Simple Sequence Repeat (SSR) markers show greater similarity among morphologically diverse Date palm (Phoenix dactylifera L.) cultivars grown in Pakistan

Date palm is among the top three fruit crops of Pakistan which is grown throughout the country except the northern highlands. This study was conducted for evaluation of morphological, chemical and molecular diversity of date palm cultivars in Pakistan. Twenty two morphological parameters of fruit, leaf and trunk of forty five locally adapted cultivars were studied for this purpose. Proximate analysis of the date fruit was also done. Morphological traits of trunk, leaves and spines had no significant correlation with fruit traits. Seven components were found to explain 81% variability in the data set by principal component analysis. Length and width of leaf, weight, volume, diameter and length of fruit, weight of pulp, weight of seed, height of perianth, total soluble solids, % moisture, % total sugars and % reducing sugars largely contributed to variability among the cultivars. Forty six simple sequence repeat markers were used to find genetic diversity in date palm cultivars under study. Only two out of forty six SSR markers showed polymorphism with amplification of five amplicons in total. Only two of the tested markers amplified

Plants show variation in yield, vegetative traits and morphological properties of fruits and seeds in response to environmental changes [5,6].Phoenix dactylifera L. is different from other plants with respect to genetic diversity due to several reasons.Firstly it is vegetatively propagated with long life and is able to pass its genes to many generations.Secondly a single female can be selected on the basis of phenotype for further cloning.Thirdly due to cross breeding, heavy selection and transportation to long distance, its history is more complicated [7].Adapted cultivars are a result of natural and human selection.Human selection is based mainly on fruit traits and resistance to biotic and abiotic stresses [8].In date palm cultivars there exist a high degree of variation especially in the reproductive characters so cultivars are usually identified on the basis of fruit characteristics [9].Phenotypic markers have been in use for characterization of date palm germplasm by researchers in different countries [10][11][12][13][14][15][16].In date palm the different naming of the cultivars in different growing regions, introduction of new varieties and their distribution in different geographical regions has made the identification of varieties difficult [17].Long generation cycle and the time it takes in showing the reproductive characteristics is the main hindrance in date palm breeding [18].Thus a reliable technique of characterization would be very useful for the improvement of such crops.This will make the identification of the tree very easy even at its juvenile stage [19].Simple sequence repeats which are abundantly present throughout the higher eukaryotic genomes are highly polymorphic than other genetic markers.These are helpful in identification of cultivars, analysis of pedigree, germplasm characterization and genetic mapping study [20].Principal component analysis, a statistical tool used for determining variation in samples, has been used for assessment of variation in apricot

Morphological analysis
Morphological data were scored for 45 date palm cultivars (Table 1) in triplicates.Each replication was represented by a single tree of the cultivar selected randomly for sampling.Twenty two parameters including leaflength and width (cm), leaf base width (cm), spine and pinnae number, midrib length (cm), length of midrib with spines (cm), length of midrib with pinnae (cm), length of the top pinnae (cm), fruit volume and dimensions (length and width), pulp weight (gm), seed weight (gm), Perianth height (mm) and trunk diameter (cm) were selected from standard descriptors established for date palm by Rizk and Sharabasy [25].Trunk circumference was measured and then diameter was calculated with formula: Diameter = Circumference/ [26] Data were scored on mature leaves of selected trees for leaf length and width, midrib length, midrib length with spines, midrib length with pinnae and length of the top pinnae [12].1).Morphological and proximate data were converted to interval data and similarity matrices were computed using Euclidean coefficient and these matrices were then used to draw dendrogram.Each polymorphic band with SSR markers on the gel was considered as an allele.Bands on gel were scored as present (1) or absent (0) to form binary data matrix.Data were then computed with NTSYSpc version 2.10j software to produce similarity matrix using Jaccard's coefficient to detect the polymorphism among the cultivars on the basis of size of alleles.The matrix was then computed to form dendrogram.

Different
strategies for diversity evaluation of date palm on phenotypic, molecular and proximate composition basis were used.Phenotypic data were subjected to a multivariate technique called principal component analysis using Minitab software version 16.The data obtained were used to draw dendrograms using Euclidean coefficient and Jaccard's coefficient respectively.

Morphological and proximate analysis
The present study was conducted to evaluate the genetic and morphological variation in forty five cultivars of date palm in Pakistan (Table 1) and to find traits that can discriminate among the cultivars.The descriptive statistics of data showed large variation among characteristics of cultivars studied (Table 2).Thirteen traits including leaf length and width, fruit weight, volume, diameter and length, pulp weight, seed weight, perianth height, % moisture, total soluble solids, % total sugars and % reducing sugars were found to be more variable and important for characterization of date palm cultivars.

Principal component analysis
Principal component analysis of the obtained data was done.First seven components contributed 81% variability in the data.The first component had an Eigen value of 5.48 and contributed 24% variability in the data (Table 3).Five variables including leaf length, weight, volume and length of fruit and pulp weight had more weight than other variables in this component.Leaf length, weight, volume and length of the fruit and pulp weight are the traits that mainly accounted for 24% variability in PC1 (Table 4).The second component had an Eigen value of 3.85 and explained 17 % variability in the data (Table 3).In second component the three variables including total soluble solids, % reducing sugars, and % total sugars had greater weight than other variables (Table 4).PC3 with Eigen value of 2.73 explained 12% variability in the data and represented two variables, fruit diameter and % moisture.Eigen values of the next three components were 1.78, 1.56 and 1.26 and 1.15 respectively and thus each represented one variable.These variables are leaf base, seed weight and perianth height respectively (Table 3 & 4).Correlation study among the traits of date palm showed that vegetative traits were positively correlated with other vegetative traits but had no significant correlation with fruit traits.Similarly some fruit traits were found to be positively correlated with other fruit traits but fruit traits had no significant correlation with vegetative traits.Positive correlation between leaf length and midrib length and pinnae number and pinnated part length was also reported by [10] for Moroccan date palm cultivarsand this can be explained as the long leaf can have a long midrib with greater number of pinnae thus accounting for a greater pinnated part length.The dendrogram based on combined morphological and proximate data (Fig 2 ) clustered Peeladora and Kerbaline along with other 9 cultivars in one cluster but these two cultivars were grouped in different clusters from the rest of the cultivars (Pathri, Makhi, Kokna, Eedal Shah, Basrawali, Sufaidah, Gajjarwali, Kur, Fasli, and Shakri) in dendrogram based on molecular data.Makran, Haleni, Baidhar and Kupra were clustered at one point with closely related Saib and distantly related Champa kali at one point in one cluster and Berahmi, Khudrawi, Zirin, Daanda, Rachna, Shamran , Zahidi, and Kozanabad in another cluster by the dendrogram based on molecular data while all these cultivars were grouped together in one cluster by combined morphological and proximate data.Begum Jhangi, Neelum, Zerdo, Shado and Wahan wali were grouped in one cluster with Angoor, Sanduri, Halwain and Taarwali by combined morphological and proximate data while the later were grouped in different cluster by molecular data.Aseel and Halwai were placed in one cluster in both the dendrograms while Jansohar, Peelisunder and Chohara were placed in different clusters by molecular data.

Table 2. Descriptive statistics of traits of date palm cultivars studies
Clustering of cultivars at one point on the basis of morphological traits may be due to the local naming where one cultivar is given different names or multiple cultivars may be given the same name.Unrelated cultivars may be grouped together due to the similarity in fruit characteristics.This is also observed by [33] while studying the biodiversity of dates in the Saltanate of Oman.The dendrogram formed by morphological and proximate data shows similar relationship among the cultivars as shown by the score plot of the principal component analysis.

Conclusion
The studied date palm cultivars grown in Pakistan showed great variation with respect to morphological characteristics and proximate composition of the fruit whereby some traits like length and width of leaf of date palm tree, length, diameter, weight and volume of date fruit, weight of fruit pulp and seed, height of perianth, % moisture content, total soluble solids, % total sugars and %reducing sugars were found as important parameters for characterization of date palm cultivars but microsatellite SSR markers have shown greater genetic similarity among these cultivars.Hence it is concluded from this study that although great morphological and compositional variation exists among the studied cultivars but SSR markers used in this study were unable to reflect that polymorphism correctly.The reason may be that the studied markers do not represent those genetic regions where variation exists.Thus to look for genetic basis of these variations more robust sequence based molecular markers like single nucleotide polymorphism markers should be used to underpin the genetic regions causing these variations.
Introduction Date Palm (Phoenix dactylifera L.), a member of the Arecaceae family is a monocotyledonous perennial woody plant which is cross breeding in nature.Almost 5000 cultivars of date palm exist in the world which are developed by date palm growers through selection [1].Date palm covers an area of 896, 000ha in Pakistan that produces 524.6 metric tons of dates [2].Major date producing regions in Pakistan are Khairpur, Makran, Bahawalpur and D.I. Khan [3].Aseel, shakri, BegumJhangi, Fasli, Dhaki, Halawi and Zahidi are some of the important commercial cultivars [4].

[21], maize [ 22 ]
and date palm [11].Little work is done on morphological characterization of date palm cultivars in Pakistan.Some cultivars were characterized morphologically and others on the basis of physico-chemical traits of their fruit.[14, 23, 24].Date palm is prone to the loss of genetic diversity due to continuous use of offshoots from few genetic material of the gene pool.Therefore to evaluate and preserve the genetic diversity are important for germplasm conservation of date palm [19].This study aimed at diversity evaluation and morphological, molecular and chemical characterization of the local date palm cultivars.

Figure 1 .
Figure 1.Score plot based on morphological and proximate data together and closely related to each other while distantly related to Wahanwali and Taarwali in the same cluster.Cluster 3 had 16 cultivars grouped further in 3 sub clusters.The first sub cluster had 4 cultivars including Haleni, Kupra, Kozanabad and Zahidi.The second sub cluster also had four cultivars, Baidhar and Champakali paired together and closely related to Shamran and distantly related to Makran.The third subcluster had eight cultivars including Berahmi, Zirin, Khudrawi, Kohraba and Daanda.Rachna and Saib were paired together while Dhaki was placed in the same cluster at a distance from the rest of the cultivars.Cluster 4 was the largest cluster with two sub clusters having thirteen cultivars grouped mostly in pairs.The first sub cluster had only three cultivars Saib, Akhrot and Makhi.The second sub cluster had the remaining ten culitvars including Kerbaline, Peeladora, Kokna, Eedalshah, Basrawali, Sufaidah, Gajjarwali, Kur, Fasli and Shakri.

Figure 2 .
Figure 2. Tree plot based on combined morphological and proximate data using euclidean coefficient

Figure 4 .Figure 5 .
Figure 4. PCR amplification of mpdCIRO25 in45 date palm cultivars on 2% agarose gel M=100bp plus DNA ladder Authors' contributions Conceived and designed the experiments: A Muhammad, GM Ali, MZ Hyder & H Rahman, Performed the experiments: N Faqir, Analyzed the data: N Faqir & A Shezad, Contributed reagents/ materials/ analysis tools: A Muhammad, GM Ali, A Shehzad, MZ Hyder, Wrote the paper: N Faqir.

Table 4 . Correlation among the variables studied and the first six components
Abbreviations: TD; Trunk diameter, LL; Leaf length, LBW; Leaf base width, SN; Spine number, MLS; Midrib length with spines, ML; Midrib length, PN; Pinnae number, MLP; Midrib length with pinnae, LTP; Length of top pinnae, F.

Table 5 . Correlation matrix of the traits studied Dendrogram based on morphological and proximate data
The dendrogram (Figure 2) based on morphological data of trunk, leaves, spines, fruit and proximate composition data of date fruit shows clustering of studied cultivars into four groups.Cluster1 grouped six cultivars Aseel, Halawi, Jansohar, Dhadi, Chohara and Peelisunder together.Cluster 2 included 10 cultivars mostly paired together.Qantar and Begum Jhangi, Neelum and Zerdo, Shado and Angoor, Sanduri and Halwain were paired