an article



ISHS Contact


  Fruits 72 (5) 292-305 | DOI: 10.17660/th2017/72.5.5
ISSN 0248-1294 print and 1625-967X online | © ISHS 2017 | Fruits, The International Journal of Tropical and Subtropical Horticulture | Original article

Exploiting DNA-based molecular tools to assess genetic diversity in pomegranate (Punica granatum L.) selections and cultivars

A. Giancaspro2, A. Mazzeo1, L.S. Giove2, D. Zito2, I. Marcotuli2, A. Gallotta1, P. Colasuonno2, D. Nigro1, A. Blanco1, M. Aradhya3, A. Gadaleta2 and G. Ferrara1,a
1 Department of Soil, Plant and Food Sciences, University of Bari ‘Aldo Moro’, Via G. Amendola 165/A, 70126 Bari, Italy
2 Department of Environmental and Territorial Sciences, University of Bari ‘Aldo Moro’, Via G. Amendola 165/A, 70126 Bari, Italy
3 National Clonal Germplasm Repository, USDA-ARS, University of California, One Shields Ave, Davis, CA 95616, USA

Introduction – Nowadays the demand for pomegranate (Punica granatum L.) as fresh fruit and derived products (arils, juice, jam, etc.) has been considerably rising due to increased awareness about its nutritive value and nutraceutical properties. Consequently, genetic improvement efforts are focused on the identification of the most productive and nutritionally valuable genotypes for commercial production. Evaluation of pomegranate germplasm has been usually based on morpho-pomological traits such as yield, fruit size, seed-hardness, juice sweetness, rind and aril color, antioxidant activity, fatty acids, polyphenols and anthocyanin content, whereas genetic studies received less attention. Materials and methods – Microsatellite (SSR) markers have been employed to estimate genetic diversity and determine the genetic structure in a collection of pomegranate accessions including both selections and cultivars with different origins/disseminations and pomological traits. Results and discussion – The overall genetic diversity analysis was able to group pomegranate germplasm into clusters according to both geographical origin/diffusion and pomological traits, such as juice sweetness, skin and aril color. Moreover, pomegranate accessions from different geographical areas appeared more similar in comparison with those accessions within the same origin. The SSR classification was consistent with either the morphological description (juice taste and skin/aril color) or the geographical origin/diffusion, thus confirming the intense germplasm flow since ancient times from Asia to Mediterranean areas such as Southern Italy, Spain and Northern Africa. Conclusion – Microsatellites were able to establish genetic relationships among the different pomegranate selections and cultivars and allowed to identify synonyms and homonyms. They proved powerful tools for genetic improvement programs combining complementary traits addressing the new market needs.

Exploiter des outils moléculaires à base d’ADN pour évaluer la diversité génétique dans les sélections et les cultivars de grenade (Punica granatum L.).
Introduction – De nos jours, la consommation de grenade (Punica granatum L.) en fruits frais et produits dérivés (arilles, jus, confitures, etc.) a considérablement augmenté en raison d’une prise de conscience accrue de sa valeur nutritive et de ses propriétés nutraceutiques. En conséquence, les efforts d’amélioration génétique pour la production commerciale ont été axés sur l’identification des génotypes les plus productifs et les plus nutritifs. L’évaluation des ressources génétiques de grenade est habituellement basée sur des traits morpho-pomologiques tels que le rendement, la taille des fruits, la dureté des graines, la douceur des jus, la couleur de l’écorce et de l’arille, l’activité anti-oxydante, les teneurs en acides gras, polyphénols et anthocyanines, alors que les études génétiques ont reçu moins d’attention. Matériel et méthodes – Les marqueurs microsatellites (SSR) ont été utilisés pour estimer la diversité génétique et déterminer la structure génétique dans une collection d’accessions de grenade comprenant à la fois des sélections et des cultivars de différentes origines/distributions et caractéristiques des fruits. Résultats et discussion – L’analyse globale de la diversité génétique a permis de grouper les ressources génétiques de grenade en clusters selon les origines géographiques / la diffusion de matériel génétique et les caractéristiques des fruits, telles que la douceur des jus, la peau et la couleur arille. De plus, les accessions de différentes régions géographiques sont apparues plus semblables entre elles que les accessions de même origine. Ces similitudes confirment le flux intense de ressources génétiques depuis l’Antiquité en provenance d’Asie vers les régions méditerranéennes telles que le sud de l’Italie, l’Espagne et l’Afrique du Nord. Conclusion – Les microsatellites ont pu établir des relations génétiques entre différentes sélections et cultivars de grenade et ont permis d’identifier synonymes et homonymes. Ils se sont révélés des outils puissants pour les programmes d’amélioration génétique combinant des caractères complémentaires répondant aux nouveaux besoins du marché.

Keywords pomegranate, Punica granatum, genetic diversity, germplasm management, microsatellites (SSR), population structure

Mots clés grenade, Punica granatum, diversité génétique, gestion des ressources génétiques, microsatellites (SSR), structure de la population

Significance of this study

What is already known on this subject?

  • Pomegranate includes a very huge number of cultivated, wild and ornamental genotypes, differing for several morpho-chemical traits such as yield, fruit size, flowering and ripening time, seed-hardness, juice sweetness, rind and aril color, antioxidant activity, fatty acid, polyphenol and anthocyanin contents.
What are the new findings?
  • Pomegranate accessions from different geographical areas (Italy, Israel, Turkmenistan, USA, Japan) appeared more similar with respect to accessions within the same country. The SSR classification was consistent with either the morphological description (juice taste and skin/aril color) or the geographical origin/diffusion. It confirmed the intense flow of genetic materials from Persia towards different countries since ancient times.
What is the expected impact on horticulture?
  • The SSR markers are able either to discriminate between synonyms and homonyms or to identify suitable accessions to be used in breeding programs. Genetic diversity assessment with microsatellites can be used in breeding to select superior pomegranate cultivars for improved nutritional properties, disease resistance and agronomical traits (flavor, size, color, etc.).

Download fulltext version
    subscribers & pay-per-view - check for available options and price details
How to cite this article       Export citation to RIS format      



  • Ajal, E.A., Jbir, R., Legua, P., Martínez, J.J., Martinez, R., Hannachi, A.S., and Haddioui, A. (2015). Genetic diversity of Moroccan pomegranate (Punica granatum L.) cultivars using AFLP markers. Austr. J. Crop Sci. 9(1) 22–29.

  • Alamuti, M.K., Zeinalabedini, M., Derazmahalleh, M.M., RoodbarShojaie, T., Irandoost, H.P., Zahravi, M., Vazifehshenas, M., Ebrahimi, M.A., Nekouei, S.M.K., Salekdeh, G.H., and Mardi, M. (2012). Extensive genetic diversity in Iranian pomegranate (Punica granatum L.) germplasm revealed by microsatellite markers. Sci. Hortic. 146, 104–114.

  • Billotte, N., Lagoda, P.J.L., Risterucci, A.M., and Baurens, F.C. (1999). Microsatellite-enriched libraries: applied methodology for the development of SSR markers in tropical crops. Fruits 54, 277–288.

  • Bossu, A., Bertaudière-Montès, V., Dubut, V., and Manel, S. (2014). Microsatellite primers in Parietaria judaica (Urticaceae) to assess genetic diversity and structure in urban landscapes. Applic. in Plant Sci. 2(9), 1400036.

  • Brown, S.M., Hopkins, M.S., Mitchell, S.E., Senior, M.L., Wang, T.Y., Duncan, R.R., Gonzalez-Candelas, F., and Kresovich, S. (1996). Multiple methods for the identification of polymorphic simple sequence repeats (SSRs) in sorghum [Sorghum bicolor (L.) Moench] Theor. and Appl. Gen. 93, 190–198.

  • Chandra, R., Babu, D.K., Jadhav, V.T., and Teixeira da Silva, J.A. (2010). Origin, history and domestication of pomegranate. In Pomegranate, R. Chandra ed., Fruit Veg. Cereal Sci. Biotechnol., Vol. 4, Special Issue 2, p. 1–6.

  • Currò, S., Caruso, M., Di Stefano, G., Gentile, A., and La Malfa, S. (2010). New microsatellite loci for pomegranate Punica granatum (Lythraceae). Am. J. of Botany 97(7), e58–60.

  • Decroocq, V., Favé, M.G., Hagen, L., Bordenave, L., and Decroocq, S. (2003). Development and transferability of apricot and grape EST microsatellite markers across taxa. Theor. and Appl. Gen. 106(5), 912–922.

  • Ebrahimi, S., Sayed-Tabatabaei, B.E., and Sharifnabi, B. (2010). Microsatellite isolation and characterization in pomegranate (Punica granatum L.). Iran. J. Biotechnol. 8, 156–163.

  • Evanno, G., Regnaut, S., and Goudet, J. (2005). Detecting the number of clusters of individuals using the software Structure: A simulation study. Mol. Ecol. Notes 14, 2611–2620.

  • Falush, D., Stephens, M., and Pritchard, J.K. (2003). Inference of population structure using multilocus genotype data: linked loci and correlated allele frequencies. Genetics 164(4), 1567–1587.

  • Ferrara, G., Giancaspro, A., Mazzeo, A., Giove, S.L., Matarrese, A.M.S., Pacucci, C., Punzi, R., Trani, A., Gambacorta, G., Blanco, A., and Gadaleta, A. (2014). Characterization of pomegranate (Punica granatum L.) genotypes collected in Puglia region, Southeastern Italy. Sci. Hortic. 178, 70–78.

  • Ferrara, G., Mazzeo, A., Pacucci, C., Matarrese, A.M.S., Tarantino, A., Crisosto, C., Incerti, O., Marcotuli, I., Nigro, D., Blanco, A., and Gadaleta, A. (2016). Characterization of edible fig germplasm from Puglia, Southeastern Italy: Is the distinction of three fig types (Smyrna, San Pedro and Common) still valid? Sci. Hortic. 205, 52–58.

  • Hajiahmadi, Z., Talebi, M., and Sayed-Tabatabaei, B.E. (2013). Studying genetic variability of pomegranate (Punica granatum L.) based on chloroplast DNA and Barcode genes. Mol. Biotechnol. 55(3), 249–259.

  • Hasnaoui, N., Buonamici, A., Sebastiani, F., Mars, M., Trifi, M., and Vendramin, G.G. (2010). Development and characterization of SSR markers for pomegranate (Punica granatum L.) using an enriched library. Conserv. of Gen. Res. 2, 283–285.

  • Hasnaoui, N., Buonamici, A., Sebastiani, F., Mars, M., Zhang, D., and Vendramin, G.G. (2012). Molecular genetic diversity of Punica granatum L. (pomegranate) as revealed by microsatellite DNA markers (SSR). Gene 493, 105–112.

  • Holland, D., Hatib, K., and Bar-Ya’akov, I. (2009). Pomegranate: botany, horticulture, breeding. Hortic. Rev. 35, 127–191.

  • Hvarleva, T.Z., Russanov, K., and Atanassov, I. (2014). Microsatellite markers for characterization of grape genetic resources and identification of QTLs for important agronomical traits. Biotechnol. & Biotechnol. Equipm. 19(3), 116–123.

  • Jbir, R., Hasnaoui, N., Mars, M., Marrakchi, M., and Trifi, M. (2008). Characterization of Tunisian pomegranate (Punica granatum L.) cultivars using amplified fragment length polymorphism analysis. Sci. Hortic. 115, 231–237.

  • Jbir, R., Zehidi, S., Hasnaoui, N., Ben Dhiaf, A., Mars, M., and Sahli Hannachi, A. (2012). Microsatellite polymorphism in Tunisian pomegranates (Punica granatum L.): Cultivar genotyping and identification. Biochem. Syst. and Ecol. 44, 27–35.

  • Jian, Z.H., Liu, X.S., Chen, Y.H., and Feng, J.C. (2012). Mining microsatellite markers from public expressed sequence tag sequences for genetic diversity analysis in pomegranate. Indian Acad. Sci. 91(3), 353–358.

  • Kader, A.A. (2006). Postharvest biology and technology for pomegranates. In Pomegranates Ancient Roots to Modern Medicine, N. Seeram, R. Schulman, and D. Heber, eds. (Boca Raton, FL.: Taylor and Francis), p. 211–220.

  • Kalia, R.K., Rai, M.K., Kalia, S., Singh, R., and Dhawan, A.K. (2011). Microsatellite markers: an overview of the recent progress in plants. Euphytica 177(3), 309–334.

  • Lihua, Z., Mingyabg, L., Guangze, C., Tiianchun, P., and Chenghai, S. (2013). Assessment of the genetic diversity and genetic relationships of pomegranate (Punica granatum L.) in China using RAMP markers. Sci. Hortic. 151, 63–67.

  • Melgarejo, P., Martínez, J.J., Hernández, F., Martínez, R., Legua, P., Oncina, R., and Martínez-Murcia, A. (2009). Cultivar identification using 18S–28S rDNA intergenic spacer-RFLP in pomegranate (Punica granatum L.). Sci. Hortic. 120, 500–503.

  • Mena, P., Gironés-Vilaplana, A., Moreno, D.A., and García-Viguera, C. (2011). Pomegranate fruit for health promotion: myths and realities. Funct. Plant Sci. and Biotechnol. 5(2), 33–42.

  • Miah, G., Rafii, M.Y., Ismail, M.R., Puteh, A.B., Rahim, H.A., Islam, Kh.N., and Latif, M.A. (2013). A review of microsatellite markers and their applications in rice breeding programs to improve blast disease resistance. Int. J. Mol. Sci. 14(11), 22499–22528.

  • Moslemi, M., Zahravi, M., and Khaniki, G.B. (2010). Genetic diversity and population genetic structure of pomegranate (Punica granatum L.) in Iran using AFLP markers. Sci. Hortic. 126(4), 441–447.

  • Ono, N.N., Britton, M.T., Fass, J.N., Nicolet, C.M., Lin, D., and Tian, L. (2011). Exploring the transcriptome landscape of pomegranate fruit peel for natural product biosynthetic gene and SSR marker discovery. J. Integr. Plant Biol. 53(10), 800–813.

  • Ophir, R., Sherman, A., Rubistein, M., Eshed, M., Schwager, M.S., Harel-Beja, R., Bar-Ya’akov, I., and Holland, D. (2014). Single Nucleotide Polymorphism markers from de-novo assembly of the pomegranate transcriptome reveal germplasm genetic diversity. Plos One 9(2), e88998.

  • Pareek, S., Valero, D., and Serrano, M. (2015). Postharvest biology and technology of pomegranate. J. Sci. Food and Agric. 95(12), 2360–2379

  • Parvaresh, M., Talebi, M., and Sayed-Tabatabaei, B.-E. (2012). Molecular diversity and genetic relationship of pomegranate (Punica granatum L.) genotypes using microsatellite markers. Sci. Hortic. 138, 244–252.

  • Peakall, R., and Smouse, P.E. (2006). GenAlEx 6: genetic analysis in Excel Population genetic software for teaching and research. Mol. Ecol. Notes 6(1), 288–295.

  • Peakall, R., and Smouse, P.E. (2012). GenAlEx 6.5: genetic analysis in Excel Population genetic software for teaching and research-an update. Bioinformatics 28(19), 2537–2539.

  • Pirseyedi, S.M., Valizadehghan, S., Mardu, M., Ghaffari, M.R., Mahmoodi, P., Zahravi, M., Zeinalabedini, M., and Nekoui, S.M.K. (2010). Isolation and characterization on novel microsatellite markers in Pomegranate (Punica granatum L.). Int. J. Mol. Sci. 11, 2010–2016.

  • Pritchard, J., Stephens, M., and Donnelly, P. (2000). Inference of population structure using multilocus genotype data. Genetics 155, 945–959.

  • Sarkhosh, A., Zamani, Z., Fatahi, R., and Ebadi, A. (2006). RAPD markers reveal polymorphism among some Iranian pomegranate (Punica granatum L.) genotypes. Sci. Hortic. 111, 24–29.

  • Sarkhosh, A., Zamani, Z., Fatahi, R., and Ranjbar, H. (2009). Evaluation of genetic diversity among Iranian soft-seed pomegranate accessions by fruit characteristics and RAPD markers. Sci. Hortic. 121, 313–319.

  • Smith, J.M., Smith, N.H., O’Rourke, M., and Spratt, B.G. (1993). How clonal are bacteria? Proc. Nat. Acad. Sci. USA 90, 4384–4388.

  • Teixeira da Silva, J.A., Rana, T.S., Narzary, D., Verma, N., Meshram, D.T., and Ranade, S.A. (2013). Pomegranate biology and biotechnology: a review. Sci. Hortic. 160, 85–107.

  • Vieira, M.L.C., Santini, L., Diniz, A.L., and de Freitas Munhoz, C. (2016). Microsatellite markers: what they mean and why they are so useful. Gen. and Mol. Biol. 39(3), 312–318.

  • Yuan, Z., Yin, Y., Qu, J., Zhu, L., and Li, Y. (2007). Population genetic diversity in Chinese pomegranate (Punica granatum L.) cultivars revealed by fluorescent-AFLP markers. J. Genet. and Genom. 34(12), 1061–1071.

  • Zamani, Z., Sarkhosh, A., Fatahi, R., and Ebadi, A. (2007). Genetic relationships among pomegranate genotypes by RAPD markers and morphological characters of fruit. J. Hortic. Sci. and Biotechnol. 82, 11–18.

Received: 14 July 2017 | Accepted: 22 August 2017 | Published: 28 September 2017 | Available online: 28 September 2017

previous article     Volume 72 issue 5     next article