Corresponding author: Marco Ballardini (
Academic editor: K. Jordaens
The genus
The genus
The taxonomy, phylogeny and evolution of the genus itself have been assessed using morphological and molecular approaches. According to
The cultivated date palm
Added to the common hybridization process between
However, despite all efforts, no locus (alone or in combination), has proven to be 100% efficient as universal DNA barcode in plants at the species level.
The first DNA barcoding analysis in palms (
Investigating the taxonomic status of
Given the potential of the
The sequenced cpDNA
One hundred and thirty-six individuals, belonging to 13
For each sample, genomic DNA was extracted from 40 mg of freeze-dried leaf tissue which was first grinded using a bead-mill homogenizer Tissuelyser (Qiagen, France). Extraction was performed using the DNeasy Plant Mini Kit protocol along with the QIAcube robotic workstation for DNA automated purification (Qiagen, France). Extracted DNA was quantified by means of a Nanodrop ND1000 spectrophotometer (Thermo Fisher Scientific Inc., USA) and visualized on 1% agarose gels stained with ethidium bromide.
The PCR amplification was carried out using the monocotyledoneous universal primers
The chromatograms obtained with the forward and reverse primers were combined and edited with SeqMan II 5.00 software (DNASTAR Inc., USA), to generate consensus sequences, which were aligned in BioEdit (
To assess the potential of the
The amplification of the plastid target region
The analysis of the intra- and interspecific variation within the sequenced region by direct observation of the sequence alignment showed four mutation types that contributed to the separation of
Distribution of observed polymorphisms in the region
Substitutions |
9 bp deletion |
Minisatellite |
Homo-polymer |
Species |
||||
---|---|---|---|---|---|---|---|---|
36607 | 36754 | 37099 | 37183 | 37190 | 36795-36803 | 37050-37098 | 37128-37139 | |
Haplotypes recorded in a single species |
||||||||
C | T | G | A | T | absent |
|
7 C + 5 A | |
C | T |
|
A | T | absent |
|
|
|
C | T | G | A | T | absent |
|
7 C + 5 A | |
C | T | G |
|
T | absent | 6M1+1M2 |
7 C + 5 A | |
C | C | G | A |
|
absent | 2M1+1M2 |
7 C + 5 A | |
A | C | G | A | T | absent | 3M1+1M2 |
6 C + 6 A | |
A | C | G | A | T | absent | 2M1+1M2 |
7 C + 5 A | |
C | C | G | A | T | absent | 2M1+1M2 |
6 C + 6 A | |
C | T | G | A | T | present | 2M1+1M2 |
6 C + 6 A | |
C | T | G | A | T | present | 4M1+1M2 |
|
|
C | T | G | A | T | present | 3M1+1M2 |
|
|
C | T | G | A | T | absent | 4M1+1M2 |
7 C + 5 A | |
C | T | G | A | T | absent | 2M1+1M2 |
|
|
Haplotypes shared by two species (5.1%) | ||||||||
C | T | G | A | T | absent | 6M1+1M2 |
7 C + 5 A | |
Haplotypes shared by three species (14.8%) | ||||||||
C | T | G | A | T | absent | 3M1+1M2 |
7 C + 5 A | |
a Position in the complete chloroplast genome of
b Number of individuals analysed for each species in parentheses (total sampling of 136 specimens).
c Number of repetitions of the 12 bp minisatellite units, including number of units of motif 1 (M1) and motif 2 (M2) as represented in
d Species-specific mutations in bold.
(1–7) Minisatellites haplotypes as reported in
The minisatellite located in the
Structure and variation of the minisatellite in the
Additional deletions and SNPs were detected in some of the analysed species, both upstream and downstream of the minisatellite (
The TaxonDNA pairwise comparison analysis of the 121 samples retained resulted in 115 sequences with a closest match at 0%. There were 18 allospecific matches at 0% (15.65%). At the individual level, the Best Match test, and the Best Close Match test with a threshold of 3%, resulted in 82.64% correct identifications, 14.87% ambiguous identifications and 2.47% incorrect identifications. At the species level, however, only
The haplotype sequences used, and the new ones obtained during this study, are deposited in GenBank under accessions:
In this study, we tested the usefulness of the
TaxonDNA unambiguously identified a single species,
The 9 bp-deletion, shared by
Regarding the 12 bp minisatellite, our results revealed much more complexity than previously reported (
In total, considering all mutation types, our results allowed us to efficiently identify eight out of 13 species. This indicates that the locus
Nevertheless, in order to increase resolution, other DNA regions should be examined, in search of characters allowing the identification of all taxa. Given their proven utility in palms, the
We wish to thank the following persons and institutions for their valuable help during the various phases of our study: Rita Bregliano†, Paolo Curir, Laura De Benedetti, Federica Nicoletti (CRA-FSO, Sanremo, Italy); Frédérique Aberlenc-Bertossi, Natalie Chabrillange, Nora Scarcelli (IRD Montpellier, France); Muriel Latreille, Sylvain Santoni (AMM at INRA SupAgro, Montpellier, France); Patrizia Martini (IRF, Sanremo, Italy); Mauro Roggero (Cooperativa “Il Cammino”, Sanremo, Italy); Giancarlo Pignatta (“U Risveiu Burdigotu” Association, Bordighera, Italy); the Natta family (Bordighera, Italy); Robert Castellana (CRP, Nice, France), Salwa Zehdi (University of Tunis, Tunisia). We also thank the three anonymous reviewers and the scientific editor for their useful comments and suggestions.