Morphological Markers as a Tool for Investigating Genetic Diversity of Some Lime Genotypes

Document Type : Research Article


1 Assistant Professor, Tea Research Center, Horticultural Sciences Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Lahijan, Iran

2 Associate Professor, Citrus and Subtropical Fruits Research Center, Horticultural Sciences Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Ramsar, Iran


Introduction: Citrus fruits are the most common semi-tropical crops in the world. Acid lime (Citrus aurantifolia Swingle) is an important commercial fruit crop, cultivated in the south of Iran. High variation of acid lime fruits is observed in the south of Iran due to crossing within the other citrus species and frequent bud mutation. Recently, Witch’s Broom Disease of Lime (WBDL) become a major limiting factor for lime production in the South of Iran  as well as the main threating factor for lime industry in Iran. Having knowledge about the genetic of this plant is helpful for designing citrus breeding program. Therefore, in this research morphological traits were used to understand the genetic relationships and diversity of this gene pool.
Materials and Methods: Thirty citrus samples, including 23 undefined local and native genotypes and seven known cultivars were collected from four regions in Iran (Darab (54.53E, 28.75N), Manojan (57.49E, 27.40N), Minab (57.07E, 27.14N) and Ramsar (50.64E, 36.92N)). Forty-one (32 qualitative and nine quantitative) traits were investigated using leaves, fruits and seeds. The selection of morphological traits were made based on IPGRI descriptors. The similarity was calculated by simple matching coefficient and dendrogram was designed based on UPGMA algorithms. Principal components analysis was performed.
Results and Discussion: Genetic diversity of 23 lime genotypes and seven commercial cultivars were investigated by using 41 morphological characters. Based on gained data similarity matrix (Jaccard, Dice and Simple maching) were calculated and dendrogram based on UPGMA algorithms were designed. To finding better similarity coefficient, cophenetic test was done, it showed that if used SM coefficient 79 percent of data of similarity matrix was shown in designed cluster based on UPGMA algorithm. According to results, range of similarity was between 0.141 until 0.683. Maximum similarity was observed between two lime cultivars (Persian lime and Mexican lime). Average of similarity was calculated 0.39. In cluster analyses of studied samples at 32 level of similarity samples were divided into four main groups. First group has only one member and it was pomelo, as it is one of the ancestors of citrus fruits, this is quite acceptable. The notable point for second main group that it included all examined samples of Minab. As in this region most of cultivated citrus are lime, there is little gene mixing with other citrus. The third group consist of two cultivars sweet orange and Citon that intended in present investigation. Fourth group which was the biggest created groups that included more than half the samples (56%) was more complex because it consists of samples of limes and lemons that intended in investigation as control and samples of Darab and Manojan. This main group at level of 0.38 divided into three sub-groups, at first, Lisbon lemon was separated from other samples and next Manojan samples created their special sub-group and finally third sub-group which consist of 11 members (samples of Darab, MA6 from Manojan and three commercial cultivars, Persian lime, Mexican lime and Rough lemon). Principle Component Analysis (PCA) showed the first five principal components, which contributed 59.01% of the total variability of investigated samples. Maximum variability was contributed by the first component (22.77%) followed by the second component (12.54%), and the third component (9.85%). A two‐dimensional plot (2D plot) generated from PCA showed three groups. This grouping was roughly in line with the distribution of the samples in the resulting cluster analysis based on SM coefficient and UPGMA algorithm. Principle Component Analysis using 41 descriptors showed that 26 of 41 descriptors were informative and contributes significantly to the variation present in the germplasm.
Conclusion: This study described and estimated the extent of phenotypic variation present among the samples of limes from Iran germplasm. Morphological analyses among 23 genotypes and seven commercial cultivars from four regions of Iran were successfully used to calculate genetic diversity and genetic relationships. According to our results, it was confirmed that morphological analyses in limes and other Citrus species were exploit to determine genetic diversity and relationship, successfully. Characterization by using morphological descriptors based on 41 characters, revealed significant diversity in traits of leaf, fruit and seed. This investigation display the use of morphological characters to study genetic diversity of Iranian lime genotypes from four different regions that their relationships were somewhat clarified. The results of this study also opened a door to tackle the long standing problem of citrus classification and identification in Iran. But, we suggest that this type of study needs to be continued due to Iran has a very large and numerous citrus germplasm. In south and central regions of Iran, it is being propagated by seed which gives researchers a chance to find new genotypes that need to be classified, investigated and introduced as a new cultivar.


  1. 1-       Abedinpour H., Ranjbar Gh.A., Babaein Jelodar N.A., and Golein B. 2014. Evaluation of genetic diversity in citrus genotypes by IRAP molecular marker. International Journal of Farming and Allied Sciences 3: 230-234.

    2-       Al-Anbari A., Kanawapee N., Al-Kazragi T.A., Al-Jewari H., Al.Mashhadani A., Barusrux S., Pornpongrungrueng P., and Theerakulpisut P. 2014. Genetic diversity of citrus (Rutaceae) in Iraq based on random amplified polymorphic DNA (RAPD) markers, African Journal of Agricultural Research 9(11): 1112-1019.

    3-       Barrectt H.C., and Rhodes A.M. 1976. A numerical taxonomic study of affinity relationships in cultivated Citrus and its close relatives. Systematic Botany 1: 105–136.

    4-       Campos E.T., Espinosa M.A.G., Warburton M.L., Varela A.S., and Monter A.V. 2005. Characterization of Mandarin (Citrus spp.) using morphological and AFLP markers. Interciencia 30(11): 687-693.

    5-       Cooper W.C., Reece P.C., and Furr J.R. 1962. Citrus breeding in Florida-past, present and future. Florida State Horticultural Society 75: 5-13.

    6-       Davies F.S., and Albrigo L.G. 1994. Taxonomy cultivars and breedin, In: Davis, F.S., and Albrigo, L.G. (Eds) Citrus, Wallingford, CAB Internationa.

    7-       Dorji K., and Yapwattanaphun Ch. 2011. Morphological identification of mandarin (Citrus reticulate Blanco) in Bhutan. Kasetsart Journal (Natural Science) 45: 793–802.

    8-       Fatahi R., Ebadi A., Vezvaei A., Zamani Z., and Ghanadha M.R. 2004. Relationship among quantitative and qualitative characters in 90 grapevine (Vitis vinifera) cultivars. P.275-282. In XXVI International Horticultural Congress: Viticulture-Living with Limitations 640, 31 August 2004. International Society for Horticultural Science, Toronto, Canada.

    9-       Ferguson A.R. 2006. The need for characterisation and evaluation of germplasm: kiwifruit as an example. Euphytica 154: 371–382

    10-   Fotohi ghazvini R., and Fatahi Moghadam J. 2010. Citrus cultivation in Iran, Guilan University, Rast. (In Persian)

    11-   Gulsen O., and Roose M.L. 2001a. Lemons: diversity and relationships with selected Citrus genotypes as measured with nuclear genome markers. American Society for Horticultural Science 126(3): 309-317.

    12-   Gulsen O., and Roose M.L. 2001b, Chloroplast and Nuclear Genome Analysis of the Parentage of Lemons, American Society for Horticultural Science 126(2): 210–215.

    13-   IPGRI. 2000. Descriptores para los citricos Citrus spp. Instituto Nacional de Recursos Fitogeneticos, Roma, 75

    14-   Jahangirzadeh Khiavi Sh., Hamidoghli Y., Golein B., and Sabouri A. 2016. Study of genetic diversity of some Iranian acid lime (Citrus aurantifolia Swingle) genotypes using AFLP marker, Journal of Plant Production Research 23(3): 81-96. (In Persian with English abstract)

    15-   Johnson R.A., and Wichern D.W. 2002. Applied multivariate statistical analysis. Upper Saddle River, New Jersey Prentice hall.

    16-   Karp A., Kresovich S., Bhat K.V., Ayad W.G., and Hodgkin T. 1997. Molecular tools in plant genetic resources conservation: a guide to the technologies. IPGRI Technical Bulletin No. 2. International Plant Genetic Resources Institute, Rome, Italy.

    17-   Khankahdani H.H., Rastegar S., Golein B., Golmohammadi M., and Jahromi A.A. 2017. Genetic diversity in Persian lime (Citrus latifolia Tanaka) accessions using morphological and molecular markers. Poljoprivreda i Sumarstvo 63(3): 221-231.

    18-   Khiavi S.J., Hamidoghli Y., Golein B., and Sabouri A. 2015. Evaluation of genetic diversity in acid lime ('Citrus aurantifolia'swingle) genotypes using AFLP markers. Australian Journal of Crop Science 9(10): 996-1002.

    19-   Khiavi S.J., Hamidoghli Y., Golein B., and Sabouri A. 2016. Assessment of lime genetic diversity in three regions of Iran using morphological and ISSR markers. Agricultural Communications 4(3): 18-29.

    20-   Koehler-Santos P., Dornelles A.L.C., and Freitas L.B. 2003. Characterization of mandarin citrus germplasm from southern Brazil by morphological and molecular analyze. Pesquisa Agropecuária Brasileira 38: 797-806.

    21-   Kumar S., Jena S.N., and Nair N.K. 2010. ISSR polymorphism in Indian wild orange (Citrus indica Tanaka, Rutaceae) and related wild species in North-east India. Scientia Horticulturae 123: 350–359.

    22-   Malik S.K., Rohini M.R., Kumar S., Choudhary R., Pal D., and Chaudhury R. 2012. Assessment of Genetic Diversity in Sweet Orange [Citrus sinensis (L.) Osbeck] Cultivars of India Using Morphological and RAPD Markers. Agricultural Research 1(4): 317–324.

    23-   Naghavi M.R., Ghareyazie B., and Hosseini Salekdeh Gh. 1388. Molecular Markers. University of Tehran press, Tehran.

    24-   Nei M. 1973. Analysis of gene diversity in subdivided populations. Proceedings of the National Academy of Sciences 70(12): 3321-3323.

    25-   Pal D., Malik S.K., Kumar S., Choudhury R., Sharma K.C., and Chaudhury R. 2013. Genetic Variability and Relationship Studies of Mandarin (Citrus reticulata Blanco) Using Morphological and Molecular Markers. Agricultural Research 2(3): 236–245

    26-   Raheb S., Ghasemnezhad M., Golein B., Golmohammadi M., and Sabouri A. 2018, Variability analysis of lime (Citrus sp.) genotypes using morphological markers in the south of Iran. Journal of Research in Ecology 6(1): 1400-1411

    27-   Rajapakse S., Belthoff,L.E., He G., Estager A.E., Scorza R., Verde I., Ballard R.E., Baird W.V., Callahan A., Monet R., and Abbott A.G. 1995. Genetic linkage mapping in peach using morphological, RFLP and RAPD markers. Theoretical and Applied Genetics 90(3-4): 503-510.

    28-   Roose M.L., and Close T.J. 2008. Genomics of Citrus, a Major Fruit Crop of Tropical and Subtropical Regions. p. 187-201. In: Moore P.H., Ming R. (eds) Genomics of Tropical Crop Plants. Plant Genetics and Genomics: Crops and Models, vol 1. Springer, New York, NY.

    29-   Şahin-Cevik M., and Moore G.A. 2012. Quantitative trait loci analysis of morphological traits in Citrus. Plant Biotechnology Reports 6: 47–57.

    30-   Salimpour A., Ebadi A., Moghaddam M.F., and Bihamta M. 2012. An evaluation of genetic diversity in some almond genotypes using morphological traits. Iranian Journal of Horticultural Science 42(4): 319-327. (In Persian with English abstract)

    31-   Shahsavar A.R., Izadpanah K., Tafazoli E., and Seyed Tabatabaei B.E. 2007, Characterization citrus including unknown variants by inter-simple sequence repeat (ISSR) markers, Scientia Horticulturae 112: 310-314.

    32-   Shahsavar A., Izadpanah K., Tafazoli E., and Sayed Tabatabaei B.E. 2005. Evaluation of genetic variability of limes and lemons in the Fars province by morphological trats and inter-simple sequence repeat (ISSR) markers. Iranian Journal of Horticultural Science and Technology 5(4): 177-188. (In Persian with English abstract)

    33-   Smith J.S.C., and Smith O.S. 1989. The description and assessment of distances between inbred lines of maze: the utility of morphological, biochemical and genetic descriptors and a scheme for the testing of distinctiveness between inbred lines. Maydica 46: 190-193.

    34-   Soost R.K., and Roose M.L. 1996. Citrus. p. X. In Okie W.R., and Weinberger J.H. (ed.) Fruit Breeding, Tree and Tropical Fruit. John Wiley and Sons.

    35-   Zandkarimi H., Talaie A., Fatahi R., and da Silva J.A.T. 2011. Evaluation of Some Lime and Lemon Accessions by using Morphological Characterization in Hormozgan Province (Iran). Fresh Produce 5(1): 69-76.