Physiology and Molecular Biology of Plants

, Volume 25, Issue 5, pp 1211–1223 | Cite as

Conserved DNA-derived polymorphism, new markers for genetic diversity analysis of Tunisian Pistacia vera L.

  • Meriem Aouadi
  • Karim Guenni
  • Donia Abdallah
  • Marwa Louati
  • Khaled Chatti
  • Ghada Baraket
  • Amel Salhi HannachiEmail author
Research Article


Pistachio trees (Pistacia vera L.) have been cultivated in Tunisia for decades and the plantation was extended mostly in the center of the country contributing to the economic growth of marginalized areas. Herein we used conserved DNA derived polymorphism (CDDP) technique, which target specifically conserved sequences of plant functional genes, to assess the genetic diversity and construct genetic relationships among 65 Tunisian pistachio trees. A set of nine primers were used and 157 CDDP markers were revealed with an average of 17.44 showing a high degree of polymorphism (99.37%). The average of polymorphism information content of CDDP markers was of 0.86, which indicates the efficiency of CDDP primers in the estimation of genetic diversity between pistachios. UPGMA dendrogram and the principal component analysis showed four clusters of analyzed pistachios trees. Our results showed that the genetic structure depends on: (1) the gene exchanges between groups, (2) the geographical origin and (3) the sex of the tree. The same result was revealed by the Bayesian analysis implemented in STRUCTURE at K = 4, in which the pistachio genotypes of El Guettar, Kasserine and Sfax were assigned with more than 80% of probability. Our results prove polymorphism and efficiency of CDDP markers in the characterization and genetic diversity analysis of P. vera L. genotypes to define conservation strategy.


Pistacia vera L. CDDP markers Genetic diversity Structure analysis 



Analysis of molecular variance


Conserved DNA-derived polymorphism


Deoxyribonucleic acid


Principal component analysis


Polymerase chain reaction


Polymorphism information content


Percentage of polymorphic bands


Quantitative trait locus


Restriction site-associated DNA


Random amplified polymorphic DNA


Sequence-characterized amplified region


Sequence-related amplified polymorphism


Simple sequence repeats


Unweighted pair group method with arithmetic mean



The authors would like to thank the Regional Commissions for Agricultural Development (CRDA), the officials of two experimental stations: Chenchou (Gabès) and Taous (Sfax), and technicians.


This research was supported by grants from the Tunisian Ministry of Higher Education, Scientific Research.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Ahmad R, Ferguson L, Southwick SM (2003) Identification of pistachio (Pistacia vera L.) nuts with microsatellite markers. J Am Soc Hortic Sci 128:898–903CrossRefGoogle Scholar
  2. Ahmad R, Ferguson L, Southwick SM (2005) Molecular marker analysis of pistachio rootstocks by simple sequence repeats and sequence-related amplified polymorphisms. J Hortic Sci Biotechnol 80:382–386CrossRefGoogle Scholar
  3. Al-Saghir M, Abu Baker S, Pusok R (2014) Effective method to resolve the chromosome numbers in Pistacia species (Anacardiaceae). Am J Plant Sci 5:2913–2916. CrossRefGoogle Scholar
  4. Andersen JR, Lübberstedt T (2003) Functional markers in plants. Trends Plant Sci 8(11):554–560. CrossRefPubMedGoogle Scholar
  5. Anai T, Miyata M, Kosemura S, Yamamura S, Tsuge T, Matsui M, Uchida H, Hasegawa K (1997) Comparison of abp1 primary sequences from monocotyledonous and dicotyledonous species. J Plant Physiol 151(4):446–449CrossRefGoogle Scholar
  6. Atia MAM, Sakr MM, Adawy SS (2017) Assessing date palm genetic diversity using different molecular markers. Methods Mol Biol 1638:125–142CrossRefPubMedGoogle Scholar
  7. Behboudian MH, Walker RR, Törökfalv E (1986) Effects of water stress and salinity on photosynthesis on pistachio. Sci Hortic 29:251–261CrossRefGoogle Scholar
  8. Carbonell-Barrachina AA, Memmi H, Noguera-Artiaga L, Gijon-Lopez Mdel C, Ciapa L, PerezLopez D (2015) Quality attributes of pistachio nuts as affected by rootstock and deficit irrigation. J Sci Food Agric 95(14):2866–2873CrossRefPubMedGoogle Scholar
  9. Chatti K, Choulak S, Genni K, Hannachi AS (2017) Genetic diversity analysis using morphological parameters in Tunisian pistachio (Pistacia vera L.). J Res Biol Sci 02:29–34Google Scholar
  10. Chelli-Chaabouni A, Ouni S, Trad M, Ouerghi I, Ben Hamda H (2016) Morphometric features of local and foreign female pistachio cultivars and ecotypes in north east Tunisian conditions. In: Kodad O, López-Fran cos A, Rovira M, Sociasi Compan y R (eds) XVI GREMPA meeting on Almonds and Pistachios (Options Méditerranéennes: Série A. Séminaires Méditerranéens; n.119). CIHEAM, Zaragoza, pp 37–41Google Scholar
  11. Choulak S, Rhouma-Chatti S, Marzouk Z, Said K, Chatti N, Chatti K (2015) Chloroplast DNA analysis of Tunisian pistachio (Pistacia vera L.): sequence variations of the intron trnL (UAA). Sci Hortic 191:57–64. CrossRefGoogle Scholar
  12. Choulak S, Chatti K, Rhouma Chatti S, Guenni K, Salhi Hannachi A, Said K, Chatti N (2019) Microsatellite (SSR) markers reveal genetic diversity and population structure in Tunisian pistachio. Fruits 74(2):73–78. CrossRefGoogle Scholar
  13. Collard BCY, Mackill DJ (2009) Conserved DNA-derived polymorphism (CDDP): a simple and novel method for generating DNA markers in plants. Plant Mol Biol Rep 27:558–562. CrossRefGoogle Scholar
  14. Development Core Team R (2013) R: a language and environment for statistical computing. Version 3.0.1. R Foundation for Statistical Computing, ViennaGoogle Scholar
  15. Earl DA, VonHoldt BM (2012) STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Conserv Genet Resour 4(2):359–361. CrossRefGoogle Scholar
  16. Ehsani V, Amirteimoury M, Taghipour Z, Shamsizadeh A, Bazmandegan G, Rahnama A, Khajehasani F, Fatemi I (2017) Protective effect of hydroalcoholic extract of Pistacia vera against gentamicin-induced nephrotoxicity in rats. Ren Fail 39(1):519–525. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Esfandiyari B, Davarynejad G, Shahriari F, Kiani M, Mathe A (2012) Sex determination in Pistacia species using molecular markers. Euphytica 185(2):227–231. CrossRefGoogle Scholar
  18. Evanno G, Regnaut S, Goudet J (2005) Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol Ecol 14(8):2611–2620. CrossRefPubMedGoogle Scholar
  19. FAOSTAT (2017) Food and agriculture organization, FAO STAT data base. Accessed 16 Mar 2018
  20. Farès K, Guasmi F, Touil L, Triki T, Ferchichi A (2009) Genetic diversity of pistachio tree using inter-simple sequence repeat markers ISSR supported by morphological and chemical markers. Biotechnology 8:24–34CrossRefGoogle Scholar
  21. Ferguson L, Sanden B, Grattan S, Epstein L, Klueger B (2005) The orchard. Pistachio rootstocks. In: Ferguson L, Beede RH, Freeman MW, Haviland DR, Holtz BA, Kallsen CE, Coviello J (eds) Pistachio production manual, 4th edn. Fruit and Nut Research and Information Center, University of California, Davis, pp 67–73Google Scholar
  22. Ghrab M, Ben Mimoun M, Triki H, Gouta H (2002) Evaluation of the performances of seventeen male pistachio-tree specimens. Acta Hortic 591:473–477CrossRefGoogle Scholar
  23. Ghrab M, Ben Mimoun M, Gouta H (2004) Pistachio production in Tunisia. Nusis FAO Newsl 12:19–21Google Scholar
  24. Ghrab M, Zribi F, Chelli-Chaabouni A, Gouta H, Ben Mimoun M (2010) Genetic diversity of pistachio in Tunisia. Options Méditerranéennes: Série A. Séminaires Méditerranéens 94:221–228Google Scholar
  25. Guenni K, Aouadi M, Chatti K, Salhi Hannachi A (2016) Analysis of genetic diversity of Tunisian pistachio (Pistacia vera L.) using sequence-related amplified polymorphism (SRAP) markers. Genet Mol Res. CrossRefPubMedGoogle Scholar
  26. Gupta PK, Rustgi S (2004) Molecular markers from the transcribed/expressed region of the genome in higher plants. Funct Integr Genomics 4(3):139–162. CrossRefPubMedGoogle Scholar
  27. Gutterson N, Reuber TL (2004) Regulation of disease resistance pathways by AP2/ERF transcription factors. Curr Opin Plant Biol 7(4):465–471CrossRefGoogle Scholar
  28. Hajibarat Z, Saidi A, Hajibarat Z, Talebi R (2015) Characterization of genetic diversity in chickpea using SSR markers, start codon targeted polymorphism (SCoT) and conserved DNA-derived polymorphism (CDDP). Physiol Mol Biol Plants 21(3):365–373. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Hormaza JI, Dollo L, Polito VS (1994a) Identification of a RAPD marker linked to sex determination in Pistacia vera using bulked segregant analysis. Theor Appl Genet 89:9–13. CrossRefPubMedGoogle Scholar
  30. Hormaza I, Dollo L, Polito S (1994b) Determination of relatedness and geographical movements of Pistacia vera (Pistachio; Anacardiaceae) germplasm by RAPD analysis. Econ Bot 48(4):349–358. CrossRefGoogle Scholar
  31. Hormaza J, Pinney K, Polito V (1998) Genetic diversity of pistachio (Pistacia vera. Anacardiaceae) germplasm based on randomly amplified polymorphic DNA (RAPD) markers. Econ Bot 52:78–87CrossRefGoogle Scholar
  32. Jakobsson M, Rosenberg NA (2007) CLUMPP: a cluster matching and permutation program for dealing with label switching and multimodality in analysis of population structure. Bioinformatics 23(14):1801–1806. CrossRefPubMedGoogle Scholar
  33. Jiang L, Zang D (2018) Analysis of genetic relationships in Rosa rugosa using conserved DNA-derived polymorphism markers. Biotechnol Biotechnol Equip 32(1):88–94. CrossRefGoogle Scholar
  34. Jiang C, Gu X, Peterson T (2004) Identification of conserved gene structures and carboxy-terminal motifs in the Myb gene family of Arabidopsis and Oryza sativa L. ssp.indica. Genome Biol 5(7):R46CrossRefPubMedPubMedCentralGoogle Scholar
  35. Kafkas S (2006) Phylogenetic analysis of the genus Pistacia by AFLP markers. Plant Syst Evol 262:113–124CrossRefGoogle Scholar
  36. Kafkas S, Çetiner S, Perl-Treves R (2001) Development of sex-associated RAPD markers in wild Pistacia species. J Hortic Sci Biotechnol 76(2):242–246. CrossRefGoogle Scholar
  37. Kafkas S, Ozkan H, ErolAk B, Acar I, Alti HS (2006) Detecting DNA polymorphism and genetic diversity in a wide pistachio germplasm: comparison of AFLP, ISSR, and RAPD marker. J Am Soc Hortic Sci 131(4):522–529CrossRefGoogle Scholar
  38. Kafkas S, Khodaeiaminjan M, Güney M, Kafkas E (2015) Identification of sex-linked SNP markers using RAD sequencing suggests ZW/ZZ sex determination in Pistacia vera L. BMC Genom 16:98. CrossRefGoogle Scholar
  39. Kalendar R, Flavell AJ, Ellis TH, Sjakste T, Moisy C, Schulman AH (2001) Analysis of plant diversity with retrotransposon-based molecular markers. J Hered 106(4):520–530. CrossRefGoogle Scholar
  40. Katsiotis A, Hagidimitriou M, Drossou A, Pontikis C, Loukas M (2003) Genetic relationships among species and cultivars of Pistacia using RAPDs and AFLPs. Euphytica 132:279–288CrossRefGoogle Scholar
  41. Kebour D, Boutekrabt A, Mefti M (2012) Using ISSR markers to study genetic polymorphism of pistachio (Pistacia vera L.) in Algeria. J Biotechnol Pharm Res 3(3):47–53Google Scholar
  42. Li YY (2013) Screening of CDDP molecular marker primers for Tree Peony and analysis of its polymorphism. J NuclAgricSci 27(8):1099–1105Google Scholar
  43. Li T, Guo J, Li Y, Ning H, Sun X, Zheng C (2013) Genetic diversity assessment of chrysanthemum germplasm using conserved DNA-derived polymorphism markers. Sci Hortic 162:271–277. CrossRefGoogle Scholar
  44. Lim J, Moon YH, An G, Jang SK (2000) Two rice MADS domain proteins interact with OsMADS1. Plant Mol Biol 44(4):513–527CrossRefPubMedGoogle Scholar
  45. McDermott JM, McDonald BA (1993) Gene flow in plant pathosystems. Annu Rev Phytopathol 31:353–373. CrossRefGoogle Scholar
  46. Nadernejad N, Ahmadimoghadam A (2013) Effect of different rootstocks on PAL activity and phenolic compounds in flowers, leaves, hulls and kernels of three pistachio (Pistacia vera L.) cultivars. Trees 27:1681–1689CrossRefGoogle Scholar
  47. Nei M (1977) F-statistics and analysis of gene diversity in subdivided populations. Ann Hum Genet 41(2):225–233. CrossRefPubMedGoogle Scholar
  48. Peakall R, Smouse PE (2012) GenAlEx 6.5: genetic analysis in Excel Population genetic software for teaching and research—an update. Bioinformatics 28(19):2537–2539. CrossRefPubMedPubMedCentralGoogle Scholar
  49. Prevost A, Wilkinson MJ (1999) A new system of comparing PCR primers applied to ISSR fingerprinting of potato cultivars. Theor Appl Genet 98(1):107–112. CrossRefGoogle Scholar
  50. Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155(2):945–959. CrossRefPubMedPubMedCentralGoogle Scholar
  51. Rosenberg NA (2004) DISTRUCT: a program for the graphical display of population. Mol Ecol Notes 4(1):137–138CrossRefGoogle Scholar
  52. Sambrook J, Fritsch F, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold spring Laboratory, Cold Spring HarborGoogle Scholar
  53. Shanjani PS, Mardi M, Pazouki L, Hagidimitriou M, Avanzato D, Pirseyedi SM, Ghaffari MR, KhayamNekoui SM (2009) Analysis of the molecular variation between and within cultivated and wild Pistacia species using AFLPs. Tree Genet Genomes 3:447–458CrossRefGoogle Scholar
  54. Smith JSC, Chin ECL, Shu H, Smith OS, Wall SJ, Senior ML, Kresovich S, Ziegle J (1997) An evaluation of the utility of SSR loci as molecular markers in maize (Zea mays L.): comparisons with data from RFLPs and pedigree. Theor Appl Genet 95(1–2):163–173. CrossRefGoogle Scholar
  55. Stracke R, Werber M, Weisshaar B (2001) The R2R3-MYB gene family in Arabidopsis thaliana. Curr Opin Plant Biol 4(5):447–456CrossRefPubMedGoogle Scholar
  56. Sun Q, Yang X, Li R (2014) SCAR marker for sex identification of Pistacia chinensis Bunge (Anacardiaceae). Genet Mol Res 13:1395–1401. CrossRefPubMedGoogle Scholar
  57. Talebi M, Kazemi M, Sayed-Tabatabaei BE (2012) Molecular diversity and phylogenetic relationships of Pistacia vera, Pistacia atlantica sub sp. Mutica and Pistacia khinjuk using SRAP markers. Biochem Syst Ecol 44:179–185. CrossRefGoogle Scholar
  58. Talebi R, Nosrati S, Etminan A, Naji AM (2018) Genetic diversity and population structure analysis of landrace and improved safflower (Cartamus tinctorious L.) germplasm using arbitrary functional gene-based molecular markers. Biotechnol Biotechnol Equip 32(5):1183–1194. CrossRefGoogle Scholar
  59. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739. CrossRefPubMedPubMedCentralGoogle Scholar
  60. Tavalleli V, Rahemi M (2007) Effects of rootstock on nutrient acquisition by leaf, kernel and quality of pistachio (Pistacia vera L.). J Agric Environ Sci 2(3):240–246Google Scholar
  61. Topçu H, Çoban N, Kafkas S (2016) Novel microsatellite markers in Pistacia vera L. and their transferability across the genus Pistacia. Sci Hortic 198:91–97. CrossRefGoogle Scholar
  62. Turkeli Y, Kafkas S (2013) First genetic linkage map in pistachio constructed using an interspecific cross between Pistacia vera L. and monoecious Pistacia atlantica Desf. Sci Hortic 151:30–37. CrossRefGoogle Scholar
  63. Wang X, Fan H, Li Y, Sun X, Sun X, Wang W, Zheng C (2014) Analysis of genetic relationships in tree peony of different colors using conserved DNA-derived polymorphism markers. Sci Hortic 175:68–73. CrossRefGoogle Scholar
  64. Xie Z, Zhang ZL, Zou X, Huang J, Ruas P, Thompson D, Shen QJ (2005) Annotations and functional analyses of the rice WRKY gene superfamily reveal positive and negative regulators of abscisic acid signaling in aleurone cells. Plant Physiol 137(1):176–189CrossRefPubMedPubMedCentralGoogle Scholar
  65. Yakubov B, Barazani O, Golan-Goldhirsh A (2005) Combination of SCAR primers and Touchdown PCR for sex identification in Pistacia vera L. Sci Hortic 103:473–478. CrossRefGoogle Scholar
  66. Yeh FC, Yang RC, Boyle T (1999) POPGENE Version 1.32: microsoft window-based freeware for population genetics analysis. University of Alberta, EdmontonGoogle Scholar
  67. Zohary M (1952) A monographical study of the genus Pistacia. Palest J Bot Jerus Ser 5:187–228Google Scholar

Copyright information

© Prof. H.S. Srivastava Foundation for Science and Society 2019

Authors and Affiliations

  1. 1.Laboratoire de Génétique Moléculaire, Immunologie et Biotechnologie (LR99ES12), Faculté des Sciences de TunisUniversité de Tunis El ManarTunisTunisia
  2. 2.Laboratoire de Génétique, Biodiversité et Valorisation des Bioressources (LR11ES41), Institut Supérieur de Biotechnologie de MonastirUniversité de MonastirMonastirTunisia

Personalised recommendations