Investigation of polymorphism in different acid lime (Citrus aurantifolia Swingle) genotypes with free witches broom disease by molecular markers of SSR and ISSR

Document Type : Full Paper


1 Former Ph. D. Student, Faculty of Agriculture, University of Guilan, Rasht, Iran

2 Professor, Faculty of Agriculture, University of Guilan, Rasht, Iran

3 Associate Professor, Citrus and Subtropical Fruits Research Center, Ramsar, Iran

4 Assistant Professor, Citrus and Subtropical Fruits Research Center, Ramsar, Iran

5 Assistant Professor, Faculty of Agriculture, University of Guilan, Rasht, Iran


Limes have been consumed as fresh fruits and processing products, due to high quality and acidity in fruit juice. In recent years, the incidence of Witches Broom disease resulted in destroy of thousands Limes trees in the south of Iran. The best method for controlling this disease is planting tolerant genotypes. Therefore, in this study, genetic diversity of 33 different lime genotypes which didn’t showed Witches Broom disease in the field and preliminary test, with 10 commercial citrus cultivars as a control was evaluated by 17 pair primers of SSR and 11 primers of ISSR. According to results with Simple Sequence Repeat (SSR) markers, the highest Polymorphic Information Content (PIC) were observed in TAA1 and TC26 with 0.72 and 0.68, respectively and by using ISSR markers were in ISSR1, 809 and 810 with 0.49 which was identified as a the most appropriate distinguishing markers. Results showed that there were differences between Limes and other types of citrus based on kind of studied markers. On the other hand, all of Lime genotypes had high affinity with Mexican Lime and Persian Lime and grouped in a cluster. Therefore, most of acid limes in south of Iran belonged to Limes group.


Main Subjects

  1. Abdollahi, B., Sadigh, P., Azizi, H., Piri, Y., Nasri, SH. & Arzhangh, S. (2015). Comparative assessment of   IRAP, REMAP, ISSR and SSR markers for evaluation of genetic diversity of alfalfa. Scientific Technology, 17, 991-1010.
  2. Asadi Abkenar, A., Mardi, M., Salehi Abarghoei, M. & Shafiee, M. (2012). Lime breeding to producing the varieties of disease resistant of Witches Broom, 3rd National conference on agriculture and food sciences, 16 Dec., Fasa University. (in Farsi)
  3. Asadi Abkenar, A., Sharafi, A. A., Tarang, A. R., Seyghalani, R., Masaeli, M. & Rfia, P. ( 2013). Use of ISSR molecular markers for evaluating genetic variation in a number of northern citrus accessions, 8th symposium on biotechnology and 4th biosafety national conference, 15-17 Oct., Tehran University, pp. 853-857. (in Farsi)
  4. Bakhshipoor, H., Mehrgan, A., Golein, B. & Saadatmand, S. (2013). Evaluation of Genetic diversity of unknown and commercial citrus varieties using SSR marker. Journal of New Cellular and Molecular Biotechnology Journal, 4(16), 17-25. (in Farsi)
  5. Barrect, H. C. & Rhods, A. M. (1976). A numerical taxonomic study of affinity relationship in cultivated Citrus and its closed relatives. Systematic Botany, 1, 105-136.
  6. Bove, J. M., Danet, J. L., Bananej, K., Hassanzadeh, N., Taghizadeh, M., Salehi, M. & Garnier, M. (2000). Witches Broom Disease of Lime (WBDL) in Iran. Fourteenth IOCV Conference, Insect-Transmitted Procaryotes, 207-212.
  7. Davies, F. S. & Albrigo, L. G. (1994). Taxonomy cultivars and breeding. In: Davis, F. S., and Albrigo, L. G. (Eds) Citrus, Wallingford, CAB International. pp. 12-23.
  8. Edwards, K., Johnstone, C. & Thompson, C. A. (1991). Simple rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Research, 19(6), 1349.
  9. Fang, D. Q. & Roose, M. L. (1997). Identification of closely related Citrus cultivars with intersimple sequence repeat markers. Theoretical and Applied Genetics, 95, 408-417.
  10. Federici, C. T., Fang, D. Q., Scora, R. W. & Roose, M. L. (1998). Phylogenetic relationships within the genus Citrus (Rutaceae) and related genera as revealed by RFLP and RAPD analysis, Theoretical and Applied Genetics, 96, 812-822.
  11. Food and Agriculture Organization of the United Nations, FAOSTAT database. (2013). From
  12. Moore, G. A. (2001). Oranges and lemons: clues to the taxonomy of Citrus from molecular markers. TRENDS in Genetics, 17(9), 536-540.
  13. Gmitter, F. G., Grosser, J. W. & Moore, A. G. (1992).Hammerschlag, F. A. & Litz, R. E. (eds.). Citrus. In: Biotechnology of Perennial Fruit Crops. (pp. 335-369). CAB International, Wallingford, Oxon.
  14. Golein, B. & Adouli, B. (2011). Citrus (cultivation). Novin Pooya. (Pp 160). (in Farsi)
  15. Golein, B., Bigonah, M., Azadvar, M. & Golmohammadi, M. (2012). Analysis of genetic relationship between ‘Bakraee’ (Citrus sp.) and some known Citrus genotypes through SSR and PCR-RFLP markers, Scientia Horticulturae, 148, 147-153.
  16. Jahangirzadeh, SH., Hamidoghli, Y., Golein, B. & Sabouri, A. (2015). Investigation of genetic relationships and genome study of some Lime genotype by morphological and genetical markers. Ph.D. Thesis. University of Guilan, Iran.
  17. Mohammadi, S. A. (2006). Molecular analysis of genetic diversity of perspectives. In: Proceeding of 9th Iranian Crop Sciences Congress.27-29 August, Tehran. (in Farsi)
  18. Morton, J. F. (2004). Fruits of warm climates, Creative Resource Systems, pp: 2997.
  19. Nematollahi, A. K. H., Golein, B. & Vahdati, K. (2013). Analysis of the genetic diversity in citrus (Citrus spp.) species using SSR markers. Journal of Plant Physiology and Breeding, 3(2), 39-47.
  20. Nicolosi, E., Deng, Z., Gentile, A. & La Malfa, S. (2000).Citrus phylogeny and genetic origin of important species as investigated by molecular marker, Theoretical and Applied Genetics, 100, 1155-1166.
  21. Novelli, V. M., Cristofani, M., Souza, A. A., Marcos, A. & Machado, M. A. (2006). Development and characterization of polymorphic microsatellite markers for the sweet orange (Citrus sinensis L., Osbeck). Genetics and Molecular Biology, 29(1), 90-96.
  22. Ollitrault, F., Terol, J., Pina, J. A., Navarro, L., Talon, M. & Ollitrault, P. (2010). Development of SSR markers from Citrus clementina (Rutaceae) BAC end sequences and interspecific transferability in Citrus. American Journal of Botany, 97(11), e124-e129.
  23. Ovesna, J., Polakova, K. & Leisova, L. (2002). DNA Analyses and their Applications in Plant Breeding. Czech Journal of Genetics and Plant Breeding, 38(1), 29-40.
  24. Rohlf, F. J. (1998). NTSYS-pc, Numerical Taxonomy and Multivariate Analysis System Version 2.02. Exeter Publications, Setauket, NY.
  25. Roldain-Ruiz, I., Calsyn, E., Gilliand, T. J., Coll, R., Van-Eijk, M. J. T. & De-Loose, M. (2000). Estimating genetic conformity between related ryegrass (Lolium) varieties, 2 AFLP characterization. Molecular Breeding, 6, 593-602.
  26. Salehi, M., Hasanzadeh, H., Faghihi, M. & Zakerifard, A. (2012). Evaluation the effects of inter stock in citrus varieties on distribution of the causal agent of Witches Broom of Lime, (Final Report). Iran Citrus Research Institute. 20. (in Farsi)
  27. Shahsavar, A. R., Ezadpanah, K. A., Tafazoli, A. A. & Seyed Tababaie, B. A. (2004). Evaluation of genetic variability of limes and lemons in the Fars province by morphological traits and inter-simple sequence repeat (ISSR). Journal of Horticultural Science and Technology, 5 (4), 177-188. (in Farsi)
  28. Shrestha, R. L., Dhakal, D. D., Gautum, D. M., Paudyal, K. P. & Shrestha, S. (2012). Genetic Diversity Assessment of Acid Lime (Citrus aurantifolia Swingle) Landraces in Nepal, Using SSR Markers. American Journal of Plant Sciences, 3(12), 1674-168.
  29. Tripolitsiotis, C., Nikoloudakis, N., Linos, A. & Hagidimitriou, M. (2013). Molecular characterization and analysis of the Greek citrus germplasm. Notuae botanicae hortiagrobotanici, 41 (2), 463-471.
  30. Nicolosi, E., Deng, Z., Gentile, A. & La Malfa, S. (2000). Citrus phylogeny and genetic origin of important species as investigated by molecular marker. Theoretical and Applied Genetics, 100,
  31. Scora, R. W. (1975). On the history and origin of Citrus. Bulletin of the Torrey Botanical Club, 369-375.
  32. Zhang, D. & Mabberley, D. J. (2016). ????. Citrus Linnaeus, 2, 782- 1753.
  33. Zhang, D. & Mabberley, D. J. (2008). Citrus. In Flora of China, Edited by Z. Y. Wu, P. H. Raven, and D. Y. Hong, pp. 90–96. Science Press-Missouri Botanical Garden Press, Beijing-St. Louis, China-USA.