Molecular Phylogeny Of Qatari Date Palm Genotypes Using Simple Sequence Repeats Markers

MATERIALS AND METHODS

Plant material: Fifteen Qatari Date palm cultivars derived from mature in vitro culture were used in this study (Table 1). These cultivars are now 10 years old and are grown in Qatar University Experimental Farm at the North part of Doha. They are considered to be the most common Date palm cultivar in Qatar. One tree representing each cultivar was chosen randomly and used for leaf sampling.

DNA extraction: Young leaf samples were cleaned carefully to remove the waxy layer. Two hundred to three hundred milligrams of leaf sample was cut in small pieces and grinded in liquid nitrogen in a cold Mortar and Pestle (kept at -20 °C over night).

Dneasy Plant Mini kit (Qiagen) was used to extract DNA from the Qatari Date palm leaf samples according to the manual instructions of the kit (DNeasy Plant Handbook). Obtained DNAs were quantified and qualified by using agarose gel electrophoresis. Two microliter of DNA from each sample were applied to 0.85% Agarose gel and electrophoreses was done at 100V for 30 min. The gels were stained in Ethidium bromide and visualized under UV light.

PCR and Microsatellites (SSR) amplification: A set of 16 date-palm specific SSR primer pairs developed by Billotte et al. (2004) (Table 2) was tested. PCR reactions were performed in a total reaction mixture of 25 μL containing: 20-30 ng of total genomic DNA (1 μL) as template, buffer (GeneAmp, Applied Biosystems), 0.2 mM of dNTP PCR mix (GeneAmp, Applied Biosystems), 0.625 U of Taq DNA polymerase (AmppliTaq, Applied Biosystems) and 0.2 mM of primers. Amplifications were performed in a GeneAmp PCR System 9700 Thermocycler, with the following conditions: a denaturation step of 5 min at 95 °C followed by 35 cycles of 30 sec at 95 °C, 90 sec at 52-60 °C and 90 sec at 72 °C and a final extension step at 72 °C for 7 min. The amplified DNA fragments were separated on 2% agarose gel and stained with ethidium bromide. The amplified pattern was visualized on a UV transilluminator and photographed using Gel documentation system.

Data analysis: Microsatellite bands were precisely measured by Gel documentation System software and scored for each genotype. Each reproducible polymorphic DNA band at particular position on the gel was treated as a separate character and scored as present (1) or absent (0) to generate a binary data matrix.

Data were then computed with the SPSS program to produce a genetic distance matrix which assesses the similarity between any two populations on the basis of the number of generated bands using Jaccard’ s similarity coefficient (Jaccard, 1908). The matrix was then computed to draw phylogenetic diagrams.

Table 1:	Names of the studied fifteen qatari date palm genotypes

Table 2:	List of SSR primers used in this study developed for Date palm by Billotte et al. (2004)

RESULTS AND DISCUSSION

Among the 16 primer pairs tested for their ability to generate expected SSR banding patterns in Qatari data-palm genotypes, 10 primers successfully produced clear single bands in most of the studied genotypes. So, far, six SSR primers did not amplify clear bands in our genetic materials even using different PCR conditions.

The amplified SSR band sizes ranged from 100-300 bp. A total of 40 alleles with a mean of 4 alleles per locus were scored. The number of alleles per locus varied from 3 (primer 3) to 6 (primer 5). All the 16 SSR primers were checked for amplification using four Date palm DNA genotypes as a first screening. Figure 1 shows an example for first screening using primers 9 and 10 and four different Qatari date palm genotypes. Subsequently, primers that showed clear bands, used to fingerprint the all 15 Qatari date palm genotypes as shown in Fig. 2 for primer 5 as an example.

Among the sixteen SSR tested primers, only ten were used to assess genetic relationships in the tested accessions. These primers which have revealed polymorphic products that were consistently and unambiguously scorable were identified as: mPdCIR016, mPdCIR025, mPdCIR032, mPdCIR035, mPdCIR050, mPdCIR057, mPdCIR078, mPdCIR085, mPdCIR090 and mPdCIR093.

Interestingly, twenty distinct unique bands were obtained to represent nine date palm cultivars. Four out of seven different sizes bands obtained from primer 3 were appeared in Hatamy, Barhee, Khadraway and Thuri. Each band represent one cultivar. In the other hand, some cultivars could be represented with single bands amplified with different primers. For example, Zahidi was represented with single bands obtained from Primer 4, 5 and 15 in 185, 302 and 155 bp, respectively. Six cultivares (Hatamy, Helaly, Sheshy, Khadrawy and Thuri) had two unique single bands from two different primers. However, one unique single bands were shown in Succary, Abu Main, Barhee, Naboot Saif and Khanezy.

Fig. 1:	First check for SSR primers No. 9 and No. 10 using four date palm DNA samples of Zahidi, Hatamy, Helaly and Khalas. M: 100 base Ladder marker

Band pattern data was converted into a binary data in excel work sheet and was analyzed using SPSS program to calculate similarity coefficient values according to Jaccard (1908). A similarity matrix between Qatari date palm cultivars (Table 3) showed an average similarity coefficient range from 0.000-0.750. The cultivars studied here were highly divergent at the DNA level. The highest similarity coefficient value was observed between Barhee and Sultana cultivars (0.750) which seem to be the nearest two varieties and can be closely regrouped. The similarity coefficient value of 0.000 was obtained between Abu Main and each of Barhee, Sultana, Khush Zabad, Khanezy and Thuri cultivars, indicating that how far is the relationship between Abu Main cultivar and those cultivars. All the other cultivars displayed low levels of similarity but still were grouped with each others.

Table 3:	Similarity values matrix among the 15 Qatari date palm cultivars, based on SSR data calculated using Jaccard’ s similarity coefficient

Fig. 2:	Examples of SSR polymorphism banding patterns in a subset of 15 Qatari date palm genotypes using primers No. 5. M: 50 bp Standard ladder marker; Lanes (1-15): Qatari date palm genotypes described in Table 1

The Jaccard similarity coefficient matrix was computed to cluster the data and to draw the precise relationships among the fifteen studied Qatari date palm genotypes. The Dendrogram shown in Fig. 3, illustrates the divergence between the studied Qatari date palm cultivars and suggests their tree branching.

Abu Main cultivar was in a separate far group compared to the rest of cultivars. Barhee and Sultana cultivars were very closed to each other and could be considered one cultivar with different name. The following cultivars: Anbara- khadrawy, Khanezy-Thuri, Hatamy-Khalas, Zahidi-Helaly may constitute paired clusters (Fig. 3).

Fig. 3:	Dendrogram of 15 Qatari date-palm cultivars based on Jaccard genetic similarity coefficient using SSR data

DNA markers are powerful tool to provide information on the relatedness of various clones or varieties that are difficult to distinguish morphologically, thus helping in the management of plant accessions and in breeding programs. Simple sequence repeat DNA markers (SSR, or microsatellite markers) is considered the method of choice due to their abundance, polymorphism and reliability compared to other types of DNA markers. However, it was only with the development of SSR markers for date palm (Billotte et al., 2004) that reliable, co-dominant and comparable molecular data on date palm populations could be generated.

The highest levels of polymorphism for SSRs system compare to other systems also reported in earlier studies (Belaj et al., 2003; Russel et al., 1997; Gomes et al., 1998; Maguire et al., 2002; Palombi and Damiano, 2002; Rajora and Rahman, 2003; Ferreira et al., 2004). This high level of polymorphism, associated with SSR markers, is to be expected because of the unique mechanism responsible for generating SSR allelic diversity by replication slippage. Replication slippage is thought to occur more frequently than single nucleotide mutations and insertion\deletion events, which generated the polymorphisms detected by RAPD analysis (Powell et al., 1996). The codominant nature of SSR markers also permits the detection of a high number of alleles per locus and contributes to higher levels of expected heterozygosity being reached than would be possible with RAPD markers.

CONCLUSION

In this study, SSR markers have been used to asses the molecular characterization and the phylogenic relationships of Qatari date palm cultivars. Present results provide evidence of a genetic diversity among the studied Qatari date Plam cultivars and the ability of SSR markers to detect the genetic diversity in date palm. We may conclude that all date-palm ecotypes are interrelated in spite of their agronomic divergence. Genetic similarities and dendrogram could re-group the Qatari date palm cultivars in a way that one cultivar (Abu Main) was excluded from the group due to its dissimilarity with the other cultivars. Two cultivars (Barhee and Sultana) were much closed and could be considered as they came from one origin. Some cultivars were grouped in different similar pairs.

ACKNOWLEDGMENTS

This research project was made possible by a grant from the Qatar National Research Fund and Qatar University Research Fund No. CAS07031. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the Qatar National Research Fund.