Genetic Variation of Lime (Citrus sp.) Accessions Using Flow Cytometry Technique, Morphological Characteristics and Molecular Markers

Document Type: Research paper


1 The Former Ph.D Student of Fruit Trees Physiology and Breeding, Horticulture Department of Agricultural and Natural Resource Faculty, Hormozgan University and Horticulture Research Department, Agricultural and Natural Resources Research and Education Center, AREEO, Bandar Abbas, Hormozgan, Iran.

2 Assistant Professor of Horticulture Department of Agricultural and Natural Resource Faculty, Hormozgan University, Iran.

3 Associate and Assistant Professor of Citrus and Sub Tropical Fruits Institute, Horticultural Science Research Institute, AREEO, Ramsar, Mazandaran, Iran, Respectively.

4 Horticulture Department, Jahrom Branch, Islamic Azad University, Jahrom, Fars, Iran.


Because of sexual propagation of Mexican lime in southern regions of Iran, there are many lime accessions (lime biotypes) in Mexican lime gardens in which appear some variation in fruit and tree shape. However, most of these accessions are susceptible to witches broom disease of lime (WBDL). Persian lime (C. latifolia Tanaka) is a triploid WBDL-tolerant species. Considerable number of un-described lime biotypes have been cultivated by farmers as Persian lime with no documented evidence related to their origin and genetic background. To unveil systematic modality of the lime accessions, we investigated 18 lime accessions collected from Fars, Hormozgan and Mazandaran provinces (Iran) using flow cytometry (FCM) approach, morphological characteristics, ISSR and SSR markers in 2016. The results of FCM revealed that 14 out of 18 lime accessions were triploid. Triploid accessions showed higher leaf dimension values together with lower stomata and secretory sacs density compared with diploid accessions. Application of SSR markers confirmed the obtained results of ploidy level determination using FCM and morphological analysis. Results of morphological and ISSR markers demonstrated that four out of 18 accessions were different from others and it was in correspondence with the results of FCM. SSR results also grouped the accessions in line of FCM. In conclusion, it has been shown that IFJKh, Cucumber-shaped lime and IFJKMes accessions, are diploid and their cultivation is not recommended in the infected regions to WBDL phytoplasma.


Afshar-Mohammadian M, Omidi Z, Purakbari-Kasmaei R, Asadi-Abkenar A. 2013. The effect of polyploidy on some anatomical and antioxidant characteristics of Citrus aurantifolia. Plant Research (Iranian Journal of Biology) 26(3), 238-246. (In Persian)

Ahmad R, Struss D, Southwick S.M. 2003. Development and characterization of microsatellite markers in Citrus. Journal of American Society of Horticultural Science 128(4), 584-590.

Cantuarias-Avilés T, Filhoa F.A, Stuchi E.S, da-Silva S.R, Espinosa-Nuneza E, Neota H.B. 2012. Rootstocks for high fruit yield and quality of ‘Tahiti’ lime under rain-fed conditions. Scientia Horticulturae 142, 105-111.

Curk F, Ollitrault F, Garcia-Lor A, Luro F, Navarro L, Ollitrault P. 2016. Phylogenetic origin of limes and lemons revealed by cytoplasmic and nuclear markers. Annals of Botany 1-19.

Edwards K, Johnstone C, Thompson C. 1991. Simple rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Research 19(6), 1349.

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.

Golein B, Rabiei V, Mirabbasi F, Fifaei R, Hallaji-Sani M.F. 2015. Effect of salinity stress on physiology and biochemistry characteristics of citrus genotypes. Horticultural Science 29(3), 416-425. (In Persian)

Gu X.F, Yang A.F, Meng H, Zhang J.R. 2005. In vitro induction of tetraploid plants from diploid Zizyphus jujube Mill. cv. Zhanhua. Plant Cell Report 24, 671-676.

Hosseini H.R, Chehrazi M, Nabati-Ahmadi D, Mahmoodi-Soorestani M. 2015. Induction of auto-tetraploidy in Madagascar periwinkle (Catharanthus roseus cv. Rosea) by Colchicine treatment in order to induce diversity of morph-physiological and phenology traits. Plant Process and Function 3(9), 1-10. (In Persian)

IPGRI. 1999. Descriptors for Citrus. International Plant Genetic Resources Institute, Rome, Italy. Available at

Lee J.H, Ma Y, Wako T, Li L.C, Ki K.Y, Park S.W, Uchiyama S, Fukui K. 2004. Flow karyotypes and chromosomal DNA contents of genus Triticum species and rye (Secale cereale). Chromosome Research 12, 93-102.

Padoan D, Mossad A, Chiancone B, Germana M.A, Valli Khan P.S. 2013. Ploidy levels in Citrus clementine affect leaf morphology, stomatal density and water content. Theoretical and Experimental Plant Physiology 25(4), 283-290.

Salehi M, Nejat N, Tavakoli A.R, Izadpanah K. 2005. Reaction of Citrus cultivars to Candidatus Phytoplasma aurantifolia in Iran. Plant Disease 41, 363-376. (In Persian)

Santos M.G, Passos O.S, Filho W.S, Girardi E.A, Gesteira A.S, Ferreira C.F. 2013. Variability analysis of Persian acid lime tree selections using agronomic and molecular markers. Genetic and Molecular Research 12(4), 4604-4614.

Seker M, Tuzcu O, Ollitrault P. 2003. Comparison of nuclear DNA content of citrus rootstock populations by flow cytometry analysis. Plant Breeding 122, 169-172.

Shahriari-Ahmadi F, Dehghan E, Farsi M, Azizi M. 2008. Tetraploid induction of Hyoscyamus muticus L. using Colchicine treatment. Pakistan Journal of Biological Sciences 11, 2653-2659.

Sharafi A, Asadi-Abkenar A, Sharafi A, Masaeli M. 2016. Genetic variation assessment of acid lime accessions collected from south of Iran using SSR and ISSR molecular markers. Physiology and Molecular Biology of Plants doi: 10.1007/s12298-016-0336-4.

Sharma N, Dubey A.K, Srivastav M, Singh B.P, Singh A.K, Singh N.K. 2015. Assessment of genetic diversity in grapefruit (Citrus paradisi Macf.) cultivars using physico-chemical parameters and microsatellite markers. Australian Journal of Crop Science 9(1), 62-68.

Tripolitsiotis C, Nikoloudakis N, Linos A, Hagidimitriou M. 2013. Molecular characterization and analysis of the Greek citrus germplasm. Notulae Botanicae Horti Agrobotanici 41(2), 463-471.

USDA. 2002. Citrus variety collection: Egyptian lime. University of California Riverside, CRC 2883 PI 185427.

Watanabe M, Yamamoto T, Ohara M, Nishitani C, Yahata S. 2008. Cultivar differentiation identified by SSR markers and the application for polyploidy loquat plants. Journal of Japanese Society of Horticultural Science 77, 388-394.

Ye Y.M, Tong J, Shi X.P, Yuan W, Li G.R. 2010. Morphological and cytological studies of diploid and Colchicine-induced tetraploid lines of crape myrtle (Lagerstroemia indica L.). Scientia Horticulturae 124, 95-101.

Zhu S.P, Song J.K, Hu Z.Y, Tan B, Xie Z.Z, Yi H.L, Deng X.X. 2009. Ploidy variation and genetic composition of open-pollinated triploid citrus progenies. Botany Study 50, 319-324.