
  21. Mattheij, W.M., R. Eijlander, J.R.A. de Koning, and K.M. Louwes. 1992. Interspecific hybridization between the cultivated potato Solanum tuberosum subspecies tuberosum L. and the wild species S. circaeifolium subsp. circaeifolium bitter exhibiting resistance to Phytophthora infestans (Mont.) de Bary and Globodera pallida (stone) Behrens. 1. Somatic hybrids. Theoretical and Applied Genetics 83: 459–466.CrossRefGoogle Scholar
  22. Murashige, Toshio, and Folke Skoog. 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15: 473–497. Scholar
  23. Ochatt, S.J., E.M. Patat-Ochatt, and A. Moessner. 2011. Ploidy level determination within the context of in vitro breeding. Plant Cell, Tissue and Organ Culture 104: 329–341. Scholar
  24. Ordonez, B. 2014. Assessment of Ploidy By Chloroplast Count in Stomatal Guard Cells. Potato Center (CIP), Lima, Peru.: 4.Google Scholar
  25. Ozias-Akins, Peggy, and Robert L. Jarret. 1994. Nuclear DNA content and ploidy levels in the genus Ipomoea. Journal of the American Society for Horticultural Science 119: 110–115.CrossRefGoogle Scholar
  26. Pehu, E., A. Karp, K. Moore, S. Steele, R. Dunckley, and M.G.K. Jones. 1989. Molecular, cytogenetic and morphological characterization of somatic hybrids of dihaploid Solanum tuberosum and diploid S. brevidens. Theoretical and Applied Genetics 78: 696–704. Scholar
  27. Pijnacker, L.P., K. Sree Ramulu, P. Dijkhuis, and M.A. Ferwerda. 1989. Flow cytometric and karyological analysis of polysomaty and polyploidization during callus formation from leaf segments of various potato genotypes. Theoretical and Applied Genetics 77: 102–110. Scholar
  28. Puite, Klaas, W.I.L.T.E.N. Broeke, and J.A.N. Schaart. 1988. Inhibition of cell wall synthesis improves flow cytometric sorting of potato heterofusions resulting in hybrid plants. Plant Science 56: 61–68.CrossRefGoogle Scholar
  29. Ramulu, K.S., and P. Dijkhuis. 1986. Flow cytometric analysis of polysomaty and in vitro genetic instability in potato. Plant Cell Reports 5: 234–237.CrossRefGoogle Scholar
  30. Rasmussen, J.O., and O.S. Rasmussen. 1995. Characterization of somatic hybrids of potato by use of RAPD markers and isozyme analysis. Physiologia Plantarum 93: 357–364. Scholar
  31. Sari, N., K. Abak, and M. Pitrat. 1999. Comparison of ploidy level screening methods in watermelon: Citrullus lanatus (Thunb.) Matsum. And Nakai. Scientia Horticulturae 82: 265–277.CrossRefGoogle Scholar
  32. Singsit, Chongkhohao, and Richard E. Veilleux. 1991. Chloroplast density in guard cells of leaves of anther-derived potato plants grown in Vitro and in Vivo. HortScience 26: 592–594.CrossRefGoogle Scholar
  33. Veilleux, R.E., J. Booze-Daniels, and E. Pehu. 1985. Anther culture of a 2 n pollen producing clone of Solanum phureja Juz. & Buk. Canadian Journal of Genetics and Cytology 27: 559–564. CrossRefGoogle Scholar
  34. Visser, Richard G.F., Christian W.B. Bachem, Jan M. de Boer, Glenn J. Bryan, Swarup K. Chakrabati, Sergio Feingold, Robert Gromadka, Roeland C.H.J. van Ham, Sanwen Huang, Jeanne M.E. Jacobs, Boris Kuznetsov, Paulo E. de Melo, Dan Milbourne, Gisella Orjeda, Boris Sagredo, and Xiaomin Tang. 2009. Sequencing the potato genome: Outline and first results to come from the elucidation of the sequence of the world’s third most important food crop. American Journal of Potato Research 86: 417–429. Scholar
  35. Zonneveld, B.J.M., and F. Van Iren. 2001. Genome size and pollen viability as taxonomic criteria: Application to the genus Hosta. Plant Biology 3: 176–185. Scholar

Copyright information

© The Potato Association of America 2019

Authors and Affiliations

  1. 1.Department of Plant, Soil and Microbial SciencesMichigan State UniversityEast LansingUSA
  2. 2.Horticulture Department College of AgricultureAl-Qasim Green UniversityBabylonIraq

Personalised recommendations