научная статья по теме MOLECULAR CHARACTERIZATION OF SYRIAN DATE PALM CULTIVARS USING PLASMID-LIKE DNA MARKERS Биология

Текст научной статьи на тему «MOLECULAR CHARACTERIZATION OF SYRIAN DATE PALM CULTIVARS USING PLASMID-LIKE DNA MARKERS»

ГЕНЕТИКА, 2012, том 48, № 2, с. 270-274

КРАТКИЕ СООБЩЕНИЯ

УДК 575.1:582.545.2

MOLECULAR CHARACTERIZATION OF SYRIAN DATE PALM CULTIVARS

USING PLASMID-LIKE DNA MARKERS

© 2012 г. N. Haider, I. Nabulsi

Department of Molecular Biology and Biotechnology, AECS, P.O. Box 6091, Damascus, Syria

e-mail: ascientific@aec.org.sy Received February 24, 2011

Date palm (Phoenix dactylifera L.) is one of the most important domesticated fruit trees in the Near East and North African countries. This tree has been, for several decades, in serious threat of being completely destroyed by the "Bayoud" disease caused by Fusarium oxysporum f. sp. albedinis. In this study, 18 Syrian date palm cultivars and four male trees were analyzed according to the identity of mitochondrial plasmid-like DNAs. A PCR strategy that employs plasmid-like DNAs-specific primer pair was used. These primers amplify a product of either 373-bp or 265-bp that corresponds to the S- (Bayoud-susceptible) or the R-plasmid (Bayoud-resistant), respectively. Generated data revealed that only six cultivars ('Medjool', 'Ashrasi', 'Gish Rabi', 'Khineze', and yellow- and red-'Kabkab') have the S-plasmid, suggesting their susceptibility to the fusariosis, while the remaining 12 cultivars and the four male trees contain the R-plasmid, suggesting their resistance to the fusariosis. The PCR process applied here has been proved efficient for the rapid screening for the presence of the S and R DNAs in Syrian date palm. PCR markers developed in this study could be useful for the screening of date palm lines growing in the field. The availability of such diagnostic tool for plas-mid characterization in date palm would also be of great importance in establishing propagation and breeding programs of date palm in Syria.

Dates are dioecious and perennial monocotyledon fruit trees that belong to the family of Arecaceae [1]. Date palm (Phoenix dactylifera L.) (2n = 2x = 36) is one of the most important domesticated fruit tree in the Near East and North African countries [2]. It is mainly cultivated in arid regions of the Middle East, where it has been domesticated for at least 5000 years [3]. Date palm covers a surface of about 800000 ha and is important for the life of about 100 million inhabitants [4], and has therefore a great socio-economic importance [5]. Date palm is an important tree in Syria, where the cultivation area exceeds 369 ha in 2009 (FAO statistics) and the number of productive trees reached 72.600 trees [6]. It is mainly distributed in the North Eastern regions and some inland areas of Syria.

Date palm has been, for several decades, in serious threat of being completely destroyed by the "Bayoud" disease [2]. The organism that causes this disease is an imperfect filamentous microscopic fungus, Fusarium oxysporum f. sp. albedinis (Killian and Maire) Gordon, which belongs to the mycoflora of the soil [7]. Since Bayoud disease was first reported, in Zagora-Morocco in 1870, it destroyed and still destroying the world's most renowned cultivars that are susceptible to the disease, particularly those which produce high quality and quantity fruits (e.g. 'Medjool' and 'Deglet Noor') [8], particularly in North African countries [9].

Molecular markers that are correlated with date palm resistance to Bayoud have been reported based on isozymes [e.g. 10], polyphenolics [e.g. 11] and plasmid-like DNAs (discussed below, e.g. [9]).

In addition to the main mitochondrial genome, one or more kinds of small plasmid-like DNAs (circular or linear double stranded DNA molecules) have been identified and characterized in the mitochondria of some higher plants such as rice, sugarbeet, Vicia fa-ba (see [12]) and Paramecium caudatum [13]. The significance of plasmid-like DNAs to the plant is not known, and they can be lost without obvious effects on viability or appearance [14].

Benslimane [15] identified and characterized two minicircular plasmid-like DNAs, in date palm mitochondrial DNA (hereafter mtDNA). The authors revealed that these DNAs did not show any homology to nuclear, chloroplastic or main mitochondrial genomes, and that no major homology was observed between them and the other higher plant plasmid-like DNAs reported so far. Although those plasmids, referred to as the S- (1454-bp) and R-plasmids (1345-bp), are of 98.8% sequence similarity [9], Benslimane et al. [16] revealed that a 109-bp sequence is only present in the S-plasmid. The two plasmids were found in the mitochondria of two Moroccan cultivars, the first cultivar was Bayoud-susceptible and contained the S-plasmid, while the second contained the R-plasmid and was Bay-oud-resistant. This suggested that the S- and R- plasmids could be correlated with date palm susceptibility/resistance to Bayoud disease [2].

Quenzar et al. [9] also proposed that the presence of either plasmid in the mtDNA could be considered as an early molecular marker linked to susceptibility or resistance to Bayoud disease in date palm. Therefore,

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they developed a PCR strategy that involved two oligonucleotide primers (olil and oli2) which hybridize to sequences located 128 nucleotides upstream of and 137 nucleotides downstream from the 109-bp sequence to amplify either a 373-bp fragment, in the case of the S-plasmid, or a 265-bp fragment, in the case of the R-plasmid (Fig. 1).

Trifi et al. [18] demonstrated that there is a great correlation between the type of the mitochondrial plasmid-like DNAs and the date palm trees comportment within Bayoud. The use of the PCR strategy developed by Quenzar et al. [9] allowed a rapid, reliable and efficient identification of selected Bayoud-resis-tant individuals of Tunisian [2] and Mauritanian [17] date palm cultivars. Therefore, an attempt was made in this study to analyze, for the first time, 18 date palm cultivars and four male trees of the Syrian date palm germplasm with respect to the identity of hosted plas-mid-like DNA (R or S) using the PCR strategy developed by Quenzar et al. [9].

A set of 18 native and introduced Syrian date palm cultivars and the best performing four native male palm trees (Table) was screened for plasmid-like DNA-based markers. The plant material consisted of young leaves collected from adult trees in Al-Boukamal date palm production center for all male trees and cultivars except for 'Deglet Noor', that was obtained from a private orchid in Al-Boukamal city. The leaves were washed three times with sterile distilled water, immersed in liquid nitrogen, and kept at —60°C until use.

Total cellular DNA was extracted from the frozen leaves according to the CTAB method of Doyle and Doyle [19]. Integrity of extracted DNAs was detected through the electrophoresis of those DNAs on 1.8% agarose (Q. BlOgene) gel. DNA was quantified using a Gene Quant spectrometer (Amersham Biociences, UK) and a working concentration of each DNA sample was set at 10 ng/pl.

In order to evidence the R- and S-plasmids in date palm samples analyzed, PCR was performed in 0.2 ml microtubes (Greiner Bio-One, USA) using primers oli1 and oli2 (5'-ccttataccagtcgtgctt 3'- and 5'-aaggca-gatataatcgga 3', respectively). Each reaction contained 10x PCR buffer (Eurobio), 10x MgCl2 (50 mM) (Eurobio), forward and reverse primers (50 pM each) (In-vitrogen), dNTPs (1 mM) (Mix Roche), and Taq polymerase (1.5 U) (Eurobio). DNA was added to each PCR reaction at a rate of 40 ng, and the total volume was adjusted with dd H2O to 25 pi. Using Eppendorf thermocycler, PCR reactions were subjected to a cycle of 94°C for 5 min, followed by 35 cycles each of which consisted of 94°C for 30 s, 50°C for 1 min and 72°C for 2 min. A final extension cycle was performed at 72°C for 2 min. At least ten replicates were prepared for each PCR reaction.

Aliquots of10 pl of each PCR product were loaded and electrophoresed on 1.8% ethidium bromide (Flu-ka)-stained agarose gels in 0.5x Tris Borate EDTA

S amplimer (373 bp)

3'-3'

5'-5'

PCR

t

P2

S-plasmid

109 bp--

3' 5'

P1

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R-plasmid 53''

P1

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R amplimer (256 bp)

Fig. 1. Illustration of the designed strategy to amplify the region of interest in the R- and S-plasmid-like DNAs in the mitochondria of date palm [17]. The 109-bp stippled box delimits the region ofthe S-plasmid which is not found in the R-plasmid. P1 and P2 refer to the forward and reverse primers, respectively.

(TBE). A 100-bp DNA Ladder (Fermentas) was used to estimate the approximate molecular weight of amplification products. Electrophoresis was performed at 85 V for 2.30 h, and amplification profiles generated were photographed under UV light.

Names of Syrian date palm cultivars and male trees used, their geographical origin (as provided by the Ministry of Agriculture) and types of plasmid-like DNAs they possess

Number Male/cultivar name Geographical origin Plasmid type

1 Deglet Noor Tunisia R

2 Medjool (Mujhoolah) Morocco S

3 Zahidi Syria, Iraq, Iran R

4 Birbin Syria, exists in Iraq R

5 Ashrasi Syria class 1 S

6 Maktoom Syria R

7 Khastawi Syria R

8 Barhee KSA R

9 Khalas KSA R

10 Khadrawy KSA R

11 Nabtat-seyf KSA R

12 Lolo UAE R

13 Gish Rabi UAE S

14 Khineze UAE S

15 Zaghloul Egypt R

16 Shahabi Iran R

17 Kabkab (yellow) Iran S

18 Kabkab (red) Iran S

19 Male 1 Syria R

20 Male 2 Syria R

21 Male 3 Syria R

22 Male 4 Syria R

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Fig. 2. Amplification products generated for S- (373-bp) and R-plasmids (265-bp) from total cellular DNA of the 18 date palm cultivars and the four male trees analyzed. Arrows indicate to the position of the R- and S-plasmids.

When DNA samples were electrophoresed on agarose gels, their integrity was confirmed since high molecular mass bands without any contamination or degradation were ob

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