Studies on Oman elite date palm varieties and preliminary establishment of identity through SSR marker

2.1 Materials

Date palm leaf from Khasab (Sinaw, Bahla and Wadibanikhaled) Naghal (Sinaw, Samail and Bahla), Barni (Sinaw, Tawa and Alkamel), Khenezi (Bidiya, Tawa and Alkamil), Madloki (Ibra, Tawa and Bidiya), Nasho (Ibra, Tawa and Alkamil) and Fard (Nizwa, Samail and Sinaw), primers, DNA extraction kit, PCR kit, electrophoresis apparatus, PCR, spectrophotometer. The Date palm cultivar varieties used in the current study fruit morphology, location and specifications were specified in Table 2 and the information is from Oman encyclopedia.

3.1 Isolation of genomic DNA

Two methods were used to extract genomic DNA from seven different date palm by following the Edwards method (Edwards et al., 1991) and QIAGEN Maxi Kit method (DNEasy Plant Maxi Kit, Thermo Scientific).

3.2 Edwards’s method

DNA extraction was carried out from date palm leaf sample by following Edwards’s method by taking 20 g of plant tissue in eppendorf tubes. The plant tissue is grounded with pellet pestle by adding little sterile sand and 400 μL of extraction buffer (200 mM Tris-HCl pH 7.5, 250 mM NaCl, 25 mM EDTA, 0.5 % SDS). Grind on ice with pellet pestle until completely ground up. The mixture is centrifuged in cooling centrifuge at 4 °C for 2 min. The supernatant was collected to new Eppendorf tube (about 250 μL). One volume of ice-cold isopropyl alcohol was added and incubated in icebath for 30 min. Further Centrifuged at room temperature for 5 min. Remove the upper layer and pellet was dried. Resuspended the lower portion in 100 μL of TE buffer and stored at −20 °C.

3.3 QIAGEN Maxi Kit method

DNA extraction was carried out from date leaf sample using Maxi protocol. From each of the test sample 1 gm of leaf was weighed and grounded in liquid nitrogen using mortar and pestle. Pinch of sterilized sand was added to it. After that, total mixture is transferred in centrifuge tube, 5 ml of AP1 buffer and 10 µL of RNase is added to it. Then, the Eppendorf tubes were placed in the hot water bath for 30 min at 65 °C. After that, the sample was incubated in the ice for 5 min. 1.8 ml of AP2 was added to hold and keep DNA in upper layer. Then, it was transferred to the refrigerator for 5 min. Next, the sample was centrifuged for 5 min and the supernatant was transferred to the filter tube. Then, it was centrifuged for 5 min. Then, the supernatant was transferred to Eppendorf tube. The volume of supernatant was measured and the amount of AP3 was calculated by using this equation (volume of supernatant *1.5) and added to the Eppendorf. The sample was centrifuged for 5 min. Then, the liquid was removed and 12 ml of AW buffer was added to the filter with supernatant. After that, it was centrifuged for 10 min. Then, it was transferred to new filter tube, and it was kept open to allow the ethanol to evaporate at room temperature for 20 min. Next, 1000 μl of AE buffer it was added to the filter tube and then it was centrifuged for 5 min. Finally, the sample was stored in the refrigerator. Quantity of sample was determined by the Nano drop device and the standard of the sample was determined by gel electrophoresis.

3.4 PCR amplification

For PCR amplification the total volume of 20 μL was prepared by mixing: 12.9 μL of H2O, 50 ng of total cellular DNA (2 μL) as template, 2 μL/PCR buffer, 1 μL of 0.2 mM dNTP PCR mix, 0.1 μL (0.625 U) of Taq DNA polymerase and 1 μL of 0.2 mM of each selected primer. The PCR procedure as follows: “Denaturation step was carried out at 95 °C for 5 min followed by 35 cycles of 30 s, 1 min at 48–56 °C (Primer annealing temperature) and 1 min at 72 °C, and a final extension step at 72 °C for 7 min”. Amplified products were separated by AGE (1.2 %) based to their MW and visualized by staining with EtBr as described by (Sambrook et al., 1989). The amplified DNA was visualized using ultraviolet trans illuminator and documented by using a gel doc system. Amplifications were performed for twice and only reproducible products were considered for further data analysis.

3.5 Data analysis

For genome lab fragment analysis, Sample was prepared as following steps: 21.5 μl of sample loading solution, 2 μl standard, 1ul PCR products and drop of mineral oil were mixed together and centrifuged for few seconds. Then, it was placed in the plate and applied to the genome lab fragment analysis. Finally, data was analyzed by using the software GenAlEX to identify the percentage of dissimilarity and the “phylogenetic relationship among the genotypes on the basis of the allele’s size”.

4.1 Genetic diversity analysis

The morphological descriptors are used traditionally to detect the genetic variations in date palm germplasm. However, these descriptors are often not satisfactory and ambiguous due to various environmental factors involved in plant developmental stages (Elhoumaizi et al., 2002a,b). Further, the traditional method of detecting germplasm is also a time taking process. The advanced SSRs and SNPs, are very effective than other methods therefore they become a tool of primary consideration with many advantages with most of the population (Brumfield et al., 2003). In addition, the SNPs and SSR markers are quickly and easily used through bioinformatics tools and can be used in the detection of alleles involved in disease resistance, genomic imprinting, genetic diversity analysis, paternal assessment, forensic studies and influence of population history (Gong et al., 2011; Ashkani et al., 2012; Katti et al., 2001). SSRs are dominant molecular markers, because of their genetic codominance, large volume, present in the whole genome, multiallelic dissimilarity, high robustness and level of polymorphism (Tóth et al., 2000; Schlötterer, 2000). The high level of polymorphism is because of mutations that affects repeating unit’s numbers. Using SSR markers as tool has many advantages than other types like more number of SSR alleles can be identified by a simple PCR based screening at a single locus, minute amount of DNA is enough, and analysis is manageable in automated allele detection (Mortimer et al., 2005). There are evidences strongly recommends that some SSRs in exon sites are also can be used in analysis (Jubrael et al., 2005). In the present analysis, the binary data was prepared using excel work sheet based on SSR banding pattern and was further analysed using geneAlex is used to evaluate variance in coefficient data. The variance between seven samples is identified to be 26 %. Also, it indicates the percentage of variance between Omani cultivars and variance within tested individuals is 74 %. The Date palm varieties tested were showed highly diverse in their Genome sequence. The present study shows that, the similarity of date palm individuals tested ranged between 0.00 and 0.74. The remaining cultivars showed differences in their dissimilarity but still they were grouped with each other's. A variance grid between tested cultivars (Table 1) were found a mean variance range from 0.098 to 0.423. The lowest dissimilarity number was found in between Chair and Nashi cultivars (0.098). Based on this finding it is clear that these two varieties can be close to each other therefore they can be regrouped. The variance 0.423 was obtained between Barny and Far. All the other cultivars displayed low level of variance but still they have been grouped with each other (Table 3).

The microsatellite genetic makeup was used to identify the genetic variation and genotypic relationship among 7 different cultivars grown widely in the Sultanate of Oman. Our findings evidences that the Omani date palm associated genomic variation, principal coordinate analysis for seven cultivars of date palm from three different regions results are represented in Fig. 1. As we observe in the Fig. 1 that, the Khasab (KHB) date palm which collected from (Senaw 1a, Bahla 2a and Wadibani Khaled 3a) are close to each other and its morphological and DNA analysis are comparable with each other and seems to be single cultivar. The similar results are seen for Fard (FARD), Khenezi (KHZ) and Madloki (MD) where morphological identification and SSR DNA sequence agree with each other. In addition, Ibra-1e and Tawa-2e of Madloki species are identical to each other. Whereas the analysis for Naghal from Samael-2b is closer to Khasab than Naghal which are from Senaw-1b and Bahla-3b. Also, Nasho (NASH) from Alkamel-3f is closer to the Khenezi. Barni from Senaw-1c is closer to the Khasab than Barni. Based on that five primers which were used in this study, we indicate that Naghal from Samael is maybe not Naghal. Barni from Senaw-1c is seemed to be Khasab and Nasho from Alkamel- 3f is Barni. Sedra et al. (1998) suggested that there might be a general genetic information in the tested varieties but there are chances of variation in their fruit nature and tree structure. There are similar findings in Tunisian date palm cultivars (Zehdi et al., 2004; Wrigley, 1995) have been reported. Studies suggest that the date palm domestication is a long history with the unknown origin (Wrigley, 1995), while the type of cultivation might have played a key role in the genetic composition of cultivars (Elshibi and Korpelainen, 2008). Novel date palm varieties are the results of the selective screening with selective breeding performed by farmers through seed propagation. Interchange of cultivars takes place between farmers which are the mixture of seed and vegetatively propagated date palm varieties. All of these reasons may results mixed genome in date palm in the studied region (Elshibi and Korpelainen, 2008). The present study using SSR analysis revealed the evidence that high level of polymorphism among the tested Omani date palm cultivars. However, classification and nomenclature of date palm in Oman still based on fruit nature like, structural, physical appearance, and chemical composition. In conclusion, Omani cultivated germplasm needs further analysis and application of specific and latest techniques that may help in identifying and characterizing the varieties which are economically and agronomically important (Table 4).

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Fig. 1

Principle coordinate analysis among the seven different cultivars between the three different selective locations.

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