ABSTRACT
Date palm (Phoenix dactylifera L.) is widely cultivated in Saudi Arabia, with annual production estimated to be above one million tons. Of the 450 cultivars that are commonly used, 70 cultivars are found in the Eastern province and 20 cultivars are well known and common in the Al-Ahsa oasis. In this study, thirty seven date palm samples representing nine cultivars collected from six locations in the Al-Ahsa oasis, Saudi Arabia. Total proteins were extracted and subjected to analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results revealed several differences within the cultivars. Overall polymorphism ranged between 12 and 76%, with the highest being 76% within Helali cultivar, and the least being 12% within OmRohaim cultivar. These results support the conclusion that some palms are grown from seeds resembling a known cultivar, which are later distributed as the known cultivar. These results revealed differences in the degree of similarity between the cultivars from 0.059 to 0.579, estimated according to Jaccard`s similarity method based on the presence and absence of protein bands. Khosab and Shehel cultivars were closely related to each other with 0.579 degrees of similarity, both separated by phylogenetic dendrogram in mini-cluster, which have been confirmed by the 1st principal components. The mini-cluster was related to OmRohaim and Khonaizi cultivars within a larger cluster. Shishi, Garrah and Helali were related cultivars, with degrees of similarities ranging between 0.250 and 0.455, and were separated in one cluster by phylogenetic dendrogram. The close relationship between the cultivars may indicate that they originated from the same ancestor. Further molecular and biochemical studies are needed to enrich our knowledge about the relationships among date palm cultivars.
Key words: Components, dendrogram, patterns, principal, protein.
Date palm (Phoenix dactylifera L.) is a dioecious species belonging to the Arecaceae family that includes 225 genera and 2600 species (Corner, 1966). It is cultivated in the Middle East and North Africa (Hamza et al., 2011), where it is considered a valuable fruit crop (Racchi et al., 2014). The annual global production of dates is estimated to be about 7.4 million tons, while Saudi Arabia accounts for more than one million tons (FAOSTAT, 2013). In Saudi Arabia, about 450 cultivars are cultivated with more than 70 cultivars being grown for centuries in the eastern province (Asif et al., 1982), and ~ 20 cultivars are well known and common in the Al-Ahsa oasis (Al-Baker, 1972), which is the largest date palm oasis of Saudi Arabia (Al-Abdolhadi et al., 2012), with an estimated three millions date palms (Al-Abbad et al., 2011). In general, date palms produce inferior fruits when propagated by seeds, therefore offshoots are preferred for conventional propagation, as offshoots produce fruit identical to the mother tree (Abass, 2013; Kenna and Mansfield, 1997).
Utilizing DNA markers is most accurate than protein patterns in studying similarities between cultivars, but still protein profiles characterized by high stability and reproducibility, which produces reliable results to verify the differences within and between species and cultivars (Dakhil et al., 2013; Rouholamin and Saei, 2016; Alege et al., 2014; George et al., 2013), electrophoretic analysis of protein bands has been used as an effective tool in recognizing the origin of cultivated plants (Ladizinsky and Hymowitz, 1979). It is a fast and inexpensive tool (Smykal et al., 2008; Rouholamin and Saei, 2016). Electrophoretic protein analysis has been used traditionally in higher plants to study genetics, P. dactylifera L. (Dakhil et al., 2013), Punica granatum L (Rouholamin and Saei, 2016), taxonomy, Trifolium L. (George et al., 2013), physiology, P. dactylifera L. (Al-Helal, 1994) and for the study of phylogenetic relationships, P. dactylifera L. (Attaha et al., 2013), as well as for the genotoxicity of different chemicals on date palm callus (Abass et al., 2017). Phylogenetic electrophoresis has been used to study the genetic relationships between and within date palm cultivars (Attaha et al., 2013), and in identifying the cultivars (Stegemann et al., 1987), and other plants (Barta et al., 2003). Date palm leaflets have been used extensively as a source of proteins in cultivar identification and phylogenetic studies (Ahmed and Al Qaradhawi, 2009; Attaha et al., 2013), therefore, thirty seven leaflet samples representing nine different cultivars, namely, Khosab, Shishi, Shehel, Helali, Hatemi, Khalas, OmRohaim, Khonaizy and Garrah, were collected from six locations in the Al-Ahsa oasis, namely, Al Oyoon (OY), Al Hafof (HF), Al Kelabiah (KL), Al Menaizlah (MN), Ash shgaig (SH) and Ass Sabatt (SB) (Table 1 and Figure 1). The purpose of the present study was to verify that the palms of each cultivar are identical and to analyze the phylogenetic relationships between the cultivars using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Young green leaflets were collected from date palm trees of age 30 years and above. The samples were snap frozen immediately in liquid nitrogen, and then stored at -20°C.
Protein extraction
The samples were cut into small pieces, and ground in a mortar with liquid nitrogen. Total proteins were extracted according to Al-Helal (1994) with some modifications, homogenizing 200 mg of ground leaflet powder in 1.2 ml extraction buffer [0. 5 M Tris/HCl (pH 6.8) + Glycerol 10% (v/v) + PVP 4% (w/v)] using a Bullet Blender Homogenizer. The extracts were incubated overnight at 4°C, vortexed, boiled at 95°C for 5 min, and centrifuged at 12500 rpm for 10 min, after that loaded in 20 µl.
SDS-PAGE Electrophoresis
Discontinuous vertical electrophoresis (SDS-PAGE) was carried out according to Laemmli (1970) with some modifications. 12.5% resolving gels were prepared with 6.67 ml acrylamide-bis acrylamide (30/2.6%) + 4 ml 1.5 M tris/HCl buffer (pH 8.8) + 5.05 ml distilled water (DW) + 0.16 ml 10% SDS + freshly prepared 0.12 ml 10% amm. persulfate (NH4)2S2O8 + 0.008 ml TEMED and stacking gels were prepared with 0.95 ml acrylamide-bis acrylamide (30%\2.6%) + 1.25 ml 0.5 M tris/HCl buffer pH 6.8 + 2.72 ml DW + 0.05 ml 10% SDS + freshly prepared 0.025 ml 10% ammonium persulfate + 0.005 ml TEMED. The running buffer was prepared from 3.0 g Tris + 14.1 g glycine + 1 g SDS, dissolved in 1 L DW, the pH adjusted by hydrochloric acid to 8.3. Electrophoresis was carried out at 100 to 150 V using Bio-Rad Broka 0.75 mm mini electrophoresis system and Bio-Rad PowerPac Basic.
Staining and destaining
The gels were washed with 100 ml of gel fixing solution (Ethanol: Acetic Acid: Distilled Water, 5:1:4%, respectively GFS) for 1 h with agitation at room temperature. The gels were then covered with 100 ml of gel washing solution (Methanol: Acetic Acid: Distilled Water, 5:1:4%, respectively GWS) overnight with agitation at room temperature. The gels were incubated with 100 ml Coomassie brilliant blue R-250 stain, prepared by dissolving 0.4 g of Coomassie Brilliant Blue R-250 in 200 ml methanol (40%) and 200 ml acetic acid for 3 to 4 h with agitation at room temperature The gels were then rinsed with several changes of 100 ml gel washing solution.
Band scoring and analysis
The electrophoresis was carried out three times and the gel with best resolution of protein bands was analyzed using a Gel Analyzer 2010a densitometer software and was scored according to the presence (I) or absence (O) of protein bands (Gel Analyzer, 2010). Molecular weights of the protein bands were calculated, with reference to the standard protein marker, starting at the top with 250 KDa band followed by 150, 100, 75, 50, 37, 25, 20, 15 and 10 KDa band, using a Gel Analyzer 2010a densitometer software. The results obtained from protein patterns were analyzed statistically. Jaccard’s similarity was determined based on the presence (I) or absence (O) of protein bands (Ahmed and Al Qaradhawi, 2009), hierarchical clustering was performed, and principal components analysis was conducted using IBM SPSS Statistics for Windows software (IBM Corp., 2010).
Total protein extracts from date palm cultivars were subjected to SDS-PAGE analysis. Electrophoretic protein bands varied in intensity, with some bands showing very low resolution. Upon analyzing the protein patterns (Figures 2 and 3) with densitometer software, low resolution protein bands with raw volumes below 10 were excluded.
The number of protein bands ranged between 15 in Hatemi cultivar (Table 2) to 25 in Helali cultivar (Table 3).
The percentages of polymorphism observed was higher 76% (Table 4) in Hel cultivar for 25 electrophoretic bands representing six locations because the electrophoretic bands varied between 12 bands in OY and SB locations to 20 bands in KL location (Table 3).
High polymorphism ratio 70% (Table 4) in Khl cultivar for 20 electrophoretic bands representing six locations because the electrophoretic bands varied between 11 bands in SH location and 16 bands in KL location (Table 5); high polymorphism ratio 68% (Table 4) in Shi cultivar for 22 electrophoretic bands representing five locations because the electrophoretic bands varied between 12 bands in KL and MN locations and 16 bands in HF (Table 6); low polymorphism ratio 44% (Table 4) in Grr cultivar for 16 electrophoretic bands representing five locations because the electrophoretic bands varied between 11 bands in SB location and 13 bands in HF location (Table 7); low polymorphism ratio 40% (Table 4) in Hat cultivar for 15 electrophoretic bands representing three locations because the electrophoretic bands varied between 11 bands in MN and SH locations and 14 bands in OY location (Table 2); low polymorphism ratio 38% (Table 4) in Shl cultivar for 24 electrophoretic bands representing four locations because the electrophoretic bands varied between 16 bands in OY location and 22 bands in HF location (Table 8); low polymorphism ratio 37% (Table 4) in Khn cultivar for 19 electrophoretic bands representing three locations because the electrophoretic bands varied between 14 bands in MN location and 18 bands in HF location (Table 9); low polymorphism ratio 25% (Table 4) in Khs cultivar for 20 electrophoretic bands representing three locations because the electrophoretic bands varied between 17 bands in KL location and 19 bands in HF location (Table 10); and very low polymorphism ratio 12% (Table 4) in OmR cultivar for 17 electrophoretic bands representing two locations because the electrophoretic bands varied between 15 bands in SB location to 17 bands in HF location (Table 11).
Phylogenetic analysis of protein patterns according to nearest neighbor single linkage hierarchical clustering dendrogram based on Jaccard’s similarity, regarding the presence and absence of all bands for all the cultivars locations, showed that the different cultivar palms of the different locations were grouped within clusters on the phylogenetic tree (Figure 4).
The similarity between protein profiles according to Jaccard’s similarity method, ranged between 0.059 and 0.579 degrees of similarity (Table 12).
The highest similarity value 0.579 was observed between Khs and Shl cultivars, and the least similarity value 0.059 was observed between Hat and Grr cultivars (Table 12). Phylogenetic analysis of protein patterns according to nearest neighbor single linkage hierarchical clustering dendrogram based on Jaccard’s similarity, regarding the presence and absence of common bands for all of the cultivar locations (Figure 5) showed two clusters, in addition to two cultivars Khl and Hat, which were separated out singly.
The first cluster was composed of a sub-cluster, in addition to one line, the line represented by Khn cultivar. The sub-cluster was composed of a mini-cluster for two closely related cultivars, Khs and Shl, in addition to OmR cultivar, which was separated out individually. The second cluster composed of Shi, Grr and Hel cultivars.
Principal component analysis (PCA) revealed six components while two components were targeted, according to the variance results summarized in Table 14, because the first component represented 42.580% of the total variations, the second component represented 41.621% of the total variations, hence the cumulative value for the targeted components
represented 84.201 % of the total variations. The results of Kaiser-Mayer-Olkin (KMO) and Bartlett’s test for adequacy and sphericity were 0.761 and 0.025, respectively (Table 13).
However, the first component was characterized by three variables; 134, 122 and 82 KDa bands, while the second component was characterized by three variables; 180, 86 and 50 KDa bands (Table 15) because the component variables were mostly positioned close to each other, on the 2-dimensional scatter gram (Figure 6).
It is known locally that some palms of the same cultivar differ in the quality of their fruits, for example, Khalas cultivar palms at the Ash-Shgaig and Al Hafof locations, produce higher quality dates, which command higher prices in comparison with Khalas dates produced in the other locations, additionally, some Shishi cultivar dates were known locally as the surrounding dates, since the surrounding part at the base of the date, continue at the bisser stage, not fully matured (tamer stage), which command higher prices in comparison with the regular Shishi dates, as well as, some Hilali cultivar dates were known locally as scratchy dates, since the texture was scratched, which command higher prices in comparison with the regular Hilali dates. These differences are in harmony with the results of the present study, since the polymorphism within Helali, Khalas and Shishi were 76, 70 and 68%, respectively (Table 4), even though the diversity of protein bands between varieties and within species were generally low (Hastuti et al., 2009; Khalifah et al., 2012). Due to the high polymorphism, 76% within Helali cultivar palms (Table 4), Hel at KL location and Hel at HF location were separated out of the Hel cluster (Figure 4), as well as the Hel at MN location and Hel at OY location were separated out of close related Hel at SB and SH locations, within the cluster of Hel cultivar, it could be concluded that the original Hilali cultivar is within this cluster, while it could assumed the Hel at KL location and Hel at HF location were seedling palms. The differences within Helali cultivar were reported regarding of the ripening and season of harvesting (Al-Qurashi, 2010, Awad, 2006). Due to the high polymorphism 68% within Shishi cultivar palms (Table 4), Shi at OY location and Shi at KL location were separated as close related within a sub-cluster (Figure 4), where as Shi at HF location and Shi at SH location were included within the first sub-cluster with Khn cultivar palms, while Shi cultivar at MN location was separated out singly. It could be concluded that the close related palms, Shi at OY location and Shi at KL location representing the original Shishi cultivar, whether the regular or the surrounding Shishi, while it could assumed that the rest of Shishi palms were seedlings. The results of Shishi cultivar were in agreement with the results of previous study which revealed genetic differences between palms of the same cultivar grown in different locations (Al-Issa, 2006).
Due to the high polymorphism 70% within Khalas cultivar palms (Table 4), Khl at SH location and Khl at HF location were separated as close related within a Khl cluster (Figure 4), whereas the rest of Khl palms each represented by one line within the Khalas cluster, it could be concluded that the close related palms, Khl at SH location and Khl at HF location representing the original Khalas cultivar, which agree with what is known locally that Ash-Shgaig and Al Hafof locations, produce higher quality of Khalas dates, while in a previous study, different locations were not included in present study of date palms were proposed as representative for the original Khalas cultivar; Al-Oyouni, Al Omran, Al Taraf and Ashoa’bah (Al-Issa, 2013). It could be interpreted that the Khalas cultivar is an elite cultivar, and in high demand for its fruits and offshoots, therefore, some farmers treated seedlings produced from seeds as the original cultivar if they produced fruits similar to the original cultivar. This resulted in different lines of Khalas cultivar, which differed genetically, and in the quality of their dates, being sold as the Khalas cultivar. Representation of the Khalas cultivar was 15 to 20% of all Al-Ahsa palms in 1972 (Al-Baker, 1972), which later
became more than 50% of the oasis area (Al-Abbad et al., 2011). In general these results indicate that some palms were grown from seeds, and produced fruits similar to the fruits of the original cultivar, which were then sold and distributed as the original cultivar (Devanand and Chao, 2003). The present study revealed differences between the cultivars, based on common electrophoretic bands, and these data passed the KMO and Bartlett tests for adequacy and sphericity, with 0.761 and 0.025, respectively, meaning that they are valid for principal component analysis (Andy, 2005). Khs and Shl cultivars were closely related among the studied cultivars with the degree of similarity being 0.579 (Table 12), separated by phylogenetic dendrogram in a mini-cluster (Figure 5), as well as confirmed by the first principal component with three variables 134, 122 and 82 KDa bands (Table 15), with high loading 42.580% (Table 14), which agree with Elmeer et al. (2011), who additionally revealed that the previous mini-cluster of Khs and Shl cultivars is related to OmR cultivar with degrees of similarities 0.286 and 0.227, respectively (Table 12) because they were separated by phylogenetic dendrogram in a sub-cluster (Figure 5), which is confirmed for Khs cultivar by the second principal component with three variables 180, 86 and 50 KDa bands (Table 15), with high loading 41.621% (Table 14), because the component variables collected together on the 2-dimensional scatter gram (Figure 6) agree with Al-Issa (2015). Previous studies reported that clusters constructed by phylogenetic dendrograms can be confirmed using principal component analysis (Ahmed and Al Qaradhawi, 2009; Attaha et al., 2013). The results revealed that the Khs, Shl and OmR cultivars were close related to Khn cultivar, with degrees of similarities ranging between 0.375 and 0.500 (Table 12) because they were separated in one cluster by phylogenetic dendrogram (Figure 5), which agree with Elmeer et al. (2011), but disagree with Al-Issa (2015). The results revealed that the Shi, Grr and Hel were related cultivars, with degrees of similarities ranging between 0.250 and 0.455 (Table 12) because they were separated in one cluster by phylogenetic dendrogram (Figure 5) which agree with Elmeer et al. (2011) but disagree with Al-Issa (2015). Khl cultivar was separated out individually, followed by Hat cultivar (Figure 5), which agrees with Ahmed and Al Qaradhawi (2009). It could be concluded that Khs, Shl, OmR and Khn cultivars, share a common ancestor, such results have been reported previously (Ahmed and Al Qaradhawi, 2009; Elmeer et al., 2011). Based on gene expression the results revealed an allelic polymorphism, according to the common bands; the 128 KDa band was specific for Hat cultivar, the 116 and 62 KDa bands were specific for Hel cultivar, the 95 KDa band was specific for Khl cultivar, and the 105 KDa band was specific for Khn cultivar. These bands may be suitable as genetic markers for cultivar identification, even though environmental factors have been reported to affect protein profiles (Hanna et al., 2003; Kong-mgern et al., 2005). The 193 KDa band in the Hat cultivar at OY location, 37 KDa band in the Hat cultivar at SH location, 43 KDa band in the Khl cultivar at KL location and 34 KDa band in the Shl cultivar at HF location, which appeared unique among all studied cultivars, might indicate that the cultivar palm is a mutant, and useful for breeding programs, as has been reported previously (Al-Helal, 1992). It may be concluded that these results provide helpful basic information for breeding programs to improve the fruit characteristics of date palms. However, more biochemical and molecular studies are needed to confirm the genetic relationships within and between date palm cultivars and to identify the cultivars of the Al-Ahsa oasis and surrounding areas. Differences observed in the quality of dates from some cultivars may be due to genetic differences. Hence, comprehensive studies are recommended to ascertain the true-to-type characteristics of each cultivar.
The authors have not declared any conflict of interest.
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