The date palm (Phoenix dactylifera L.) is a monocotyledon species belonging to the Arecaceae family, and is widely cultivated in North Africa, the Sahel (from the Atlantic to the Red Sea), the Middle East, and eastward to the Indus Valley. The date palm is well adapted to cultivation in arid and semiarid areas, and it has been introduced in warm and dry regions worldwide. Mainly grown for its fruits, the date palm represents an important ecological and socioeconomic resource.
Despite the increasing number of studies on date palm, there are still not enough molecular markers available for a number of applications. Most published microsatellite or simple sequence repeat (SSR) markers are dinucleotide loci from unknown noncoding regions of the genome, generally isolated from microsatellite-enriched DNA libraries (Billotte et al., 2004; Arabnezhad et al., 2012). The increasing amount of available genome sequence data offers new prospects for microsatellite marker development through in silico mining, a promising approach for date palm (Cherif et al., 2013), based on the recently published date palm genome sequence (Al-Dous et al., 2011) and expressed sequence tags (ESTs) (Zhao et al., 2012). Our aim was to develop new markers from coding sequences to ensure clear stepwise mutation patterns usable for genetic diversity, dating, and selection signature analyses, and also to facilitate transferability to other species.
METHODS AND RESULTS
In silico microsatellite mining and primer design were performed on the date palm genome draft sequence version 2 (Al-Dous et al., 2011), with the Perl script SSR_pipeline-v2.pl (Poncet et al., 2006), which incorporates three free software programs: Tandem Repeats Finder (Benson, 1999), Primer3 (Rozen and Skaletsky, 2000), and BLAST (Altschul et al., 1990). The multi-FASTA file of all 19,414 predicted genes (full and partial; PDK20.mRNA.fsa) and the multi-FASTA file with all scaffold sequences (PDK20.fsa) from version 2 of the date palm genome research program at Weill Cornell Medical College in Qatar were downloaded from http://qatar-weill.cornell.edu/research/datepalmGenome/download.html. The search identified 204 genes containing coding sequences with microsatellites, 150 of which were suitable for primer design, but only 103 had nonduplicated primer annealing sites. Among them, we retained loci having perfect trinucleotide motifs with six (excluding those without annotation) or more (with or without annotations) repeats, and hexanucleotide motifs with at least four repeats (with or without annotation).
Of the 47 primer pairs finally retained, 33 generated expected PCR amplification patterns in a preliminary test with eight P. dactylifera individuals (Table 1). The 33 loci were further tested on 16 individuals representing P. dactylifera (7), P. reclinata Jacq. (2), P. roebelenii O'Brien (2), P. rupicola T. Anderson (2), P. theophrasti Greuter (2), and the interspecific hybrid P. canadensis × P. sylvestris (Table 2). Among these loci, 15 showed consistent amplification and promising polymorphisms across the sample and were further investigated in a variable number of individuals (80–1000) of the aforementioned species, including population samplings of P. dactylifera and P. reclinata. The transferability of 10 loci was also evaluated in Chamaerops humilis L., resulting in 100% positive amplification, with eight polymorphic loci displaying two to 12 alleles among seven to 51 individuals (Table 3). Moreover, the amplification of one Hyphaene thebaica Mart. individual and one Livistona carinensis (Chiov.) J. Dransf. & N.W. Uhl individual was tested for five loci, with both species giving positive amplification results in three loci (mPdIRD25, mPdIRD31, and mPdIRD33).
Characteristics of 33 microsatellite markers developed for Phoenix species. The putative annotation was done using the BLASTX program and the UniProtKB/Swiss-Prot protein database with an E -value cutoff of 10−5
DNA from these individuals was extracted from freeze-dried or silica-dried leaf tissue. Samples were reduced into a fine powder using either an IKA A10 analytical grinder (IKA-Werke, Staufen, Germany) or a QIAGEN TissueLyser and QIAGEN DNeasy Plant Mini, Maxi, or 96-well kits (QIAGEN, Courtaboeuf, France). PCR reactions were performed in a thermocycler (Biometra GmbH, Göttingen, Germany, or Eppendorf AG, Hamburg, Germany) in a total reaction mixture of 25 µL, containing: 10 ng of total genomic DNA, 1× PCR buffer, 2 mM MgCl2, 200 µM dNTP, 0.5 U of Taq DNA polymerase, 0.4 pmol of the forward primer labeled with a 5′ M13 tail, 2 pmol of the reverse primer, and 2 pmol of the fluorochrome-marked M13 tail, plus sterile water to reach the final volume. The fluorochromes used were either 6-FAM, HEX, or TAMRA. The PCR parameters were as follows: denaturation for 2 min at 94°C; followed by six cycles at 94°C for 45 s, 60°C for 1 min, and 72°C for 1 min; then 30 cycles at 94°C for 45 s, 55°C for 1 min, and 72°C for 1.5 min; then 10 cycles at 94°C for 45 min, 53°C for 1 min, 72°C for 1.5 min; and a final elongation step at 72°C for 10 min.
Test of functionality of the 33 loci across the Phoenix genus.a
The PCR products were processed on an ABI 3130XL Genetic Analyzer (Applied Biosystems, Foster City, California, USA). Allele size scoring was performed with respect to a noncommercial ladder using GeneMapper version 3.7 software (Applied Biosystems).
Genetic analyses (number of alleles, observed and expected heterozygosities, Wright's fixation index [F IS] and its significance calculated using the permutation test) were conducted with GENETIX version 4.05 software (Belkhir et al., 2004).
Each of the 15 loci tested were polymorphic in at least one Phoenix species (Tables 2 and 3). The loci mPdIRD25, mPdIRD30, mPdIRD31, mPdIRD33, and mPdIRD40 were particularly suitable in P. dactylifera with three to eight alleles, having a clear stepwise mutation pattern in accordance with the microsatellite motif (tri- or hexanucleotide), and showing little to moderate heterozygosity deficit. The loci mPdIRD13, mPdIRD25, mPdIRD31, and mPdIRD33 were useful in Chamaerops humilis with three to 12 alleles, confirming good intergeneric transferability. In addition, mPdIRD25, mPdIRD31, and mPdIRD33 were amplified in Livistona carinensis and Hyphaene thebaica.
Polymorphism characterization for 15 loci in Phoenix and 10 loci in Chamaerops.
The loci described here are a useful addition to previously published microsatellite markers for palms. Their interspecific allelic differentiation makes them particularly suitable for hybrid and gene flow analysis within Phoenix. The most polymorphic loci can be added to other SSR loci to create marker sets for genetic diversity analysis in P. dactylifera and other species. Their transferability within the Coryphoideae subfamily will facilitate the study of species with limited molecular resources, such as Chamaerops humilis.