A Flow Cytometry-Based Assessment of the Nuclear DNA Content and Ploidy Level of Wild Sunflower Species

Abstract

Sunflower genus (Helianthus spp.), native to the North America, includes 37 perennials and 14 annual species, many of which possess valuable genes related to biotic and abiotic stress tolerance that can enhance yield and yield-related traits in cultivated sunflower. However, it is crucial to characterize wild species to incorporate these beneficial traits into breeding programs. The study aim was to determine the nuclear DNA content of 133 sunflower accessions representing 52 species of the genus by flow cytometer, and use the obtained information to determine their ploidy levels and verify their taxonomic identities. Based on flow cytometrical analysis, nuclear DNA content of the individual plants and the average values of accessions of the same species, generally showed a high degree of similarity except some cases. However, the average 2C nuclear DNA content of Helianthus species was estimated to range from 5.61 pg (H. porteri) to 27.05 pg (H. tuberosus). The differences among the species were statistically important. Based on the study results, it was determined that approximately 80% (41 species) of the wild species (15 annuals, 26 perennials) are diploid. The ploidy level of the remaining species exhibited variability and 12 species were tetraploid (4x) while 2 species were hexaploid (6x). The study results indicated that some accessions of some species were either identified incorrectly or they are progenies of natural interspecific hybrids. The results of the study confirm that flow cytometer can be used in characterization of the sunflower genetic resources before include them in breeding programs.

Keywords

• Helianthus
• Wild sunflowers
• Flow cytometry
• Nuclear DNA content
• Ploidy

References

1. Baack, E. J., Whitney, K. D., & Rieseberg, L. H. (2005). Hybridization and genome size evolution: Timing and magnitude of nuclear DNA content increases in Helianthus homoploid hybrid species. New Phytologist, 167(3), 623–630. https://doi.org/10.1111/j.1469-8137.2005.01432.x
2. Brummer, E. C., Cazcarro, P. M., & Luth, D. (1999). Ploidy determination of alfalfa germplasm accessions using flow cytometry. Crop Science, 39(4), 1202–1207. https://doi.org/10.2135/cropsci1999.0011183X003900040039x
3. Dolezel, J. (1997). Application of flow cytometry for the study of plant genomes. Journal of Applied Genetics, 38, 285–302.
4. Doležel, J., & Bartoš, J. (2005). Plant DNA flow cytometry and estimation of nuclear genome size. Annals of Botany, 95(1), 99– 110. https://doi.org/10.1093/aob/mci005
5. Gül, V., & Coban, F. (2020). Determination of yield and quality parameters of oil sunflower genotypes grown in Turkey. Turkish Journal of Field Crops, 25(1), 9-17. https://doi.org/10.17557/tjfc.609749
6. Heslop-Harrison, J. S. (1995). Flow cytometry and genome analysis. Probe, 5, 14–17. Kallamadi, P., & Sujatha, M. (2016). Ploidy analysis of Helianthus species by flow cytometry and its use in hybridity confirmation. Nucleus, 59(2), 123–130. https://doi.org/10.1007/s13237-016-0178-x
7. Kantar, M. B., Gregory, J. B., Bock, D. G., & Rieseberg, L. H. (2014). Genomic variation in Helianthus: Learning from the past and looking to the future. Briefings in Functional Genomics, 13(4), 328–340. https://doi.org/10.1093/bfgp/elu013
8. Kaya, Y. (2014). Sunflower. In A. Pratap & J. Kumar (Eds.), Alien gene transfer in crop plants (pp. 281–315). Springer. https://doi.org/10.1007/978-1-4614-9572-7_11

9. Kaya, Y., & Vasilevska-Ivanova, R. (2021). Wild sunflowers: The primary genetic resource for sunflower breeding. In M. T. Azhar & S. H. Wani (Eds.), Wild germplasm for genetic improvement in crop plants (pp. 153–186). Elsevier Academic Press. https://doi.org/10.1016/B978-0-12-822918-3.00006-9
10. Kaya, Y., Balkan Nalcaiyi, A. S., Culha Erdal, S., Arslan, O., Cicek, N., Pekcan, V., Yılmaz, M. İ., Evci, G., & Ekmekci, Y. (2016). Evaluation of male inbred lines of sunflower (Helianthus annuus L.) for resistance to drought via chlorophyll fluorescence. Turkish Journal of Field Crops, 21(2), 162–173. https://doi.org/10.17557/tjfc.15482
11. Leitch, I. J., & Bennett, M. D. (2004). Genome downsizing in polyploid plants. Biological Journal of the Linnean Society, 82(4), 651–663. https://doi.org/10.1111/j.1095-8312.2004.00349.x
12. Lu, K., Kaeppler, S. M., Vogel, K. P., Arumuganathan, K., & Lee, D. J. (1998). Nuclear DNA content and chromosome numbers in switchgrass. Great Plains Research, 8(2), 269–280.
13. Mabuza, L. M., Mchunu, N. P., Crampton, B. G., & Swanevelder, D. Z. H. (2023). Accelerated breeding for Helianthus annuus (sunflower) through doubled haploidy: An insight on past and future prospects in the era of genome editing. Plants, 12(3), 485. https://doi.org/10.3390/plants12030485
14. Makarenko, M., Usatov, A., Tatarinova, T., Azarin, K., Kovalevich, A., Gavrilova, V., & Horn, R. (2020). The investigation of perennial sunflower species (Helianthus L.) mitochondrial genomes. Genes, 11(9), 982. https://doi.org/10.3390/genes11090982
15. Michaelson, M. J., Price, H. J., Johnston, J. S., & Ellison, J. R. (1991). Variation of nuclear DNA content in Helianthus annuus (Asteraceae). American Journal of Botany, 78(9), 1238–1243. https://doi.org/10.1002/j.1537-2197.1991.tb12512.x
16. Natali, L., Ceccarelli, M., Giordani, T., Sarri, V., Zuccolo, A., Jurman, I., Morgante, M., Cavallini, A., & Cionini, P. G. (2008). Phylogenetic relationships between annual and perennial species of Helianthus: Evolution of a tandem repeated DNA sequence and cytological hybridization experiments. Genome, 51(12), 1047–1053. https://doi.org/10.1139/G08-089
17. Ohri, D. (1998). Genome size variation and plant systematics. Annals of Botany, 82(6), 751–758. https://doi.org/10.1006/anbo.1998.0753
18. Ozkan, H., Tuna, M., & Galbraith, D. W. (2006). No DNA loss in autotetraploids of Arabidopsis thaliana. Plant Breeding, 125(3), 288–291. https://doi.org/10.1111/j.1439-0523.2006.01206.x
19. Ozkan, H., Tuna, M., & Arumuganathan, K. (2003). Nonadditive changes in genome size during allopolyploidization in the wheat (Aegilops-Triticum) group. Journal of Heredity, 94(4), 260–264. https://doi.org/10.1093/jhered/esg054
20. Rayburn, A. L., Auger, J. A., Benzinger, E. A., & Hepburn, A. G. (1989). Detection of intraspecific DNA content variation in Zea mays L. by flow cytometry. Journal of Experimental Botany, 40(8), 1179–1183. https://doi.org/10.1093/jxb/40.8.1179
21. Sahin, M., Savas Tuna, G., Tuna, M., & Kaya, Y. (2022). Flow cytometrical characterization in sunflower genus. Proceedings of the 20th International Sunflower Conference, Novi Sad, Serbia, June 20–23. International Sunflower Association.
22. Savas Tuna, G., Keles, H., Gocmen, D., Guleryuz, V.,Nizam, I., Cabi, E., Yazici, A., Cakal, S. & Tuna M. (2016). Flow Sitometri ile Cok Yillik Bugdaygil Yem Bitkisi Genetik Kaynaklarinin Karakterizasyonu. Tarla Bitkileri Merkez Arastirma Enstitusu Dergisi, 25 ZEL SAYI-2), 7-12. https://doi.org/10.21566/tarbitderg.281591
23. Savaş Tuna G, Duyu G, Uzun K, Yucel G. & Tuna M (2017). Determination of nuclear DNA content and ploidy of Hypericum perforatum L. accessions collected from western Turkey. Journal of Agricultural Science 23: 395-403. https:// doi.org/10.15832/ankutbd.385863
24. Seiler, G.J. & Gulya, T.J., 2004. Exploration for wild Helianthus species in North America: Challenges and opportunities in the search for global treasures. In: Seiler, G.J. [ed.], Proc. 16th Intl. Sunflower Conf., Fargo, ND, USA, 29 August-4 September 2004, 43-68
25. Sims, L. E., & Price, H. J. (1985). Nuclear DNA content variation in Helianthus (Asteraceae). American Journal of Botany, 72(8), 1213–1219. https://doi.org/10.1002/j.1537-2197.1985.tb08374.x
26. Steel, R. G. D., & Torrie, J. H. (1960). Principles and procedures of statistics with special reference to the biological sciences (pp. 187–287). McGraw-Hill.
27. Sujatha, M., & Prabakaran, A. J. (2006). Ploidy manipulation and introgression of resistance to Alternaria helianthi from wild hexaploid Helianthus species to cultivated sunflower (H. annuus L.) aided by anther culture. Euphytica, 152(1), 201–215. https://doi.org/10.1007/s10681-006-9185-6
28. Tuna, M., Vogel, K. P., Arumuganathan, K., & Gill, K. S. (2001). DNA content and ploidy determination of bromegrass germplasm accessions by flow cytometry. Crop Science, 41(5), 1629–1634. https://doi.org/10.2135/cropsci2001.4151629x
29. Tuna, M., Deepak, K. K., Shresta, M. K., Arumuganathan, K., & Golan-Goldhirsh, A. (2004). Characterization of Dactylis populations collected from natural ranges of Thrace Region of Turkey based on ploidy and RAPD analysis. Euphytica, 135(1), 39–46. https://doi.org/10.1023/B:EUPH.0000019507.87266.f6
30. Tuna, M., Vogel, K. P., & Arumuganathan, K. P. (2005). Genome size and Giemsa C-banded karyotype of tetraploid Bromus ciliatus L. Euphytica, 146(1), 177–182. https://doi.org/10.1007/s10681-005-9005-9
31. Tuna, M., Vogel, K. P., & Arumuganathan, K. (2006). Cytogenetic and nuclear DNA content characterization of diploid Bromus erectus and Bromus variegatus. Crop Science, 46(2), 637–641. https://doi.org/10.2135/cropsci2005.05-0081
32. Vižintin, L.& Bohanec, B. (2008). Measurement of nuclear DNA content of the genus Trifolium L. as a measure of genebank accession identity. Genetic Resources and Crop Evolution, 55, 1323-1334. https://doi.org/10.1007/s10722-008-9332-9
33. Vogel, K. P., Arumuganathan, K., & Jensen, K. B. (1999). Nuclear DNA content of perennial grasses of the Triticeae. Crop Science, 39(2), 661–667. https://doi.org/10.2135/cropsci1999.0011183X003900020042x