Rapid Differentiation of Closely Related Citrus Genotypes by Fluorescence Spectroscopy


The differentiation of closely related Citrus genotypes is a meticulous, laborious, and time-consuming task that involves the assessment of complex traits such as growth, tolerance to stress, photosynthetic efficiency, yield, and many others. Such a task is generally accomplished either by analyzing specific features of adult plants or by applying molecular markers to young trees. On one hand, only after plants start yielding can distinct genotypes be differentiated by comparing their fruit sizes, shapes, taste, and the number of seeds. On the other hand, molecular markers are expensive, and demand expertise and time for the analysis of a larger number of plants. For these reasons, the development of techniques that could assist in an early, quick and accurate differen-tiation of closely related Citrus varieties is of utmost importance. In this context, laser-induced fluorescence spectroscopy (LIFS) is a promising technique, since it is rapid, highly sensitive, and inexpensive. Previous studies showed that LIFS can differentiate a variety of sweet orange. However, this new study aimed to determine LIFS accuracy in the differentiation and grouping of very closely varieties of four Sunki mandarin selections: Comum, Florida, Tropical, and Maravilha. Furthermore we compared the results with ISSR and SSR molecular markers for the same varieties. LIFS technique distinguished the four selections with accuracy greater than 70%. Only with molecular markers was possible distinguishing clearly Tropical from Maravilha, but not Comum from Florida selections. In this way the results suggest that LIFS may be a sound tool for helping the identification of closely Citrus varieties.

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Souza Santana-Vieira, D. , Pereira Milori, D. , Villas Boas, P. , Silva, M. , Santos, M. , Gaiotto, F. , Soares Filho, W. and Silva Gesteira, A. (2014) Rapid Differentiation of Closely Related Citrus Genotypes by Fluorescence Spectroscopy. Advances in Bioscience and Biotechnology, 5, 903-914. doi: 10.4236/abb.2014.511105.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Coletta Filho, H.D., Machado, M.A., Targon, M.L.P.N., Moreira, M. and Pompeu, J. (1998) Analysis of the Genetic Diversity among Mandarins (Citrus spp.) Using RAPD Markers. Euphytica, Berlin, 102133-102139.
[2] Froelicher, Y., Dambier, D., Bassene, J.B., Costantino, G., Lotfy, S., Didout, C., Beaumont, V., Brottier, P., Risterucci, A.M., Luro, F. and Ollitrault, P. (2008) Characterization of Microsatellite Markers in Mandarin Orange (Citrus reticulata Blanco). Molecular Ecology Resources, 8, 119-112.
[3] Gottwald, T.R., Graça, J.V. and Bassanezi, R.B. (2007) Citrus Huanglongbing: The Pathogen and Its Impact. Plant Management Network.
[4] Pereira, W.E., de Siqueira, D.L., Puiatti, M., Martínez, C.A., Salomão, L.C.C. and Cecon, P.R. (2003) Growth of Citrus Rootstocks under Aluminium Stress in Hydroponics. Scientia Agricola, 60, 31-41.
[5] Soares Filho, W.S., Diamantino, M.S.A.S., Moitinho, E.D.B., da Cunha Sobrinho, A.P. and Passos, O.S. (2002) “Tropical”: Uma nova seleção de tangerina Sunki. Revista Brasileira de Fruticultura, 24, 127-132.
[6] Hodgson, R.W. (1967) Horticultural Varieties of Citrus. In: Reuther, W., Webber, H.J. and Batchelor, L.D., Eds., The Citrus Industry, University of California, Berkeley, 431-459.
[7] Pompeu Jr., J. (1980) Porta-Enxertos Para Citros. In: Rodriguez, O. and Viégas, F.C.P., Eds., Citricultura Brasileira, Fundação Cargill, Campinas, 279-296.
[8] Pompeu Jr., J. (2005) Porta-Enxertos. In: Mattos Jr., D., de Negri, J.D., Pio, R.M. and Pompeu Jr., J., Eds., Citros, Instituto Agronômico e Fun-dag, Campinas, 61-104.
[9] de Carvalho, M.T., Bordignon, R., Ballvé, R.M.L., Pinto-Maglio, C.A.F. and Medina Filho, H.P. (1997) Aspectos biológicos do reduzido número de sementes da tangerina “Sunki”. Bragantia, 56, 69-77.
[10] Soares Filho, W.S., da Cunha Sobrinho, A.P., Passos, O.S. and Moitinho, E.D.B. (2003) “Maravilha”: Uma nova seleção de tangerina Sunki. Revista Brasileira de Fruticultura, 25, 268-271.
[11] Ahmad, R., Struss, D. and Southwick, S.M. (2003) Development and Characterization of Microsatellite Markers in Citrus. Journal of the American Society for Horticultural Science, 128, 584-590.
[12] Liu, Y., Liu, D.-C., Wu, B. and Sun, Z.-H. (2006) Genetic Diversity of Pummelo (Citrus grandis Osbeck) and Its Relatives Based on Simple Sequence Repeat Markers. Chinese Journal of Agricultural Biotechnology, 3, 119-126.
[13] Oliveira, A.C., Novelli, V.M., Garcia, A.N., Cristofani, M. and Machado, M.A. (2002) Identification of Citrus Hybrids through the Combination of a Leaf Apex Morphology and SSR Markers. Euphytica, 128, 397-403.
[14] Fang, D.Q. and Roose, M.L. (1997) Identification of Closely Related Citrus Cultivars with Inter-Simple Sequence Repeat Markers. Theoretical and Applied Genetics, 95, 408-417.
[15] Tusa, N., Abbate, L., Ferrante, S., Lucretti, S. and Scarano, M.T. (2002) Identification of Zygotic and Nucellar Seedlings in Citrus Interploid Crosses by Means of Isozymes, Flow Cytometry and ISSR-PCR. Cellular e Molecular Biology Letters, 7, 703-708.
[16] Shareefa, M., Singh, A.K., Srivastav, M. and Dubey, A.K. (2009) Differentiation of Nucellar and Zygotic Seedlings in Citrus Using ISSR Markers. The Indian Journal of Agricultural Sciences, 79, 884-889.
[17] Lombardo, G., Schicchi, R., Marino, P. and Palla, F. (2011) Genetic Analysis of Citrus aurantium L. (Rutaceae) Cultivars by ISSR Molecular Markers. Plant Biosystems, 146, 19-26.
[18] Weiler, R.L., Brugnara, E.C., Bastianel, M., Machado, M.A., Schifino-Wittmann, M.T., Souza, P.V.D. and Schwarz, S.F. (2009) Paternity Test and Agronomic Evaluation of Possible Hybrids of Sunki Tangerine. Scientia Agraria, 10, 429-435.
[19] Ullanh, S., Schlerf, M., Skidmore, A.K. and Hecker, C. (2012) Identifying Plant Species Using Mi-Wave Infrared (2.5 - 6 μm) and Thermal Infrared (8 - 14 μm) Emissivity Spectra. Remote Sensing of Environment, 118, 95-102.
[20] Saleh, B. (2012) Biochemical and Genetic Variation of Some Syrian Wheat Varieties Using NIR, RAPD and AFLPs Techniques. Journal of Plant Biology Research, 1, 1-11.
[21] Kim, S.W., Min, S.R., Kim, J., Park, S.K., Kim, T.I. and Liu, J.R. (2009) Rapid Discrimination of Commercial Strawberry Cultivars Using Fourier Transform Infrared Spectroscopy Data Combined by Multivariate Analysis. Plant Biotechnology Report, 3, 87-93.
[22] Lichtenthaler, H.K., Wenzel, O., Buschmann, C. and Gitelson, A. (1998) Plant Stress Detection by Reflectance and Fluorescence. Annals New York Academy of Sciences, 851, 271-285.
[23] Lichtenthaler, H.K. and Schweiger, J. (1998) Cell Wall Bound Ferulic Acid, the Major Substance of the Blue-Green Fluorescence Emission of Plants. Journal of Plant Physiology, 152, 272-282.
[24] Kancheva, R.H., Borisova, D.S. and Iliev, I.T. (2008) Chlorophyll Fluorescence Plant as a Stress Indicator. Recent Developments in Remote Sensing From Space, 5, 301-306.
[25] Pereira, F.M.V., Milori, D.M.B.P., Pereira-Filho, E.R., Venancio, A.L., de Sá Tavares Russo, M., do Brasil Cardinali, M.C., Martins, P.K. and Freitas-Astúa, J. (2001) Laser Induced Fluorescence Imaging Method to Monitor Citrus Greening Disease. Computers and Electronics in Agriculture, 79, 90-93.
[26] Cardinali, M.C.B., Villas-Boas, P.R., Milori, D.M.B.P., Ferreira, E.J., Silva, M.F.E., Machado, M.A., Bellete, B.S. and da Silva, M.F.D.F. (2012) Infrared Spectroscopy: A Potential Tool in Huanglongbing and Citrus Variegated Chlorosis Diagnosis. Talanta, 91, 1-6.
[27] Milori, D.M.B.P., Raynaud, M., Villas-Boas, P.R., Venancio, A.L., Mounier, S., Bassanezi, R.B. and Redon, R. (2013) Identification of Citrus Varieties Using Laser-Induced Fluorescence Spectroscopy (LIFS). Computers and Electronics in Agriculture, 95, 11-18.
[28] Biswas, M.K., Xu, Q. and Deng, X. (2010) Utility of RAPD, ISSR, IRAP and REMAP Markers for the Genetic Analysis of Citrus Spp. Scientia Horticulturae, 2, 254-261.
[29] Duda, R.O., Hart, P.E. and Stork, D.G. (2000) Pattern Classification. 2nd Edition, John Wiley & Sons, New York.
[30] Wold, S., Sjöström, M. and Eriksson, L. (2001) PLS-Regression: A Basic Tool of Chemometrics. Chemometrics and Intelligent Laboratory Systems, 58, 109-130.
[31] Frank, E., Wang, Y., Inglis, S., Holmes, G. and Witten, I.H. (1998) Using Model Trees for Classification. Machine Learning, 32, 63-76.
[32] Kaufman, L. and Rousseeuw, P.J. (1990) Finding Groups in Data: An Introduction to Cluster Analysis. Wiley, New York.
[33] Everitt, B.S., Landau, S. and Leese, M. (2001) Cluster Analysis. 4th Edition, Oxford University Press, Inc., New York.
[34] R Development Core Team (2012) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna.
[35] Diepeveen, D., Clarke, G.P.Y., Ryan, K., Tarr, A., Ma, W. and Appels, R. (2012) Molecular Genetic Mapping of NIR Spectra Variation. Journal of Cereal Science, 55, 6-14.
[36] Silva, C.L.S.P., Machado, M.A. and Lemos, E.G.M. (2004) Expressão protéica diferencial entre plantulas apomíticas e zigóticas de citros. Revista Brasileira de Fruticultura, 26, 1-4.
[37] Barkley, A.N., Roose, L.M., Krueger, R.R. and Federici, T.C. (2006) Assessing Genetic Diversity and Population Structure I a Citrus Germplasm Collection Utilizing Simple Sequence Repeat Markers (SSRs). Theoretical and Applied Genetics, 112, 1519-1531.
[38] Kumar, L.S., Jena, S.N. and Nair, K.N. (2009) ISSR Polymorphism in Indian Wild Orange (Citrus indica Tanaka, Rutaceae) and Related Wild Species in Northeast India. Scientia Horticulturae, 3, 350-359.

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