Flow Cytometric DNA c-Value and Ploidy Variation in some Iranian Apple Cultivars

علوم باغبانی

Article 1, Volume 30, Issue 4, November 2016, Pages 694-700   

Document Type: Research Article

DOI: 10.22067/jhorts4.v0i0.43387

Authors

Shadab Faramarzi^{* 1}; Abbas Yadollahi²; Ghasem Karimzadeh²

¹University of Hormozgan

²Trabiat Modares University

Abstract

Introduction: Apple (Malus Miller) belongs to Rosacease family and the subfamily of Pomoideae. This fruit is at first place among fruits of temperate zones. The cultivated apple (Malus × domestica Borkh.) is a complex hybrid of the apple species. Chromosomal basis of this subfamily are x = 17 and the ploidy levels have been reported for diploid (2n=2x = 34), triploid (2n =3x= 51) and tetraploid (2n= 4x= 68). Since Iran is close to the apple diversity region (Central Asia), it has a good variation of apple varieties. Despite the high levels of variation in apple cultivars and species in Iran, there is not still a database of genome size. Classification of plants according to their genome size, especially at lower taxonomic levels is important for breeders. Over the past years, several methods for estimation of nuclear DNA content (genome size) was common, but recently, the use of flow cytometry (FCM) has been increasingly used. Flow cytometry is the best method to estimate DNA c-value and ploidy levels in apples. In this study, DNA c-Value and ploidy level of Iranian apple varieties has been estimated by flow cytometry and propidum iodide staining.
Materials and Methods: Fully expanded young leaves of all apple varieties were collected in the summer 2013. Nuclear extraction was performed using Partec kit as following: 1 cm2 apple leaf and 1 cm2 parsley leaf (as internal standard) were co-chopped with razor blade after adding 500 µl of nuclear extraction buffer. Then, the extract was filtered by two kind of filters (50 and 30 um). One ml of staining buffer, 4 µl of RNAase and 4 µl ofpropidium iodide was added for 15 min at room temperature. Finally, nuclei were counted using flow cytometry (BD FACSCanto II, USA) at Tarbiat Modarres University. The genome size was estimated according to bellow formula:
DNA 2C-value sample =
Also, given the high levels of phenolic compounds in apples, treating with PVP and PVP 1% were performed to evaluate the effect of phenolic compounds on estimation of genome size. Finally, Histogram analysis and DNA c-value estimation were done with Partec Flow Max software. The difference between means was obtained by SAS software ver. 9.2 and LSD tests.
Results and Discussion: The results showed that genome size obtained from Partec Flow Max software and ranged from 1.57 pg for ‘Golab- Bastam’ to 1.73 pg for ‘Golab- Kermanshah’. Histogram analysis was demonstrated that all studied cultivars are diploid. The average genome size in this study was 1.62 pg. Research conducted on foreign apple varieties have showed that the genome size of diploid species from was obtained 1.45 for M. fusca to1.68 pg for M. ransitoria. The genome size for triploid species was ranged from 2.37 to 2.57 pg. In this study, genome size was calculated in terms of mega base pairs and was different from 748 Mbp in ‘Golab- Bastam’ to 846 Mbp in ‘Golab- Kermanshah’. Thus, the size of the genome was closed to M. ransitoria (1.68 pg). This species is native to China, which is a Crab apple and used as an ornamental tree. It has been reported that Iranian apple are M. domestica Borkh. In another study, genome size was identified in the range from1.245 pg for diploid species of M. tschonoskii to 1.653 pg for M. florentina. M. florentina species is native to Balkans and Italy, that is an ornamental tree and its genome size is close to M. domestica Borkh. (1.653 pg).
Conclusion: Classification of plants according to their genome size seems to be important, especially at lower taxonomic. Genome size, even in very close species can also be different, for example, northern corn with more heterochromatin has larger genomes than those who are located in south (less heterochromatin). This study appears the variation of DNA 2C-value in Golab cultivars, even though Golab cultivars are known clones with low genetic diversity. Therefore, it is likely that Iranian apple varieties, with the same ploidy level, have been had difference in genome size. There are various ploidy level in apple, including diploid (2n = 34), triploid (2n = 51), tetraploid (2n = 68) and hexaploid (2n = 102). Thus, it is expected that current apple M. × domestica Borkh., have been contributed some several species such as M. prunifolia (Willd.) Borkh., M. baccata (L.) Borkh., M. sieboldii (Regel) Rehder, M. sylvestris, ،M. orientalis Uglitzk and M. sieversii.

Keywords

Genetic diversity; Genome size; Germplasm; Local cultivars

References

1- Alizadeh A. 2008. Identification, collection and morphological analysis Iranian apple germplasmReport of research work (in Persian).

2-Bennett M.D., and Smith J. 1976. Nuclear DNA amounts in angiosperms. Philosophical Transactions of the Royal Society of London. Biological Sciences, 274: 227-274.

3- Doležel J., Bartos J., Voglmayr H., and Greilhuber J. 2003. Nuclear DNA content and genome size of trout and human. Cytometry. Part A: Journal of the International Society for Analytical Cytology, 51(2): 127.

4- Doležel J., Greilhuber J., and Suda J. 2007. Estimation of nuclear DNA content in plants using flow cytometry. Nature Protocols, 2(9): 2233-2244.

5-Faramarzi S., Yadollahi A.,Barzegar M., Sadraei K., Pacifico S., and Jemric T. 2014. Comparison of Phenolic Compounds’ Content and Antioxidant Activity between Some Native Iranian Apples and Standard Cultivar 'Gala'. Journal of Agricultural Science and Technology, 16: 1601-1611.

6- Gonai T., Manabe T., Inoue E., Hayashi M., Yamamoto T., Hayashi T., Sakuma F., and Kasumi M. 2006. Overcoming hybrid lethality in a cross between Japanese pear and apple using gamma irradiation and confirmation of hybrid status using flow cytometry and SSR markers. Scientia horticulturae, 109: 43-47.

7- Gustafson P., Korban S., Wannarat W., Rayburn C.M., Tatum T.C., and Rayburn A.L.2009. Genome size and nucleotypic variation in Malus germplasm. Genome, 52: 148-155.

8- Harris S.A., Robinson J.P., and Juniper B.E. 2002. Genetic clues to the origin of the apple. Trends in Genetics, 18(8): 426-430.

9- Höfer M., and Meister A. 2010. Genome size variation in Malus species. Journal of Botany, 2010.

10- Jedrzejczyk I., and Sliwinska E. 2010. Leaves and seeds as materials for flow cytometric estimation of the genome size of 11 Rosaceae woody species containing DNA-staining inhibitors. Journal of Botany, 2010.

11- Juniper B., Watkins R., and Harris S. 1998. The origins of the apple. Acta Horticulturae, 484: 27–33.

12- Kindersley D. 2008. RHS AZ Encyclopedia of GAErden Plants UK, p. 1136.

13- Korban S., and Skirvin R. 1984. Nomenclature of the cultivated apple. Hort Science, 19: 177-180.

14- Laurie D., and Bennett M.1986. Wheat× maize hybridization. Canadian Journal of Genetics and Cytology, 28: 313-316.

15- Pellicer J., and Leitch I. 2014. The Application of Flow Cytometry for Estimating Genome Size and Ploidy Level in Plants. Pascale Besse (ed.), Molecular Plant Taxonomy: Methods and Protocols, Methods in Molecular Biology. Springer Science+Business Media, New York.

16- Phipps J.B., Robertson K.R., Smith P.G., and Rohrer J.R. 1990. A checklist of the subfamily Maloideae (Rosaceae). Canadian Journal of Botany, 68: 2209-2269.

17- Phipps J.B., Robertson K.R., Rohrer J.R., and Smith P.G.1991. Origins and evolution of subfam. Maloideae (Rosaceae). Systematic Botany, 303-332.

18- Rayburn A.L., Price H.J., Smith J., and Gold J.R. 1985. C-band heterochromatin and DNA content in Zea mays. American Journal of Botany, 1610-1617.

19- Tatum T.C., Stepanovic S., Biradar D., Rayburn A.L., and Korban S.S. 2005. Variation in nuclear DNA content in Malus species and cultivated apples. Genome, 48: 924-930.

20- Way R., Aldwinckle H., Lamb R., Rejman A., Sansavini S., Shen T., Watkins R., Westwood M.N., and Yoshida Y. 1990. Apples (Malus). Acta Horticulturae, 290: 3–62.

21- YokoyaK., Roberts A., Mottley J., Lewis R., and BrandhamP. 2000. Nuclear DNA amounts in roses. Annals of Botany, 85: 557-561.

22- Zhou Z. 1999. The apple genetic resources in China: the wild species and their distributions, informative characteristics and utilisation. Genetic Researcha and Crop Evolution, 46: 599-609.