Research Article |
Corresponding author: Siham Ouchia-Benissad ( ouchiasiham@yahoo.fr ) Academic editor: Svetlana Galkina
© 2018 Siham Ouchia-Benissad, Kafia Ladjali-Mohammedi.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Ouchia-Benissad S, Ladjali-Mohammedi K (2018) Banding cytogenetics of the Barbary partridge Alectoris barbara and the Chukar partridge Alectoris chukar (Phasianidae): a large conservation with Domestic fowl Gallus domesticus revealed by high resolution chromosomes. Comparative Cytogenetics 12(2): 171-199. https://doi.org/10.3897/CompCytogen.v12i2.23743
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The development of avian cytogenetics is significantly behind that of mammals. In fact, since the advent of cytogenetic techniques, fewer than 1500 karyotypes have been established. The Barbary partridge Alectoris barbara Bonnaterre, 1790 is a bird of economic interest but its genome has not been studied so far. This species is endemic to North Africa and globally declining. The Chukar partridge Alectoris chukar Gray, 1830 is an introduced species which shares the same habitat area as the Barbary partridge and so there could be introgressive hybridisation. A cytogenetic study has been initiated in order to contribute to the Barbary partridge and the Chukar partridge genome analyses. The GTG, RBG and RHG-banded karyotypes of these species have been described. Primary fibroblast cell lines obtained from embryos were harvested after simple and double thymidine synchronisation. The first eight autosomal pairs and Z sex chromosome have been described at high resolution and compared to those of the domestic fowl Gallus domesticus Linnaeus, 1758. The diploid number was established as 2n = 78 for both partridges, as well as for most species belonging to the Galliformes order, underlying the stability of chromosome number in avian karyotypes. Wide homologies were observed for macrochromosomes and gonosome except for chromosome 4, 7, 8 and Z which present differences in morphology and/or banding pattern. Neocentromere occurrence was suggested for both partridges chromosome 4 with an assumed paracentric inversion in the Chukar partridge chromosome 4. Terminal inversion in the long arm of the Barbary partridge chromosome Z was also found. These rearrangements confirm that the avian karyotypes structure is conserved interchromosomally, but not at the intrachromosomal scale.
Barbary partridge Alectoris barbara, chukar partridge Alectoris chukar, endemic species, banding cytogenetics, high resolution chromosomes, homologies, intrachromosomal rearrangements
The Barbary partridge Alectoris barbara Bonnaterre, 1790 (Phasianidae) is the only native partridge naturally present in Algeria. This North African endemic species is found not only from Morocco to Egypt, but also in Gibraltar, Sardinia and the Canary Islands (
In addition, introduction of the exotic Chukar partridge Alectoris chukar Gray, 1830 could also lead to introgression in the wild genome of native partridge and could give rise to infertile descendants. In fact, hybridisation may occur when isolating mechanisms break down naturally or as a result of human activity as in the Alectoris partridges (
Preservation of this endemic species is a priority, which has led to a restocking programme with captive-reared Barbary partridge carried out by the Centre Cynégétique de Zéralda (36°42'06"N, 2°51'47"E). The goal of this project is to obtain strains able to reproduce in captivity, and formulate demographic monitoring after repopulation. Although the Barbary partridge is the main game-bird species in North Africa, scarce research has been reported and it concerns the reproduction and ecology of this species (
The Barbary partridge Alectoris barbara like the domestic fowl Gallus domesticus Linnaeus, 1758 belongs to the ancestral order of Galliformes which includes the most avian species whose genomes have been analysed. In fact, the domestic fowl is the best described one because of its economic importance. It is considered as a reference in phylogenetics and comparative genomics and represents the only standardised bird karyotype (
On the other hand, the chicken is the first avian genome to have been sequenced (
Although avian high resolution mapping is well advanced, reported cytogenetic studies are nevertheless partial and fewer than those of mammals despite great contribution of this discipline. In fact, classical and banding cytogenetics highlighted important features of avian karyotype as interchromosomal stability (
The aim of the present study is to describe the chromosomes of Barbary partridge Alectoris barbara and Chukar partridge Alectoris chukar at high resolution level with morphological and dynamic banding techniques. Comparison of partridges and chicken banding patterns has been conducted in order to estimate the degree of conservation and rearrangements of these species during speciation.
Barbary and Chukar partridge embryos were obtained from the Centre Cynégétique de Zéralda during the laying period (March to June). Four Barbary partridge and four Chukar partridge embryos were sampled after 5–6 days incubation at 37 °C, and kept under the same temperature and hygrometry conditions in the Laboratoire de Génétique du Développement (Faculté des Sciences Biologiques, USTHB) until at least 12 days old.
Primary fibroblast cell cultures were harvested from 6 to 12 days old embryos. The embryos were cleared from their annexes and totally ground in a trypsine solution (0.05%, Sigma). Cell suspension were incubated at 41 °C with an estimate concentration of 3×106 cells/ml in RPMI 1640 culture medium (20 mM HEPES, GIBCO) supplemented with 10% foetal calf serum (FCS, GIBCO), 1% L-Glutamine 200 mM (Sigma), 1% Penicillin, Streptomycin and Fungizone (Sigma). Trypsinisation of cells was realised to enhance division ability (adapted from
In order to increase the yield of metaphases and prometaphases cells, cultures were synchronised with a simple and double thymidine block during the S phase (
The incorporation of BrdU into the S phase lasted 6–7 hours. Meanwhile cells were continuously observed by reversed microscope until the number of mitotic round cells peaked. Cells were trypsinysed (trypsine 0.05% + 0.02% EDTA, GIBCO) and harvested in a 15 ml tube with colchicine (final concentration: 0.05 μg/ml, Sigma). After centrifugation, hypotonic treatment was undertaken during 13 min at 37 °C with diluted newborn calf serum (1:5). Intracytoplasmic structures were prefixed with 1 ml of methanol/acetic acid (3:1) at 37 °C. Fixation was finally realised at 4 °C and after centrifugation, 1 ml was let in tubes until spreading. Slides were washed, rubbed and placed in cold water. A few drops from the cell suspension were spread at 10 cm of cold slide and left to dry until staining procedures occurred (adapted from
GTG-banding (G-bands obtained with Trypsin and Giemsa) was realised following the Seabright modified method (1971). Approximately; 3 to 4 days after spreading, slides were incubated for 8–10 seconds in a trypsine solution (final concentration: 0.25%, Sigma) at room temperature. Slides were rinsed twice in PBS- (Phosphate Buffered Solution, pH=6.8) and stained in 6% Giemsa for 8–10 minutes.
RBG-FPG banding (R-bands followed by fluorochrome-photolysis) procedure was undertaken following
RHG-banding (R-bands obtained by Heat and Giemsa) was realised on A. chukar spreads. Slides were incubated in Earle’s buffer (ph=5,8) at 87 °C for 20 minutes, then rinsed and stained in 6% Giemsa solution (containing phosphate buffer) (
Slides were first observed with an optical microscope at objective magnification 10× to estimate the mitotic index (AxioZeiss Scope A1). Slides, showing a higher mitotic index, were analysed and prometaphases and metaphases, showing decondensed and dispersed chromosomes, were photographed (CoolCube1 Metasystems). The first eight macrochromosomes and Z sex chromosomes from Barbary partridge Alectoris barbara and Chukar partridge Alectoris chukar were classified in G- and R- banding as described in International System of Standardised Avian Karyotypes ISSAK (
Analyses measurements of fifteen first pairs of chromosomes were undertaken using KARYOTYPE 2.0 software (Altinordu et al. 2016). Measured parameters were: Long (q) and short (p) arms, total chromosome length (p+q) and arm ratio r: Long/short. In the Results section below, morphometry will be presented of the first eight chromosomes and the Z chromosome, which have been compared to the domestic fowl. Other microchromosomes were physically too small and did not give significant values. Partridge’s karyotypes have been established manually, considering that software used in the present work was not adapted to birds.
Primary fibroblasts cell lines were obtained a few hours after incubation and constituted a good source for obtaining chromosome preparations. The younger the embryos, the more mitotic divisions were obtained. The strict follow up of cell divisions after inhibition removal enabled the estimation of half cycle time to 7–8 hours for Barbary partridge Alectoris barbara and 6–7 hours for Chukar partridge Alectoris chukar. Important mitotic indices with high resolution chromosomes were obtained with simple synchronisation for A. barbara and double synchronisation for A. chukar during 18h. Furthermore, observation of cell cultures of both species showed that A. barbara cells were much more sensitive than A. chukar to the different drugs added during incubation. Trypsinisation and synchronisation steps caused important Barbary partridge cell death compared to Chukar partridge. In fact, we have incubated an average of 3×106 cells/ml (
Diploid numbers of Barbary partridge Alectoris barbara and Chukar partridge Alectoris chukar were estimated as 2n=78 from most metaphase plates (Fig.
Estimation of diploid number of Barbary and Chukar partridges. Major metaphase plates (10) displayed diploid number 2n= 78 chromosomes.
The authors proposed Alectoris barbara partial karyotype in GTG (Fig.
Partial karyotypes of A. barbara in GTG bands (a), A. barbara in RBG bands (b), A. chukar in GTG bands (c), and A. chukar in RHG bands (d). Gonosomes Z W are classified apart. Scale bars: 5 µm.
Observation of partridge’s spreads shows that in A. barbara an average of 45 metaphases /100 displayed break points. These breaks seem to appear in sub-terminal regions of macrochromosomes 1 and 3 (Fig.
Partridges’ metaphases showing spatial distribution of chromosomes (A. barbara on the left and A. chukar on the right). Macrochromosomes are located towards metaphases periphery, microchromosomes are confined to the central area. Arrows indicates break points in chromosomes. Bar = 5 µm.
A. barbara and A. chukar morphometry of the first eight macrochromosomes and gonosomes. Means are obtained at least from 10 prometaphases/metaphases (from 10 to 20). Chr: chromosome, q: long arm, p: short arm, t: total (p+q), r: ratio (q/p), lengths are given in micrometer (µm).
A. barbara | A. chukar | |||||||
---|---|---|---|---|---|---|---|---|
Chr | p | q | t | r | p | q | t | r |
1 | 3.78 | 5.98 | 9.76 | 1.58 | 5.63 | 8.82 | 14.45 | 1.56 |
2 | 2.89 | 4.71 | 7.6 | 1.62 | 3.83 | 6.76 | 10.59 | 1.76 |
3 | 1.03 | 5.57 | 6.6 | 5.4 | 1.2 | 7.5 | 8.7 | 6.25 |
4 | 1.02 | 4.33 | 5.35 | 4.24 | 1.15 | 6.19 | 7.34 | 5.38 |
5 | 0.75 | 2.85 | 3.6 | 3.8 | 0.78 | 4.9 | 5.68 | 6.28 |
6 | 0.68 | 2.32 | 3 | 3.41 | 0.75 | 3.35 | 4.1 | 4.46 |
7 | 0.7 | 1.7 | 2.4 | 2.42 | 0.7 | 3 | 3.7 | 4.28 |
8 | 0.53 | 1.57 | 2.1 | 2.96 | 0.63 | 2.37 | 3 | 3.76 |
Z | 2.5 | 3.1 | 5.6 | 1.24 | 3.1 | 3.5 | 6.6 | 1.12 |
W | 0.75 | 1.03 | 1.78 | 1.37 | 0.93 | 1.37 | 2.3 | 1.47 |
Partial ideograms of A. barbara and A. chukar were proposed on the basis of means of 20 metaphases plates following the International System of Standardised Avian Karyotypes (
GTG partial ideograms of (from left to right) G. domesticus (
RBG and RHG partial ideograms of (from left to right) G. domesticus (
Values summarized from partial ideograms of A. barbara and A. chukar described in GTG bands. Chr: chromosome, p: short arm, q: long arm, R: region, B: bands, LM: Landmark (all positions show negative landmarks except when (+) is added), empty boxes indicate that there is no particular landmark.
Alectoris barbara | Alectoris chukar | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Chr | P arm | Q arm | P arm | Q arm | ||||||||
R | B | LM | R | B | LM | R | B | LM | R | B | LM | |
1 | 2 | 11 | (21), (26) | 5 | 21 | (41) | 3 | 17 | (33) | 5 | 23 | (51) |
2 | 3 | 11 | (21) | 3 | 19 | (21), (31) | 3 | 13 | (31) | 3 | 21 | (31) |
3 | 1 | 3 | - | 4 | 23 | (13) (21) | 1 | 2 | - | 4 | 23 | (31), (41) |
4 | 1 | 1 | - | 4 | 19 | (21) | 1 | 2 | - | 4 | 25 | (26) + |
5 | 1 | 1 | - | 3 | 12 | (21) | 1 | 2 | - | 3 | 19 | (22) + |
6 | 1 | 1 | - | 2 | 8 | (21) | 1 | 2 | - | 3 | 9 | (22), (24) |
7 | 1 | 1 | - | 2 | 6 | - | 1 | 1 | - | 2 | 6 | (21) |
8 | 1 | 1 | - | 2 | 7 | - | 1 | 1 | - | 2 | 7 | - |
Z | 2 | 7 | (21) | 2 | 11 | (21), (22)+ | 2 | 9 | - | 2 | 11 | (15), (21) |
W | - | - | - | - | - | - | 1 | 2 | - | 2 | 5 | (11)+(22)+ |
Values summarized from partial ideograms of A. barbara and A. chukar described in RBG/RHG bands. Chr: chromosome, p: short arm, q: long arm, R: region, B: bands, LM: Landmark (all positions show negative landmarks except when (+) is added), empty boxes indicate that there is no particular landmark.
Alectoris barbara | Alectoris chukar | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Chr | P arm | Q arm | P arm | Q arm | ||||||||
R | B | LM | R | B | LM | R | B | LM | R | B | LM | |
1 | 3 | 13 | (31) | 4 | 20 | (41) (42) | 3 | 18 | (21) (31) | 4 | 25 | (13), (31), (45) |
2 | 2 | 10 | (25) | 3 | 15 | (21) | 3 | 17 | (21) (31) | 4 | 25 | (31) |
3 | 1 | 2 | - | 3 | 16 | (22)+(31) | 1 | 2 | - | 4 | 27 | (31) |
4 | 1 | 2 | - | 3 | 14 | (21) (31) | 1 | 3 | - | 4 | 21 | (14)+, (16)+ (22)+ , (24)+ |
5 | 1 | 2 | - | 3 | 8 | (21) (31) | 1 | 3 | - | 3 | 15 | (12)+, (14)+ |
6 | 1 | 2 | - | 2 | 7 | (21) | 1 | 3 | - | 2 | 8 | (21) |
7 | 1 | 1 | - | 2 | 5 | (21) | 1 | 3 | - | 2 | 7 | (14)+ |
8 | 1 | 1 | - | 2 | 5 | (13)+ | 1 | 3 | - | 2 | 7 | (21) |
Z | 2 | 7 | (21) | 3 | 9 | (24)+, (31) | 2 | 10 | (21) | 2 | 13 | (21) |
W | 1 | 2 | (12)+ | 1 | 2 | (12)+ | - | - | - | - | - | - |
Chromosome 1
P arm
Barbary partridge: two regions. 11 G bands with a visible negative band (21) which divides the p arm into two regions. A large terminal positive band is also visible (26).
Chukar partridge: three regions. 17 G bands with a predominant terminal negative band (33).
Q arm
Barbary partridge: Five regions. 21 bands, four negative bands divide the q arm into four regions with one predominant negative band (41). The centromeric region is positively banded.
Chukar partridge: Five regions. 23 G bands, with a wide terminal negative band (51).
Chromosome 2
P arm
Barbary partridge: three regions. 11 G bands with a large negative proximal band (21).
Chukar partridge: three regions. 13 G bands with large negative terminal band (31).
Q arm
Barbary partridge: three regions. 19 G bands with two wide negative bands (21 and 31).
Chukar partridge: three regions. 21 G bands with a large negative subtelomeric band (31).
Chromosome 3
P arm
Barbary partridge: one region with 3 G bands.
Chukar partridge: one region with 2 G bands.
Q arm
Barbary partridge: four regions. 23 G bands with two wide proximal negative bands (13 and 21).
Chukar partridge: four regions. 23 G bands with two large negative bands (31 and 41).
Chromosome 4
P arm
Barbary partridge: one region.
Chukar partridge: one region with 2 G bands.
Q arm
Barbary partridge: four regions. 19 G bands with a wide proximal negative band (21).
Chukar partridge: four regions. 25 G bands with a visible central positive band (26).
Chromosome 5
Q arm
Barbary partridge: three regions. 12 G bands with a wide central negative band (21).
Chukar partridge: three regions. 19 G bands with a visible central positive band (22).
Chromosome 6
Q arm
Barbary partridge: two regions. 8 G bands with a wide central negative band (21).
Chukar partridge: two regions. 9 G bands with two central positive bands (22 and 24).
Chromosome 7
Q arm
Barbary partridge: two regions. 6 G bands.
Chukar partridge: two regions. 6 G bands with a visible central negative band (21).
Chromosome 8
Q arm
Barbary partridge: two regions. 7 G bands with a wide central negative band (21).
Chukar partridge: two regions. 7 G bands with a large terminal negative band (21).
Chromosome Z
P arm
Barbary partridge: two regions. 7 G bands showing a large negative band (21).
Chukar partridge: two regions. 9 G bands with a visible negative band (21).
Q arm
Barbary partridge: two regions. 11 G bands with a large negative band (21) and a positive land mark (22).
Chukar partridge: two regions. 11 G bands with two large negative bands (15 and 21).
Chromosome W
P arm
Chukar partridge: one region. 2 G bands with terminal positive band.
Q arm
Chukar partridge: two regions. 5 G bands with one positive subcentromeric band (11) and a telomeric positive band (22)
Chromosome 1
P arm
Barbary partridge: three regions. 13 RBG bands with a large terminal negative band (31).
Chukar partridge: Three regions. 18 RHG bands with two principal negative bands (21 and 31).
Q arm
Barbary partridge: Four regions. 20 bands with two wide terminal respectively negative and positive bands (41 and 42). The centromeric region is positively banded.
Chukar partridge: Four regions. 25 bands with three large negative bands which divided the q arm (13, 31 and 45).
Chromosome 2
P arm
Barbary partridge: two regions. 10 bands with a large negative telomeric band (25).
Chukar partridge: three regions. 17 bands with large negative proximal band (21).
Q arm
Barbary partridge: three regions. 15 bands with two wide negative bands (21 and 31).
Chukar partridge: four regions. 25 bands with a large negative telomeric band (31).
Chromosome 3
P arm
Barbary partridge: one region with 2 bands.
Chukar partridge: one region with 2 bands.
Q arm
Barbary partridge: three regions. 16 bands with a central positive band (22) and a telomeric negative band (31).
Chukar partridge: four regions. 27 bands with a large submedian negative band (31).
Chromosome 4
P arm
Barbary partridge: one region and 2 bands.
Chukar partridge: one region with 3 bands.
Q arm
Barbary partridge: three regions. 14 bands with two visible negative bands (21 and 31).
Chukar partridge: four regions. 21 bands with two proximal positive bands (14 and 16) and two central positive bands (22 and 24).
Chromosome 5
P arm
Barbary partridge: one region. 2 RBG bands.
Chukar partridge: one region. 3 RHG bands.
Q arm
Barbary partridge: three regions. 8 bands with two wide negative bands (21 and 31).
Chukar partridge: three regions. 15 bands with two large proximal positive bands (12 and 14).
Chromosome 6
P arm
Barbary partridge: one region showing 2 RBG bands.
Chukar partridge: one region presenting 3 RHG bands.
Q arm
Barbary partridge: two regions. 7 bands with a wide central negative band (21).
Chukar partridge: two regions. 8 bands and a large negative band (21).
Chromosome 7
P arm
Barbary partridge: one region.
Chukar partridge: one region with 3 RHG bands.
Q arm
Barbary partridge: two regions. 5 bands showing a large distal negative band (21).
Chukar partridge: two regions. 7 bands with a central positive band (14).
Chromosome 8
P arm
Barbary partridge: one region with one band.
Chukar partridge: one region with 3 RHG bands.
Q arm
Barbary partridge: two regions. 5 bands with a central positive band (13).
Chukar partridge: two regions. 7 bands and a central negative band (21).
Chromosome Z
P arm
Barbary partridge: two regions. 7 R bands and a wide terminal negative band (21).
Chukar partridge: two regions. 10 R bands with a large negative band (21).
Q arm
Barbary partridge: three regions. 9 R bands with a positive terminal land mark (24) and a large negative band (31).
Chukar partridge: two regions. 13 R bands with a visible terminal negative band (21).
W chromosome
P arm
Barbary partridge: one region. 2 RBG bands with terminal positive band.
Q arm
Barbary partridge: one region. 2 RBG bands with a large positive telomeric band. Centromeric region is negatively stained.
Comparison of morphological and dynamic G and R banding of A. barbara and A. chukar with domestic fowl (
Schematic representation of a paracentric inversion in A. chukar (a), G. domesticus and A. barbara (b) chromosome 4. Corresponding bands are indicated by dashes. (ACH: A. chukar, GGA: G. domesticus and ABA: A. barbara).
Schematic representation of a terminal paracentric inversion in chromosome Z of A. barbara in GTG (a) and chromosome Z of G. domesticus in GTG banding (b). Corresponding bands are indicated by dashes. (ACH: A. chukar, GGA: G. domesticus and ABA: A. barbara). Rearranged ABA Z in GTG corresponds to GGA Z and ACH Z in GTG. Rearranged GGA Z in GTG corresponds to ABA in GTG and ACH in RHG.
Implementation of fibroblasts was observed in all cultures and confluence was quickly reached in all eight embryos, mainly in the youngest ones (6 days). This is due to the important mitotic power of cells at early embryonic stages (
Distribution of partridges’ macrochromosomes and microchromosomes in metaphases is similar to that reported in several studies on chicken fibroblasts and neurons nucleis (
Fortuitously, 45% of A. barbara metaphase plates show breaks on some macrochromosomes which could be identified as fragile sites (Fig.
The diploid number of Alectoris barbara and Alectoris chukar was estimated as 2n = 78. This result is concordant with the exceptional stability of avian karyotype, i.e. about 65% of karyotyped birds displayed 76 to 82 chromosomes, including 7 to 8 pairs of macrochromosomes (
Structural and dynamic R-bands obtained in the present work show similarities in pattern. However, dynamic RBG bands seem well delimited than morphological R-bands even if these latter present a higher number (Fig.
Simple and double synchronisation of partridge cell cultures have offered the possibility to obtain important rate of prometaphasic chromosomes presenting high number of bands (Table
A. barbara and A. chukar chromosome 4 is acrocentric, while in G. domesticus it is telocentric. Furthermore, comparison of bands showed conservation of patterns in A. barbara and G. domesticus but not in A. chukar. This morphological difference could suggest repositioning of the centromere during the speciation event of partridges 6 million years ago (
The morphological difference of chromosome 7 and 8 between partridges and the chicken, despite conservation of banding range, could be explained by repositioning of the centromere. However, double pericentric inversion cannot be excluded and only molecular investigations could elucidate such evolutionary events. Several studies show that chromosomes 7 and 8 are quite conserved in Galliformes and hybridize respectively to their homologous when using chicken chromosomal painting (
The Z chromosome in partridges shows a different terminal region. In fact, A. barbara Z gonosome presents an inversion of banding pattern in the terminus of long arm q compared to that of A. chukar and G. domesticus. Z gonosome of A. barbara in RBG corresponds to G. domesticus and A. chukar Z gonosome in GTG bands (Fig.
In both partridges and chicken, the W chromosome is submetacentric and highly heterochromatic as reported in other studies on partridges (
Banding cytogenetics performed on high resolution chromosomes allowed the precise description of Alectoris barbara Bonnaterre, 1790 and Alectoris chukar karyotypes. Comparative chromosomal mapping highlighted a large conservation with domestic fowl Gallus domesticus Linnaeus, 1758. However, rearrangements in acrocentric macrochromosomes 4, 7 and 8 were observed. Except for the Z chromosome, the partridge chromosomes share more similarities with the putative Galliform ancestral karyotype (Belterman and De Boer, 1984) than with chicken. Such cytogenetic studies could be of an important contribution to detect eventual chromosomal rearrangements in hybrids, given that A. barbara and A. chukar share an overlapping area. Obviously, more detailed molecular cytogenetic studies are necessary to refine the results of the present work. Indeed, we have selected clones from Wageningen chicken BAC (Bacterial Artificial Chromosomes) library (
The present work has received financial support from the Ministère de l’Aménagement du Territoire et de l’Environnement MATE (project 223), Ministère de l’Enseignement Supérieur et de la Recherche Scientifique MESRS (project 209), Ministère de l’Intérieur, in the framework of Post Graduation Specialized: Empreintes génétiques en pratique judiciaire.
Special thanks to Dr Belhamra (University of Biskra, Algeria) for the interest and contribution to this work. We would like to thank collaborators from the Centre Cynégétique de Zéralda (Direction Générale des Forêts) for providing biological material, especially Miss Zemiti and Larinouna. We would like also to thank AJZ Engineering Algérie (Zeiss company), in particular Mister LAGUEL; for the precious help. Authors are deeply grateful to Dr Ruane John (Food and Agriculture Organization of the United Nations, FAO, Italy) for his linguistic revision.