.MOLECULAR CHARACTERIZATION OF TUNISIAN DATE

PALM VARIETIES

TRIFI Mokhtar (1),BENSLIMANE AbdelAIi (2),RHOUMAAbdelmajid(3) RODE Andre(4) and MARRAKCHI Mohamed (5)

(1): Assistant Professor, Laboratoire de Genetique Moleculaire,Faculte des Sciences de Tunis, Campus Universitaire, 1060 Tunis LeBelvedere, Tunisie

(2):Professor, Faculte des Scineces Semlalia, Marrakech, Maroc

(3): Director of Centre de Recherches Phoenicicoles, INRA Degache,Tunisie

(4):Professor, IBP, Bat 630, Universite Paris -Sud, France

(5): Professor, Laboratoire de Genetique Moleculaire, Faculte desSciences de Tunis, Campus Universitaire, 1060 Tunis Le Belvedere,Tunisie

ABSTRACT

The vascular fusariosis (bayoud disease) is currently destroying theNorth African date palm plantations. Until now, Tunisian groves appearto have been spared. However, they are continuously menaced by thisdisease due to its rapid propagation into the East. Thus many strategieshave been developed aiming at the molecular characterization of Tunisiandate palm varieties and the elaboration of a preventive procedure for theirprotection by the help of RAPD and mitochondrial plasmid-like DNAsmarkers.

As a first step, we have used a large number of universal primers toamplify bzagments of each DNA. Some of them are retained as potentialmarkers for identifying Tunisian date palm varieties and ecotypes.

In a second step, we have amplified mitochondrial plasmid-likeDNAs employing to PCR procedure using appropriate oligonucleotides.It is now established that this technique could be currently used as anefficient method of screening bayoud-resistant varieties due to the greatcorrelation between these molecules and the date-palm trees comportmentwithin fusariosis.

Additional Index Words: Phoenix dactyli/era, bayoud disease, PCRand RAPD

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INTRODUCTION

For several decades, most North Atrican date palm (Phoenixdactylifera) plantations are seriously threatened by a vascular fusariosis(bayoud disease) due to the fungus: Fusarium oxysporum albedinis . InMorocco as in west Algeria, several varieties are resistant to bayouddisease. However, they have a poor date quality (Saaidi, 1992; Sedra,1992). Thus, works aiming at the screening ofbayoud-resistant varietieswith a good date quality is currently in progress aiming at the protectionof date palm groves in these countries. Until now, Tunisian plantationsappear to have been spared. However, they are continuously menaced bythis fusariosis due to its rapid spread into the East. In order to elaborate apreventive fighting strategy for Tunisian date palm varieties, manystrategies have been developed aiming at their molecular characterization.

A large number of reports have described molecular biologymethods for the detection of plant DNA polymorphism: RestrictionFragment Length Polymorphism, (RFLP) Polymerase Chain Reaction(PCR), Random Amplified Polymorphic DNA (RAPD), AmplificationFragment Length Polymorphism (AFLP), Multiple Arbitrary AmpliconPolymorphism (MAAP) (Caetano-Anolles, 1994; Devos and Gale, 1992;D'ovido et aI., 1990; Kiss et aI., 1993; Miller and Tanksley, 1990;Thormann and Osborn, 1992; Tinker et aI., 1993; Welch et aI., 1991;Williams et aI., 1992). So far, the most used techniques seems to be PCRand RAPD (Chong et aI., 1994; Lowe et aI., 1996; Saiki et aI., 1985;Weining and Langridge, 1991; Williams et aI., 1991). These procedures,due to their great sensitivity, constitute a powerful technique widely usedfor enzymatic amplification of stretches trom small amounts of DNA andprovide an alternative approach to distinguish genotypic variants.Therefore, the molecular characterization of Tunisian date palm varietieswas investigated with the help of these procedures. As a short-termobjective, it has been assumed that these procedures allow us to knowhow this molecular polymorphism is associated to bayoud-resistance.And, as a long-term objective, to know how such strategy could provide auseful early molecular marker related with bayoud-resistance. Benslimaneet aI. (1994) and Benslimane (1995) reported the presence of plasmid-likeDNAs in date palm mitochondria and molecularly characterized theseminicircular molecules called S (1 454 bp) and R (1 346 bp) plasmid-likeDNAs. Both forms were detected in bayoud-sensitive and bayoud-resistant trees, respectively. Sequence examination between Rand Sdemonstrates that S shares a very strong homology with R except for adeletion of 109 bp (trom 1 145 to 1 253). Thus, it has been suggested thatthese two plasmids seem to be correlated with the phenotype of datepalms. Up to now, correlation between this minicircular polymorphismand bayoud-resistance or sensitivity has not been clearly established.

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Within this scope, we have decided to extend a similar study on Tunisiandate palm varieties and to establish the possible correlation between thesemolecular markers and bayoud-resistance.

Here, we report the use of RAPD and PCR procedure as powerfultechniques aiming at the molecular characterization of Tunisian date palmvarieties and the screening of S and R plasmids from a set of Tunisian setof female and male date palms.

MATERIALS AND METHODS

Plant material:

We have used a set of date palm varieties characterized by theirgood fruit quality. The material (young leaves) was kindly provided bythe «Centre de Recherches Phoenicicoles, INRA, Degache», south ofTunisia. The commercial nomenclature of the varieties included in thisstudy has been conducted according to Rhouma (1994).

DNA preparation:

Nucleic acids were prepared from frozen leaves of adult trees. Totalcellular DNA was extracted according to Aitchitt et al. (1993) withseveral modifications. After purification, the DNA was quantified usingGene-Quant spectrophotometer (Pharmacia) and its quality wasdetermined by agarose minigel electrophoresis as described by Maniatiset al. (1982).

RAPD analysis:

For RAPD analysis, a large number of universal primers (decamers)purchased from Operon Technologies Inc. (California, USA) were used.These were OPA, OPB, OPD, OPF, OPG, OPI, OPK, OPM and OPN.The G+C content of all primers is about 60%.

A 25 III reaction mixture is used containing: 20 ng total cellularDNA, 50 pM (I Ill) primer, 0.25 IIIof 10X Taq DNA polymerase reactionbuffer, 1.5 U (0.3 Ill) Taq DNA polymerase (Appligene-Oncor, France),200 mM each dNTP (DNA polymerization mix, Pharmacia). Thereaction mixture was covered with 25 IIIof sterile mineral oil to avoidevaporation. Amplification was then performed in a thermal cycler (bio-med GmbH, thermocycler 60) using the following conditions: sampleswere first heated at 94°C for 5 minutes before entering 35 cycles PCRprocedure of 94°C for 30 seconds, 35°C for 1 minute and 72°C for 1minute. A final delay phase of 72°C for 5 minutes was always included.

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To reduce the possibility of cross contaminati0I!and variation in theamplification reactions, master-mixes of the reaction constituents werealways used. Standardisation between enzyme batches and experimentswas ensured by including a standard control: a first control consists ofreaction mixture excluding any DNA whilst a second control was reactionmixture including DNA without any enzyme or primer.

Amplified fragments were electrophoresed in 1.4 % TBE agarosegels and detected by ethidium bromide (1 Ilg/1)staining,accordingtoSambrook et al.,(1989). .

PCR analysis:

For PCR analysis, two appropriate primers were used. These encloseplasmid S sequence flanking the 109 bp deletion.

A 100 III PCR reaction mixture is used containinx: 20 ng (1 Ill) oftotal cellular DNA, 50 pM (2 Ill) of each primer, 10 IIIof 10 Taq DNApolymerase reaction buffer, 1.5 U (0.3 Ill) of Taq DNA polymerase(Appligene-Oncor, France), 200 mM of each dNTP (DNA polymerizationmix, Pharmacia). The reaction mixture was covered with 100 IIIof sterilemineral oil to avoid evaporation. PCR was then performed in thermalcyder (bio-med GmbH, thermocycler 60) using the following conditions:samples w~re first heated at 94°C for 5 minutes before entering 35 cyclesPCR procedure of 94°C for 30 seconds, 48°C for 1 minute and 72°C for 2minutes. A final delay phase of 72°C for 10 minutes was always included.

In each set of amplification a number of controls were also included.One was a reaction mixture excluding any DNA whilst a second controlwas reaction mixture including DNA without any enzyme.Standardisation between enzyme batches and experiments was ensured byincluding in each a standard PCR amplification ofR and/or S DNA.

PCR products were resolved by electrophoresis in 1.5 % TBEagarose gels and stained with ethidium bromide (1 Ilg/ 1)according toSambrook et al.,(1989).

RESULTS AND DISCUSSION

RAPD patterns analysis:

In this study, we have screened about 200 universal primers. Thesecould be regrouped in three different classes: the first constitutes theprimers unable to generate any stretches from total cellular DNAextracted from any date palm variety; the second is composed of primersgenerating amplified products from DNAs from several varieties only,and the third correspond to those able to amplify subfragments using

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DNAs isolated trom any variety. Our data indicated that several DNAsegments were amplified in each sample and polymorphisms wereapparent for a set of primers. These, based either on the number or thesize of the amplified DNA products, allow potential genetic markerspermitting to distinguish a given variety trom the others. Computerassisted analysis of RAPD profiles with the help of appropriate softwarewill reveal on the average linkage clusters of all varieties and ecotypes.At present, work is in progress to resolve this problem.

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Figure I: Example of DNA tragments randomly amplified using universalprimers and resolved by electrophoresis in 1.4% agarose. M: I Kb ladder(BRL) Mp!. weight standard controls included: reaction mixture withoutany DNA (Cl) and reaction mixture without any enzyme (C2). Panelsshow the amplified products using OPDI8 (1A), OPD20 (lB) and OPAOl(I C).

PCR analysis and evidence of the presence of Rand S plasmid-like in Tunisian date palm varieties:

We first attempted to determine whetlJ.erit was possible to develop aPCR analysis using plasmid-like DNAs isolated according to aminipreparation of alkaline described by Maniatis et a!. (1982). Thusexperiments were performed using for each Rand/or S DNA varyingannealing temperature (trom 38 to S2°C). It has been established that theoptimal amplification was obtained when using 48°C as annealingtemperature in our conditions.

As a rapid screening procedure for detection of amplifiedsubtragments corresponding to S or R DNAs in target DNA, total cellularDNAs was isolated trom several Tunisian date palm varieties

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characteriezed by their good date quality. Obtained results have beensummarized in table 1. The analysis of which indicates that:

* The presence of different amplified products in all varietiessuggesting that these plasmid-like DNAs are present in mitochondria ofdate palm (Phoenix dactylifera).

* 7 female varieties considered as bayoud-sensitive (Saaidi, 1992;Sedra, 1992), present S subftagment in the resultant PCR patterns. Theseare: Boufagous, Kenta, Kentichi, Ftlmi, Khouat-Ftimi, Ghondi andBesser-Hlou.

* Deglet Nour, Zehdi, Ghars-Metig and Hlaoui, . sharing Rsubfragments, constitute bayoud-resistant varieties if noting theavailability of correlation hypothesis. However, for the Deglet Nourvariety, possessing R subfragment, our data seem to be discordant in spiteof its sensitive character. So far, Deglet Nour has been considered as anintermediate variety among those which are completely bayoud-sensitiveand completely bayoud-resistant varietiess (Saaidi, 1992).

* Surprisingly, as Horra's PCR pattern, several varieties showsimultaneously both of subftagments R and S in the PCR amplifiat.

Table 1: Phenotype and plasmid patterns of the tunisian date palmvarieties included in the study. The sign of + indicate the greatness of therelative amount.

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Commerciar - Phenotype Plasmid Relative amountsname

Boufagous Sensitive SFtimi Sensitive SKhouat-Ftimi Sensitive SKenta Sensitive SKentichi Sensitive SGhondi Sensitive SBesser-Hlou Sensitive SDeglet-Bey ? SDeglet-Nour Sensitive RZehdi ? RGhars-Metig ? RHlaoui ? RHourra Sensitive SIR S.++. R.++. , .Arichti ? SIR S.+++. R.+. ,.Rhimia ? SIR S.++.R.+++. , .Tronja ? SIR S.+++.R.+. ,.

The following explanations \vere considered for these results:

(i) Both R and S plasmids could be present in an mitochondria withgreatly varying relative amounts. This fact is strongly supported: first, Sand R DNAs seem to have a common DNA ancestor suggested by theirextended sequence homology (Benslimane, 1995); second, they mayderived from each other through direct repeated sequences recombinationas established by Benslimane et aI. (1996), and third, they may bemaintained within a given variety in a variable arrangement.

(H) For the varieties sharing only S Plasmid DNA, inhibition ofrecombination events due to nuclear genes control could maintain thisshape in mitochondria. However, through direct repeated sequence,possible recombination events allow for an R minicircle and a stretch of109 Bp. This fragment, without a replication origin, will be lost. Thus,PCR analysis shares only subfragments corresponding to R plasmid.Search of subfragment corresponding to the deletion of 109 bp couldprovide evidence of this argumentation.

(Hi) The evolution of bayoud-sensitive phenotype to resistancecould be accompanied by conversion of plasmid-like S to R minicircle.Thus to reach a fixed equilibrium a great number of intermediatesituations could have occurred. Our data effectively reflect several formsregarding the different amounts of amplified subfragments Sand R froma given target DNA (Figure 2).

(iHi)Regarding the variable amounts of the generated subfragments,it seems to be ensured the hypothesis of existence of common featuresbetween the mitochondrial plasmid-like molecules and the mainmitochondrial genome in higher plants (for example, structural forms andrecombination) Arganoza and Akins, 1995; Debets et aI.; 1995 Flamandet aI., 1993).

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M Cl C2 C3 S S R S S SR. S S. RfS RfS

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Figure 2: Example of resultant PCR plasmid patterns from total cellularDNA templates extracted from good date quality varieties. M: 1 Kbladder (BRL) standard size; controls included: reaction mixture withoutany DNA (Cl) and standard PCR amplification ofR (C2) and S (C3); Rlabelling varieties possessing subfragment of R alone; S those withsubfragment of S alone, SIR varieties with R and S subfragmentssimultaneously. Fragment sizes are in bp; lanes labelled with n* indicatevarieties tested with the fungus.

CONCLUSION

In this work, we have described the importance of RAPD and PCRprocedure as current methods aiming at the molecular characterization ofa set of Tunisian date palm varieties. As a first step, a large number ofuniversal random primers were screened. It has been established thatsome of them could be retained as potential markers for date palmvarieties identification. The use of a large number of primers and othervarieties allows an efficient tool for genetic polymorphism analysis inthese varieties and ecotypes.

As a second step, for PCR analysis using appropriateoligonucleotides, we can routinely generate subfragments correspondingto Rand S DNAs. The resultant PCR products shows three differentplasmidic patterns. Several possible explanations based on recombinationwere considered for this polymorphism. Work is currently in progresseither to provide evidence of this argumentation or to elucidate theinterest of identifying plasmid-like in date palm mitochondria in relationwith bayoud-disease.

ACKNOWLEDGEMENTS

Support for this research was provided in Tunisia by grants from the« Ministere de l'Enseignement Superieur », the « Secretariat d'Etat CllaRecherche Scientifique et Clla Technologie » and in France by the CNRS(Institut de Biotechnologie des Plantes, Universite Paris-Sud) and the« Institut Franfais de Cooperation»

LITERATURE CITED

Aitchitt M., C.C. Ainsworth, and M. Thangavelu. 1993. A rapid andefficient method for extraction of total DNA from mature leaves of datepalm (Phoenix dactylifera L.). Plant Mol. BioI. Reptr. 11 (4): 317-319.

Arganoza M.T. and R.A. Akins. 1995. Recombinant mitochondrialplasmids in Neurospora composed of Varkud and a new multimericmitochondrial plasmid. Curr. Genet. 29: 34-43

190

- -- - - ---

Benslimane A.A. 1995. Plasmides mitochondriaux du palmierdattier (Phoenix dactylifera L.). Mise en evidence et caracterisationmoleculaire.These de Doctorat d'Etat, Universite Cadi Ayed,Faculte desSciences Semlalia, Marrakech, Maroc.

Benslimane A.A., A. Rode, F. Quetier, and C. Hartmann. 1994.Characterization of two minicircular plasmid-like DNAs isolated fromdate palm mitochondria. Curr. Genet. 26: 535-541.

Benslimane A.A., C. Hartmann, B. Ouenzar, and A. Rode 1996.Intramolecular recombination of a mitochondrial minicircular plasmid-like DNA of date palm medaited by a sef of short direct-repeat sequences.Curr. Genet. 29: 591-593.

Caetano-Anolles G. 1994. MAAP: a versatile and universal tool forgenome analysis. Plant Mol BioI 25: 1011-1026.

Chong D. K.X., R.C. Yang, and F.C. Yeh. 1994. Nucleotidesdivergence between populations of trembling aspen (Populustremuloides) estimated with RAPD. Curr. Genet. 00: 374 - 376.

Debets F., X. Yang, and A.J.F. Griffiths. 1995. The dynamics ofmitochondrial plasmids in Hawaiian population of Neurosporaintermedia. Curr. Genet. 29: 44-49.

Devos K.M. and M.D. Gale. 1992. The use of random amplifiedDNA markers in wheat. Theor. Appl Genet. 84: 567-572.

D' ovido R., O.A. Tanzarella, and E. Porceddu. 1990. Rapid andefficient detection of genetic polymorphism in wheat throughamplification by polymerase chain reaction. Plant Mol. BioI. 15: 169-171.

Flamand M.C., G. Duc, J.P. Goblet, L. Hong, O. Louis, M. Briquet,and M. Boutry. 1993. Variant mitochondrial plasmids of broad bean aroseby recombination and are controlled by the nuclear genome. Nuc AcidsRes. 21-23: 5468-5473.

Kiss G.B., G. Csanada, K. Kalman, P. Kalo, and L. Okresz. 1993.Construction of a basic map for alfalfa using RFLP, RAPD, isozyme andmorphological markers. Mol. Gen. Genet. 238: 129-137.

Lowe A.J., O. Hanotte, and L. Guarino. 1996. Standardization ofmolecular genetic techniques for the characterization of germaplasmcollections: the case of random amplified polymorphic DNA (RAPD).Plant genetic Ressources Newsletter 107: 50 - 54.

191

1>1aniatis S., E.F. Fritisch, and J. Sambrook. 1982. Molecularcloning: a laboratory manuel. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, New York.

Miller J.C. and S.D. Tanksley. 1990. RFLP analysis ofphylogeneticrelationships and genetic variation in the genus Lycopersicon. Theor.Appl. Genet. 80: 437- 448.

Rhouma A. 1994. Le palmier dattier en Tunisie. I. Le patrimoinegenetique. Arabesques Editions et Creations, Tunis.

Saaidi M. 1992. Comportement au champ de 32 cultivars de palmierdattier vis cl vis du bayoud: 25 annees d' observations. Agronomie 12:359-370.

Saiki R.K., S. Scharf, F. Faloona, K.B. Mullis, G.T. Horn, H.A.Erlich, and N. Rnheim 1985. Enzymatic amplification of J3-globingenomic sequences and restriction site analysis for diagnosis of sicklecell anemia. Science 239: 1350-1354.

Sambrook J., E.F. Fritisch, and T. Maniatis. 1989. Molecularcloning: a laboratory manual. 2d ed. Cold Spring Harbor Laboratory,Cold Spring Harbor, New York.

Sedra M.H. 1992. Evaluation and selection of cultivars and clones of

date palm for resistance to the bayoud disease. Arab J. Plant Prot. 10 (2):155-160.

Thormann C.E., and T.C. Osborn 1992. Use of RAPD and RFLPmarkers for germaplasm evaluation. in: Proceedings of Applications ofRAPD Technology to Plant Breeding, Minneapolis, Minn. eds. CropScience Society of America, Inc. Madison, Wis, pp: 9-11

Tinker N.A., M.G. Fortin, and D.E.Mather. 1993.Random amplifiedpolymorphic DNA and pedegree relationships in spring barley. Theor,Appl, Genet, 85:976-984.

Weining S., and P. Langridge 1991. Identification and mapping ofpolymorphism in cereals based on the polymerase chain reaction. Theor.Appl. Genet. 82: 209-216.

Welch 1., R.J. Honeycutt, M. McCelland, and B.W.S. Sobral, 1991.Parentage determination in m~ize hybrids using the arbitrary primedpolymerase chain reaction. Theor, Appl, Genet, 82: 473-476.

192

--

Williams J.G.K., A.R. Kubelik, K.J. Huak, K.J. Rafalski, and S.Y.Tingey, 1991. DNA polymorphisms amplified by arbitrary primers areuseful as genetic markers. Nucleic Acids Res, 18:653'1-6535.

Williams J.G.K., M.K. Hanafey, J.A. RafalskI, and S.Y. Tingey.1992. Genetic analysis using RAPD markers. in : Recombinant DNAvolume. Meth in Enzymol. .

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