Genetic characterization of Libyan date palm resources by microsatellite markers
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Molecular typing of 377 female date palm trees belonging to 18 Libyan cultivars and representing common genotypes in the central Libyan oasis of Al Jufrah was performed using 16 highly polymorphic microsatellite or SSR loci. A total of 110 alleles with an average of 6.88 alleles per locus were scored indicating the high level of polymorphism existing among the cultivars thus allowing their genetic fingerprinting. Moreover 28 alleles out of 110 were fixed. All the cultivars were characterized by negative values of the Fixation Index (F) due to an excess of heterozygotes with respect to HW equilibrium. The pattern of genetic diversity among cultivars was estimated by codominant genetic distances and presented by principal coordinates analysis (PCoA). The observed pattern evidences the genetic diversity existing among cultivars that allow distinguishing them easily. The average dissimilarity internal to each cultivar ranged from 0 to 21. Seven cultivars showed value zero indicating no genetic difference within cultivar in agreement with their Fixation Index (F = 1). A varietal identification key was also built using multiloci genotyping with only three microsatellite loci that identified 23 alleles in total. The possibility to attribute the unknown male plant to a cultivar was also considered and male parentage analysis was performed. Fifty-five male plants out of 63 were assigned to a definite cultivar with high confidence level. The positive result obtained in identifying males confirmed the suitability of SSR for clone fingerprinting and cultivar identification, thus opening new prospects for date palm breeding.
KeywordsDate palm SSR Identification Genetic diversity Cultivars
In this paper, we report the first data on the genetic fingerprinting using SSR of 18 Libyan date palm cultivars of Al Jufrah oasis. The objectives were (a) to investigate the genetic diversity of Libyan date palm, (b) the genotyping of cultivars, and (c) the parentage analysis of pollinator plants for the attribution to a cultivar.
Materials and methods
The desiccated leaf material was ground into a fine powder using bead-mill homogenizer TissueLyser (Qiagen, Italy). The leaf powder of each individual sample was then subjected to DNA extraction using both DNeasy Plant Maxi/Mini Kits (Qiagen, Milano Italia) and E-Z 96 Plant kit (Omega), according to manufacturer’s instructions. The resulting DNA solutions were stored at –20 °C. After purification, DNA concentration was spectrophotometrically measured using Gene Quant Pro (Amersham Biosciences) and DNA quality was verified by visualization on a 1 % ethidium bromide agarose gel.
Amplification and genotyping
Primer sequences of microsatellite loci used for genotyping of Libyan date palm cultivars
Billotte et al. (2004)
Akkak et al. (2009)
For a given locus, the forward SSR primer was 5′-end labeled with M13 extension (5′-TGTAAAACGACGGCCAGT-3′) to incorporate, via PCR step, a fluorochrome (6-FAM, VIC and NED) necessary for the detection of the PCR products on the sequencer.
Amplifications were performed in Applied Biosystem Thermocycler (AB System, Germany) with the following conditions: for Billote’s primer, a initial denaturation at 95 °C for 1 min, then 35 cycles at 94 °C for 30 s, 52 °C for 1 min and 72 °C for 2 min and a final elongation step at 72 °C for 8 min; for Akkak’s primer a initial denaturation at 95 °C for 9 min, then 28–35 cycles at 94 °C for 30 s, 55 °C for 45 s and 72 °C for 1 min and a final elongation step at 72 °C for 45 min. A negative control, with the reaction mixture excluding DNA, was also included in each experiment.
Amplification products were checked on 1.5 % agarose gel to verify the presence of a band of the expected size. PCR products labeled with various fluorescent dyes (6′-FAM, NED and VIC) were loaded on the capillary system sequencer, MegaBACE1000 (GE Healthcare), with the size standard MegaBACE ET400 (GE Healthcare). Peaks were analyzed and fragment lengths determined with the MegaBACE Fragment Profiler 2.1 software (GE Healthcare). All peaks and binning were manually checked.
Data scoring and genetic variability description
The observed heterozygosity (Hobs) was calculated as the ratio of the number of heterozygotes and the number of samples for each locus and as arithmetic average over loci. The expected heterozygosity (Hexp) assuming Hardy–Weinberg equilibrium was estimated as Open image in new window where pi is the frequency of thei-allele. The statistic was computed for single locus and as average over loci. The Unbiased Expected Heterozygosity (UHexp) takes into account the sample size n: Open image in new window.
The Fixation Index or Inbreeding Coefficient (F) was computed as Open image in new window.
Values close to zero are expected under random mating, while substantial positive values indicate inbreeding or undetected null alleles. Negative values indicate an excess of heterozygotes, due to negative assortative mating or selection (Hartl and Clark 1997).
The number of effective alleles (Ne) provides an estimate of the number of equally frequent alleles in an ideal population with homozygosity equivalent to the actual population and was computed as Open image in new window.
The matrix of distances among cultivars is obtained as average of the individual distances between couple of cultivars, while the elements of the main diagonal are the average dissimilarities for all pair-wise comparisons internal to each cultivar. The off diagonal elements were submitted to Principal Coordinate analysis (covariance standardized method) and sample Eigenvectors were used to plot the cultivar centroids.
Male trees were assigned to cultivars by a maximum-likelihood paternity assignment procedure through comparing genotypes of males and cultivars. To find the significant values of LOD scores, simulations were performed with 10,000 repeats, 0.01 as the proportion of loci mistyped and 61 individual profiles as probable cultivar candidate for each male tree. 95 % was used as strict and 80 % as relaxed confidence level as suggested by Marshall et al. (1998). The LOD score is obtained taking the natural log (log to base e) of the overall likelihood ratio. A positive LOD score means that the candidate variety is more likely to be the true variety. Negative load score can occur, when the candidate variety and query male tree share very common alleles or mismatch at one or more loci. A statistical test is performed on the base of the simulation of parentage analysis that allows determining also the confidence of parentage assignments.
Genetic variability measures and distance metrics were analyzed using GenAlEx 6.5 (Genetic Analysis in Excel; Peakall 2006; Peakall and Smouse 2012), available at: http://www.anu.edu.au/BoZo/GenAlEx. Cervus 3.0 (Kalinowski et al. 2007), available at http://www.fieldgenetics.com, was used for parentage analysis.
Genetic diversity analysis
Summary of microsatellite allele data revealed by 16 microsatellites loci in 377 female trees belonging to 18 Libyan date palm cultivars
Allelic range (bp)
Number of genotypes
Number of fixed alleles
Cultivarsa with the fixed allele
A, Be, D, Hal, O, S, Tag, Tam
A, D, Hal, S
D, Hal, O, Tag, Tal, Tam
A, D, K, N, O
Genetic diversity indices for the 18 Libyan date palm cultivars
Cultivar identification key
Identification of male plants
Assignments with positive scores of pair LOD value of parentage analysis performed on male plants sampled in Al Jufrah farms
Pair LOD score
Eighteen cultivars representing common genotypes in Al Jufrah oasis selected for their good fruit quality were analysed using 16 highly polymorphic microsatellite loci. Date palm germplasm of Libya still preserves an enormous richness attested by a great number of different accessions, widely described since the beginning of twentieth century on the base of the morphological characters of fruit and seeds. The date palm genetic resources deserve nowadays to be genetically characterized with the aim to organize their preservation, to transmit a significant genetic richness and for their exploitation. In Libya, each palm grove is typified by a distinct cultivar composition, which resulted from a local selection within the oases. As a consequence, a large number of SSR alleles have been revealed with a mean of 6.88 per locus that allowed detecting a relatively high degree of genetic variability in this crop. A high level of polymorphism was detected among cultivars as previously reported in date-palm cultivars of Algeria, Morocco, Tunisia and Sudan using both isoenzymes and SSR markers (Benaceur et al. 1991; Elhoumaizi et al. 2006; Zehdi et al. 2004a, b; Elshibli and Korpelainen 2008, 2009). Furthermore the presence of higher polymorphism within the date palm genome was evidenced by the results obtained from parallel sequencing (Al-Dous et al. 2011).
Each cultivar results from an empirical selection for heterosis carried out by the farmers based on morphological characters and fruit quality. This fact justifies both the presence of fixed alleles, 28 out of 110, due to random drift and the high level of heterozygosity maintained by clonal breeding procedure (Table 2).
For the analysis of the SSR multilocus genotypes the procedure of Smouse and Peakall (1999) was used. This method allows evaluating the genetic differences at different levels of structure, such as cultivar and locality, based on distances among the individual genetic profile of each sampled tree. The differences, if any, among plants of the same cultivars sampled in the different localities of Al Jufrah oasis were not significant (data not shown). In the case of cultivar Hamria, for example, the statistic PhiPT (ΦPT) receives the value of 0.091, which is not significant after 999 permutations and particularly in three localities out of five all the trees exhibited the same genotype.
The PCoA based on codominant genotypic distances of SSR loci (Fig. 4) represents a procedure by which the revealed genetic pattern does not assume a hierarchical structure like tree building methods consequently, it is appropriate for analysing our date palm samples, in which the assumption of a high taxonomic level is less reasonable. The results evidence the genetic diversity existing among cultivars that enable distinguishing them easily. Furthermore the codominant genetic distances procedure of Smouse and Peakall (1999) allows estimating the average dissimilarity internal to each cultivar. In seven cultivars out of eighteen absence of genetic diversity within cultivar was observed in agreement with the respective Fixation Indexes (Table 3).
The traditional practice of vegetative multiplication of plants by offshoots, which was generally performed with good skill by Al Jufrah farmers, ensures the identity and uniformity of the cultivars. Nevertheless, cases of misclassification can occur during propagation because of the difficulty faced sometimes in the identification of the cultivars on the base of morphology. The cultivar Sokeri represents an exception and seed propagation performed in some farms of Waddan resulted in the high distance values observed within this cultivar. Seed propagation of date palm has been reported to occur also in other countries. Elshibli and Korpelainen (2008) while analyzing Sudan date palm resource, because of high genetic diversity observed within groups and the weak clustering of the cultivars suggested that they are not a result of a full cloning process. The ease and rapidity of seed reproduction coupled with their large availability support the maintenance of this practice among Sudan farmers consequently, date palm plantations are a mixture of plants both clonally or seed propagated with a high genetic variability within cultivars. Clonal propagation, beyond to guarantee genetic uniformity of the cultivars, also limits the negative effect of inbreeding. In fact, it provides the maintenance of high level of heterozygosity within cultivars achieved by assorting heterotic positive characteristics as result of empirical selection of plants with good pomology features and fruit quality. The analysis of genetic variation between localities for each cultivar was performed and differences were not significant.
The strong cultivar genetic identity observed made possible to design an identification key based on a three microsatellite loci that identified 23 alleles in total and permitted the unambiguous discrimination of the date palm accessions. Consequently, the totality of Al Jufrah cultivars was univocally and easily identified on the base of their allelic profile. Similar result was previously obtained by Zehdi et al. (2006) in the analysis of 49 Tunisian accessions with three SSR loci. The effectiveness of SSR in discriminating all the accessions and cultivars examined confirms the usefulness of these markers for clonal fingerprinting and cultivar identification. Since each variety was identified by a unique profile, it is possible to generate an individual barcode useful in certification and control of origin labels of date palm products. On the contrary of other species of economic relevance, in the case of Phoenix dactilifera specific test guidelines of the UPOV system based on morphological descriptors are not requested (http://www.upov.int/test_guidelines/en). On the other hand, vegetative and fruiting traits are limited in number and influenced by the environmental conditions and their use can result in a poor discriminant power. Consequently, the introduction of molecular fingerprinting for variety protection and farmer rights is an important perspective to achieve also in the perspective of product origin certification for international trading.
The improvement of fruit yield and quality is based on the possibility to identify the genotype of male plants used as pollinators. Generally female plant pollination is carried out by mixing pollen coming from the few male plants present in the farm. Most of the time the cultivar identity of male plant is unknown because of seed propagation and the exchanges that often occur among farmers. Considering that genetic variability observed among cultivars is higher than that within cultivars, a full sib marker assisted selection procedure could be proposed starting from the cross of cultivars with different positive traits. For that purpose to attribute an unknown male tree to a cultivar becomes important. Consequently, a parentage analysis by a procedure of maximum-likelihood paternity assignment was performed through comparing genotypes of males and cultivars. The results obtained allowed to assign, with high confidence level, 55 male trees out of 63 to a distinct cultivar. Furthermore the identification key applied to the 24 male presenting positive LOD score evidenced that each male has at least one allele in common with the cultivar assigned by the parentage analysis. The methodology used do not require a priori identification of sex-specific SSR markers, but only the availability of a molecular profile representative of each candidate variety. The positive result obtained by assigning male trees further confirms the suitability of SSR for genotyping and opens new prospects for date palm breeding for yield and improved physical and chemical characteristics of the fruits.
This work was supported by funds from the Italian Ministry of Foreign Affairs DG CS; project “Improvement and development of date palm in the oasis Al Jufrah”.
Conflict of interest
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