Karyotype analysis of rye (Secale spp.) species by using the extracted enzyme from the terrestrial snail stomach (Helix lucorum L.)

Abstract

The snail (Helix lucorum L.) belongs to Helicidae family, lives in forests and moist regions and feeds from leaves, wood, and vegetable containing a lot of cellulose. The digestive system of this snail has special enzymatic components for digesting cellulosic materials and has some effects on cytoplasmic and cell wall digestion. Chromosomal studies and karyotypes analysis using different methods of staining (Aceto Carmine, Aceto Orcein, Feulgen and etc.) have some problems such as chromosomal size, amount of tannins and difficulty of contrast in chromosome detection depending on species characters. We used these components that included an active mixture of Cytase and other enzymes, for easy squashing and perfect chromosome separation in two species of rye (Secale spp.). The terrestrial snails were collected from Arasbaran humid forests (North West of Iran), and extracted the components and kept in freeze condition. The root tips of germinated seeds of two species of rye were pretreated with α-Bromonaphtalin solution and stained in Aceto-Iron Haematoxylin. Then the enzymes were used for clearing cytoplasm and softening cell walls and to obtain sufficient squashing and chromosomes spread. The using the enzymatic components of snail stomach was very effective in squashing stage, and obtaining good metaphase plates, the chromosomes were counted and distinguished very clearly. The separation of all cells, without damaging the chromosomes, was achieved easily and completely. Species of rye (Secale spp.) were diploid with 2n=2x=14 chromosomes. The satellited chromosomes were seen in both two species on short arms of chromosomes.

Keywords

• Snail (Helix),Secale,Rye,Karyotype,Cytase

Karasal Salyangoz (Helix lucorum L.) Midesinden Elde Edilen Enzim Bileşenleri Kullanılarak Çavdar (Secale spp.) Türlerinde Karyotip Analizi

Abstract

Karasal salyangoz (Helix lucorum L.), Helicidae ailesine ait, ormanlarda ve nemli bölgelerde yaşar; yaprak, ağaç kabuğu ve bol miktarda selüloz içeren bitkilerden beslenir. Karasal salyangozun sindirim sistemi, sellülözik materyali sindirebilen özel enzim bileşenlerine sahiptir. Aseto karmin, Aceto orsein, Feulgen ve farklı boyar maddeler kullanılarak yapılan kromozom ve karyotip analizi çalışmalarında, türe ait karakterlere bağlı olarak kromozom boyu, tanen miktarı ve kromozom karşılaştırmalarında sorunlar yaşanmıştır. Kara salyangozu midesinin enzimatik bileşenleri, sitoplazma ve hücre duvarı yumuşaması üzerinde etkili olmuştur. Çalışmada, çavdar (Secale spp.) bitkisine ait iki türde, en iyi ve kolay şekilde kromozomların ayrılması ve dağılması amacıyla bu bileşenler kullanılmıştır. Sitaz ve diğer enzimlerin aktif bir karışımını içeren enzimatik bileşenleri kullanmak amacıyla, karasal salyangozlar İran’ın kuzey batısındaki nemli Arasbaran Ormanları’ndan toplanmış, bileşenler elde edilmiş ve soğuk koşullarda muhafazası sağlanmıştır. İki çavdar türünün (Secale cereale L. ve S. montanum) çimlenmiş tohumlarının kök uçları α-bromonaftalin solüsyonunda ön işleme tabi tutulmuş ve aseto-iron hematoxylin boyar madde ile boyanmıştır. Daha sonra, sitoplazmayı belirginleştirmek, hücre duvarını yumuşatmak ve tatminkar bir kromozom yayılması ve ayrılması elde etmek amacıyla kara salyangozu midesinden elde edilen enzimler kullanılmıştır. Araştırma sonucunda kara salyangozu midesinin enzimatik bileşenleri kullanılarak, her iki türde kromozomlar açık bir şekilde ayırt edilmiş ve, iyi bir metafaz safhası ve dağılımı gözlemlenmiştir. Kromozomlar bozulmadan kalırken, tüm hücrelerin tamamen ayrılması kolay bir şekilde sağlanmıştır. Çavdar türleri (Secale spp.) 2n=2x=14 kromozoma sahiptir ve diploiddir. Her iki türde de satellit (uydu) kromozom vardır ve satellitler, kromozomların kısa kolları üzerinde bulunmaktadır.

Keywords

• çavdar,Secale,Karyotip,Sitaz

References

1. Agayev, Y. M., Zarifi, E., & Fernández, J. A. (2010). A study of karyotypes in the crocus sativus L. Aggregate and origin of cultivated saffron. Acta Horticulturae, 850(850), 47–54.
2. Chambers, T. C. (1955). Use of snail stomach cytase in plant cytology [13]. Nature, 175(4448), 215.
3. Dalili, G. H. (2005). Animal classification. Sokhan Gostar, Tehran (In Persian).
4. Dirihan, S., Zarifi, E., Güloğlu, D., & Sevimay, C. S. (2014). Karyotype Features of Berseem Clover (T. alexandrinum L.) and Persian Clover (T. resupinatum L.) Species Which Cultivated in Turkey. Tarım Bilimleri Araştırma Dergisi, 7(1), 49–54.
5. Dorit, R., Walker, W. F. (Warren F., & Barnes, R. D. (1991). Zoology (Internatio). Philadelphia : Saunders College Pub.
6. Fageria, N. K. (1992). Maximizing Crop Yields. Taylor & Francis. Retrieved from https://books.google.com/books?id=NbPMD99XIkEC
7. Habibi, T. (2015). General Zoology Volume II worms and molluscs. Tehran University, Tehran ( In Persian).
8. Huziwara, Y. (1962). Karyotype Analysis in Some Genera of Compositae. VIII. Further Studies on the Chromosomes of Aster. American Journal of Botany, 49(2), 116–119.

9. Karami, M. (2000). Invertebrate Zoology. Shahed University, Tehran ( In persian).
10. Khodabande, N. (2013). Cereals. Tehran University ( In Persian).
11. Khush, G. S. (1962). Cytogenetic and Evolutionary Studies in Secale. II. Interrelationships of the Wild Species. Evolution, 16(4), 484.
12. Khush, G. S. (1963). Cytogenetic and evolutionary studies in Secale III. Cytogenetics of weedy ryes and origin of cultivated rye. Economic Botany, 17(1), 60–71.
13. Kranz, A. R. (1976). Karyotype analysis in meiosis: Giemsa banding in the genus Secale L. Theoretical and Applied Genetics, 47(3), 101–107.
14. Levan, A., Fredga, K., & Sandberg, A. A. (1964). Nomenclature for Centromeric Position on Chromosomes. Hereditas, 52(2), 201–220.
15. Tang, Z. X., Ross, K., Ren, Z. L., Yang, Z. J., Zhang, H. Y., Chikmawati, T., … Gustafson, J. P. (2011). Secale. In Wild Crop Relatives: Genomic and Breeding Resources (pp. 367–396). Berlin, Heidelberg: Springer Berlin Heidelberg.
16. Meier, S., Kunzmann, R., & Zeller, F. J. (1996). Genetic variation in germplasm accessions of Secale vavilovii Grossh. Genetic Resources and Crop Evolution, 43(1), 91–96.
17. Miller, S. A., & Harley, J. P. (1992). Zoology. Wm. C. Brown.
18. Moyes, C. D., & Schulte, P. M. (2008). Principles of Animal Physiology. Pearson Education. Retrieved from http://books.google.com/books?id=f-INSQAACAAJ&pgis=1
19. Myers, F. L., & Northcote, D. (1958). A survey of the enzymes from the gastro-intestinal tract of Helix pomatia. Journal of Experimental Biology, 35(3), 639–648.
20. Negah Media. (2013). Retrieved August 31, 2018, from http://www.negahmedia.ir/media/show_pic/28306
21. Paszko, B. (2006). A critical review and a new proposal of karyotype asymmetry indices. Plant Systematics and Evolution, 258(1–2), 39–48.
22. Pruitt, N. L., Underwood, L. S., & Surver, W. (2000). BioInquiry: Making Connections in Biology. John Wiley. Retrieved from https://books.google.com/books?id=eh5FAQAAIAAJ
23. Quinn, C. J., & Rattenbury, J. A. (1972). Structural hybridity in New Zealand dacrydium. New Zealand Journal of Botany, 10(3), 427–436.
24. Rechinger, K. H. (1973). Gramineae. Flora Iranica, 30–70.
25. Reeves, A. (2001). MicroMeasure: A new computer program for the collection and analysis of cytogenetic data. Genome, 44(3), 439–443.
26. Riley, R. (1955). The cytogenetics of the differences between some Secale species. The Journal of Agricultural Science, 46(3), 377–383.
27. Russel-Hunter, W. D. (1979). A life of invertebrates. Macmillan Publishers Limited.
28. Salahi Moghaddam, A. R. (2009). Medical malacology. Hormozgan University (In persian).
29. Schmidt-Nielsen, K. (1997). Animal physiology: adaptation and environment. Cambridge University Press.
30. Sharma, A. K., & Sharma, A. (1980). Chromosome Techniques: Theory and Practice (3rd ed.). Butterworth & Co (Publishers) Ltd, London.
31. Shekoohinezhad, M., & Amin, A. (1973). Animal Biology. Amir Kabir Publishing institue, Tehran (In Persian).
32. Stebbins, L. G. (1971). Chromosomal Evolution in Higher Plants. Edward Arnold Ltd. London: Edward Arnold Ltd.,.
33. Storer, T. I. (Tracy I., NYBAKKEN, J. W., & Usinger, R. L. (Robert L. (1968). Elements of zoology (3rd ed.). McGraw-Hill.
34. Stutz, H. C. (1957). A cytogenetic analysis of the hybrid Secale cereale L. x Secale montanum Guss. and its progeny. Genetics, 42(3), 199.
35. Watanabe, K., Yahara, T., Denda, T., & Kosuge, K. (1999). Chromosomal Evolution in the Genus Brachyscome (Asteraceae, Astereae): Statistical Tests Regarding Correlation Between Changes in Karyotype and Habit Using Phylogenetic Information. Journal of Plant Research, 112(2), 145–161.
36. Zarco, C. R. (1986). A New Method for Estimating Karyotype Asymmetry. Taxon, 35(3), 526.
37. Zarifi, E., Aghyev, Y., Ghanavati, F., & Aminizadeh, Z. (2006). Cytogenetics and Evolution of Karyotype in Wormwood, Artemisia vulgaris L. Seed and Plant Improvment Journal, 22(1), 1–12.
38. Zarifi, E., & Güloğlu, D. (2016). An improved Aceto-Iron-Haematoxylin staining for mitotic chromosomes in Cornelian cherry (Cornus mas L.). Caryologia, 69(1), 67–72.