Genetic Diversity Analysis of Date Palm (Phoenix dactylifera L.) Cultivars from Morocco Using SSR Markers

In Morocco, date palm is the most important arboricultural crop and little is known about its germplasm. Thus, this work aimed at analyzing genetic diversity among 200 date palms sampled from three oases (Figuig, Tata and Zagora) of Morocco using microsatellite markers. Among these palms, 191 were females, belonging to 26 cultivars, and 9 were males. Eighteen primers were used for the analysis of their genetic diversity. Only 15 primers amplified successfully all the samples. The total number of alleles was 116 and the percentage of polymorphic loci was high and ranged between 60 and 100% with an average of 93.33%. The genetic similarity values ranged from 0.146 to 0.745. The molecular variance analysis showed 64% of variability among cultivars. The obtained dendrogram showed three groups and generally, a good structuring of cultivars. However, we noticed one case of homonymy among cultivars. In fact “Tadmant” cultivar of Figuig was different from “Tadmant” of Tata and Zagora. Males were clustered in two main subgroups.


Introduction
Date palm (Phoenix dactylifera L.) is a dioecious perennial monocotyledon plant with long generation times (a period of 4 to 5 years is necessary to reach the first flowering) that belongs to Arecaceae family [1]. It is a diploid (2n=2x=36), and the predicted genome size is estimated to be approximately between 550 and 650 Mbp [2]. Date palm has traditionally been vegetatively propagated from offshoots produced by elite individual trees. In Morocco, more than 220 clonally propagated varieties are known [3] and date palm is the most important arboricultural crop. There are 4.7 million palm trees covering a surface area of approximately 44,000 ha. More than 220 clonally propagated varieties are known [3]. All commercial varieties are female and there is no method yet of producing male palms of these varieties. However, the effects of pollen on date quality through metaxenia are well documented, and male genotypes with desirable qualities are maintained in the plantations and commonly used to hand pollinate female trees.
The most serious fungal disease threatening date palm plantations in North Africa, especially in Morocco is "Bayoud" [4]. This vascular wilt caused by Fusarium oxysporum f. sp. albedinis has recently affected about 67% of Moroccan palm trees and has continued to spread to the East, demolishing a large portion of the palm groves in its path. This disease has destroyed more than 12 million palms in Morocco [5].
Despite all these problems, little is known about Moroccan date palm genetic diversity and resistance of different cultivars to Bayoud.
Microsatellites or SSR (simple sequence repeats) or STR (short tandem repeats) are repeating sequences of 2 to 6 nucleotides in noncoding regions generally [25]. They are distributed in all regions of the genome [26][27][28][29] and are present in all eukaryotes [30,31] and in some prokaryotes [32] and are evenly spread across the genome [26][27][28][29]. Microsatellites are preferential markers used in the study of genetic diversity because they are abundant [33,34], typically co-dominant and of a high variability [35]. The objective of this work is therefore to analyze the genetic diversity of common cultivars of three date palm oases from Morocco using SSR markers.

Plant material
A set of 200 date palm samples were used in this study (Table 1). They consisted of 26 female cultivars and 9 males from 3 Moroccan oases. In Figuig oasis, 128 samples (121 belonged to 11 cultivars and 7 males) were collected; in Tata, 20 samples (18 belonged to 9 cultivars and 2 males) and in Zagora, 52 samples (belonging to 10 cultivars). Cultivars were the most common genotypes in these oases while males were less frequent as they only serve as pollinators. Young leaves of adult trees were dried and conserved in silicagel.

DNA isolation
Genomic DNA was extracted using a modified preparation procedure according to Bousquet et al. [36]. After purification, DNA yields were determined by a NanoDrop 800 spectrophotometer (PEQLAB Biotechnologie GmbH) and diluted to a working concentration of 25 ng/l.

Analysis of SSR data
The targeted fragments and allele scoring were performed by the ALFwin Fragment Analyser Software. For each marker, the average number of alleles per locus, the expected heterozygosity (He) and the observed heterozygosity (Ho) were calculated by GenAlex 6.3 software. The fixation index or F-statistic (Fis, Fst) were computed according to Wright [40]. Values of FST (fixation index) ranged from 0 (completely undifferentiated) to 1 (completely differentiated). The genetic similarity and the analysis of molecular variance (AMOVA) were also calculated using GenAlex 6.3 software [41]. DARwin 5.0 software was used to make dendrograms which showed the distribution of different individuals.

Microsatellite amplification
Among the 18 microsatellite makers used, 15 showed a net amplification of DNA fragments. MPdCIR044, MPdCIR048 and MPdCIR063 primers amplified only a few or no samples. In addition, these amplifications showed no polymorphism. Therefore, these three loci were not considered in the following statistical analyses.

Heterozygosity and fixation index
The total rate of heterozygosity (Ht) by primer for all cultivars was very high and varied between 0.696 (MPdCIR93) and 0.945 (PdCAT20) ( Table 5). The average of expected heterozygosity (MHe) ranged between 0.464 (MPdCIR16) and 0.677 (PdCAT20) and the average of observed heterozygosity (MHo) ranged between 0.816 (MPdCIR78) and 0.950 (PdCAT20) ( Table 5). For all the markers, the observed heterozygosity value was higher than the expected one. The Fis values were negative for all markers and varied between -0.895 (MPdCIR32) and -0.404 (PdCAT20) per marker with an average of -0.757 (Table 5). The Fst values for their part varied between 0.267 (MPdCIR93) and 0.472 (MPdCIR78) with an average of 0.363 (Table  5).

Pd10
Pd 15 Table 6 showed the degree of similarity between cultivars. The genetic similarity values ranged from 0.146 to 0.745. The highest similarity value was observed between "Boufeggous" et "Boufeggous gharas" while "Aguelid" and "Mejhoul" had the smallest similarity value. "Mejhoul" and "Boufeggous" had also a high genetic similarity (0.702).

Analysis of molecular variance between Moroccan cultivars
Molecular variance analysis showed 64% of variability among Moroccan cultivars (Figure 3).

Dendrogram of similarity of moroccan cultivars
The similarity dendrogram ( Figure 4) showed 3 main groups (a, b and c). The first group (c) was divided into 2 subgroups (one subgroup constituted by 2 individuals of "Aziza manzou", 1 individual of "Boufeggous" from Figuig, 6 individuals of "Tgharas", and 2 males from Figuig and one subgroup constituted by the rest of "Tgharas" individuals). The second group (b) was divided into 2 subgroups also. The first subgroup was constituted by individuals of "Boufeggous" from Zagora and from Tata, 1 individual of "Boufeggous" from Figuig, 2 individuals of "Mejhoul", 1 "Admam", 1 "Tadmant" from Figuig, 2 "Boufeggous gharas" and 2 "Ahardane". The second subgroup was constituted by 1 "Admam" and 1 "Tadmant" from Figuig. The third group (a) was also divided into 2 subgroups. The first one was constituted by the 5 individuals of "Tadmant" from Zagora, the 5 males from Figuig remaining and the 2 males from Tata. The second one was constituted by the individuals of "Aguelid", 2 "Taâbdount", 2 "Admam", "Jihel" individuals and by the rest of samples.

Discussion
MPdCIR044, MPdCIR048 and MPdCIR063 markers had a low or a lack of amplification of DNA samples. Similar observations have been reported by Zehdi et al. [18]; Billotte et al. [37] and Bodian et al. [23,24] for the first two markers.
The number of alleles found per locus ranging from 4 to 11 is the same as that found by Bodian et al. [24] who used the cultivars of Figuig only with the same markers. It is comparable to that found by Zehdi et al. [18] (ranging between 4 and 10) who recognized 7.14 alleles per locus when examining 46 Tunisian date palm accessions using 14 microsatellite loci. It is also comparable to Elmeer et al. [22] results who found between 4 and 12 alleles per locus. However it is lower to that found by Billotte et al. [37] (ranging between 5 and 18) and it is very high compared to Ahmed and Al-Qaradawi [42] studies who marked 40 different alleles with a mean of 4 alleles per locus by examining 15 Qatari date palm cultivars.
An excess of heterozygosity manifested by negative Fis values was observed. The average value of Fst equal to 0.363 means that the index of genetic differentiation was very high among all cultivars. This result is obtained by Bodian et al. [24] when examined only Figuig cultivars.
Statistical analysis showed that the genetic similarities between cultivars are fairly variable (ranging from 0.146 to 0.745). These values suggest that there are cultivars that are genetically very close and others that are very far. These genetic similarities are comparable to those found by Ahmed and Al-Qaradawi [42] (ranging between 0.00 and 0.75) and by Zehdi et al. [18] (ranging between 0.3008 and 0.7885) and by Bodian et al. [24]. The highest similarity value observed between "Boufeggous" and "Boufeggous gharas" means that they are genetically the closest. While Aguelid and Mejhoul had the smallest similarity value, that means they are the most genetically distant. "Mejhoul" and "Boufeggous" have also a high genetic similarity (0.702).
The analysis of molecular variance (64% of variability among cultivars), the average value of Fst and the values of genetic similarity suggest a variable polymorphism among Moroccan cultivars.
The dendrogram showed that "Jihel" individuals of Tata and those of Zagora were in the same subgroup and in the same level. So this cultivar was the same of one oasis to another and individuals did not show any genetic variability. This was the case of "Boufeggous" individuals of Figuig, Tata and Zagora. So "Jihel" of Tata was the same that "Jihel" of Zagora and "Boufeggous" of Figuig was also the same that "Boufeggous" of Tata and Zagora. However "Tadmant" individuals of Figuig were not in the same group with those of Zagora. Moreover, "Tadmant" individuals of Figuig showed variability while those of Zagora were identical. That could mean that "Tadmant" of Figuig was different that "Tadmant" of Zagora. They were two cultivars which had the same name but were genetically different: they could be homonyms. All males were in the same subgroup except two males from Figuig. Similar results were observed when Figuig cultivars only were analyzed [24]. Males from Tata and those from Figuig were in the same subgroup. Males were not clustered according to their geographical origin.
This study revealed the existence of genetic variation among Moroccan cultivars. So genetic differentiation was high and an excess of heterozygosity was observed. In general, cultivars were identical from one oasis to another ("Boufeggous", "Jihel"). But, one case of homonymy was noticed. In fact "Tadmant" of Figuig was genetically different from "Tadmant" of Tata and Zagora.