Article

Molecular Analysis of Genetic Variation of Potato Cultivars Infected with Viroid

Authors:
  • Faculty of Science, Benha Universityt
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Abstract

Four cultivars of the cultivated potato grown in Egypt were screened of genetic analysis using peroxidase isozymes and ISSR-PCR. The use isozyme separation to detect genetic variation among potato cultivars and to distinguish each cultivar of PSTVd infection were evaluated. DISC-PAGE isozyme separation of Cara, Nicola , Diamond and Spunta cultivars, showed 5,7,8 and 9 peroxidase isozymes in PSTVd inoculated plants compared with 7, 6, 5 and 6 peroxidase isozyme in healthy ones respectively. Similarity analysis confirmed the difference in isozyme patterns among potato cultivars. Only three ISSR primers (among 10 tested) were chosen as producing polymorphic DNA-bands differentiating the investigated cultivars. Based on these identity markers (bands), the genetic distances between cultivars were determined and their genetic relationship were estimated. The phytogenetic tree revealed that the four studied cultivars showed close similarity with the group. Although minor viroid susceptibility variations were recorded in the cultivars of some clones. The developed ISSR profiles indicated that Spunta was lowest sensitive for PSTVd infection followed by Diamond compared with Cara potato cultivars. Potato cultivars were genetic analysis for affect with Potato spindle tuber pospiviridae. Potato cultivars Cara, Nicola, Diamont and Spunta were mechanically inoculated with PSTVd. The results revealed that the external symptoms PSTVd infected potato were leaf narrow, rugosity and dark green, stem stunting and erect growth as well as, spindle and deformation of potato tubers. These results were confirmed by dot blot hybridization using DiG Labeled cDNA (specific PSTVd probe). Symptoms severity were assessed by measuring various foliage and tubers parameters. Disease severity caused by PSTVd ranged from mild severe depended on potato cultivars. Cara potato cultivar was susceptible, Nicola was mediate, Diamond and Spunta were tolerant.

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Studies on some viroids
  • Kh A El-Dougdoug
El-Dougdoug, Kh. A., 1988. Studies on some viroids, Ph.D. Thesis, Fac. Agric., Ain Shams Univ., Egypt, pp: 107.
A new system of comparing PCR promers app. To ISSR Aust
  • R Prevost
  • M J Wilkinson
Prevost, R. and M.J. Wilkinson, 1999. A new system of comparing PCR promers app. To ISSR Aust. J. Basic & Appl. Sci., 3(4): 4293-4301, 2009 4301 fingerprinting of potato cultivars. TAG theoretical and applied. Gen., 98(1): 107-112.
Molecular studies on Potato spindle tuber viroid
  • A M Sherin
  • Afifi
Sherin, A.M., Afifi, 2008. Molecular studies on Potato spindle tuber viroid. Ph.D. Thesis, Fac. Agric., Ain Shams Univ., Egypt., pp: 179.
Biological properties of CMV-SSI containing satellite RNA
  • W H Tian
  • S X Cao
  • Q Sun
  • S Q Chang
  • P Tein
Tian, W.H., S.X. Cao, Q. Sun, S.Q. Chang and P. Tein, 1985. Biological properties of CMV-SSI containing satellite RNA. Acta Phytopathologica. Sinica (China) 15: 145-149.
Response of some potato cultivars to Potato spindle tuber pospiviridae infection. 3 Nat. Conf. Of Pests and Dis. of Veg. And Fruits rd in Egypt and Arab Count
  • E K Allam
  • M A El-Afifi
  • Abou El-Nasr
  • H. Kh El-Dougdoug
Allam, E.K., M.A. Soheir El-Afifi, Abou El-Nasr and H. Kh El-Dougdoug, 1989. Response of some potato cultivars to Potato spindle tuber pospiviridae infection. 3 Nat. Conf. Of Pests and Dis. of Veg. And Fruits rd in Egypt and Arab Count. Imailia, Egypt, pp: 756-767.
Viroids and their potential danger to potatoes hot climates Canadian
  • R D Singh
Singh, R.D., 1983. Viroids and their potential danger to potatoes hot climates Canadian. Potato Disease Survey, 63(1): 13-18.
Determination of molecular and biochemical markers in resistant potato plants against some potato viruses. M.Sc. Fac Agric., Ain Shams Univ Antioxidant enzymes as biochemical markers for sharka resistance in apricot
  • A M Heba
  • J A Afifi Hernandez
  • J Cano
  • B Partillo
  • M Rubio
  • P M Gônez
Heba, A.M. Afifi, 2008. Determination of molecular and biochemical markers in resistant potato plants against some potato viruses. M.Sc. Fac. Agric., Ain Shams Univ., Cairo, Egypt, pp: 185. Hernandez, J.A., J. Cano, B. Partillo, M. Rubio and P.M. Gônez, 2006. Antioxidant enzymes as biochemical markers for sharka resistance in apricot. Biologia Plantarum, 50: 400.
Determination of molecular and biochemical markers in resistant potato plants against some potato viruses
  • A M Heba
  • Afifi
Heba, A.M. Afifi, 2008. Determination of molecular and biochemical markers in resistant potato plants against some potato viruses. M.Sc. Fac. Agric., Ain Shams Univ., Cairo, Egypt, pp: 185.