Two forms of A. halimus shrubs: erect habit ( A. halimus) and bushy habit shrub ( A. schweinfurthii) are used naturally isolated by a considerable distance from each other and occupy the same area. To explore the effect of natural isolation on the genetic basis of the two forms, Start Codon Targeted (SCoT) and the phylogenetic relationships of A. halimus by sequencing ITS1-5.8S-ITS2 regions of the ribosomal DNA are used. Significant isolation-by-distance relationship was found (r = 0.62, P = 0.001). Soil factors did not influence molecular variations. The natural isolation of A. halimus habitats restricts gene flow among the populations and the observed high within-population genetic diversity (74.19%) in this species is best explained by its outcrossing behaviour, long-lived individuals and overlapping generations. The UPGMA analysis of the SCoT results showed that all the studied populations were divided into two discrete genetic groups with significant separation of the two forms in Burg El-Arab area (Populations 1 and 2) and insignificant separation between two forms in El-Hammam area (population 5 and 6). The sequencing of the ITS1-5.8S-ITS2 rDNA regions also showed the insignificant separation of the two A. halimus forms. We conclude that gene flow depending on habitat fragmentation was the main factor affecting the population genetic differentiation. We suggest that the two forms do not merit specific rank in presence of interference between the two forms and absence of a breeding barrier fail to separate the different populations when they become sympatric.
Atriplex halimus L. (Chenopodiaceae), a C4 perennial shrub, highly outbreeding, is found in semi-arid and arid environments. This species, particularly well adapted to arid and salt-affected areas, is valued as livestock forage in low rainfall Mediterranean areas [
In recent years, changing environmental conditions and the resulting threats to the survival of existing populations have resulted in increased interest to study how genetic variation is maintained in natural populations [
The first study of genetic variability of A. halimus was made by Haddioui & Baaziz [
Recently, new marker techniques have been developed depending on gene-targeted markers. A novel marker system called Start Codon Targeted (SCoT) Polymorphism [
The present study used two forms of A. halimus shrubs: erect habit (A. halimus) and bushy habit shrub (A. schweinfurthii). The two morphotypes are used naturally isolated by a considerable distance from each other and occupy the same area to explore the effect of natural isolation on the genetic basis of the two forms using Start Codon Targeted (SCoT) and the phylogenetic relationships of A. halimusby sequencing ITS1-5.8S-ITS2 regions of the ribosomal DNA. Besides, the previous results obtained from both isozymes and RAPDs markers collected from the Mediterranean basin populations were compared with the results of the present markers.
A total of 18 accessions of A. halimus were collected from six populations growing naturally in the Western Mediterranean Desert, Egypt (
Location | Population number | Site description | Growth form | Sample size | Sample code | Sampling site longitude and latitude |
---|---|---|---|---|---|---|
Burg El-Arab | 1 | Inland typical salt marches (Ideal habitat) | Bushy habit | 4 | 1 - 4 | 30˚905' and 29˚536' |
2 | Edges of run-road-transported sand to this area | Erect habit | 3 | 5 - 7 | 30˚909' and 29˚533' | |
El-Gharbaniat | 3 | North slope, costal rocky ridge | Erect habit | 3 | 8 - 10 | 30˚929' and 29˚474' |
El-Hammam | 4 | Road run, Sandy plain | Erect habit | 2 | 11 - 12 | 30˚847' and 29˚386' |
5 | Polluted area (Cement factory) | Bushy habit | 3 | 13 - 15 | 30˚862' and 29˚478' | |
6 | Polluted area (Cement factory) | Erect habit | 3 | 16 - 18 | 30˚874' and 29˚475' |
Soil factor | Locations | |||
---|---|---|---|---|
Burg El-Arab Pop.1 & 2 | El-Gharbaniat Pop.3 | El-Hammam Pop.4 | El-Hammam Pop.5 & 6 | |
pH Conductivity mmhos/cm Sand% Silt% Clay% CaCO3 (mg/L) | 8.6 125 13 37 50 45.6 | 8 55.8 60 18 22 55.8 | 8.3 9.1 48 23 29 37 | 8.5 71.3 31 51 18 16.5 |
DNA was extracted from 2 g of young leaf tissue using DNA Plant Minipreps Kit (Bio Basic INC, Canada.), following the manufacturer instructions. All the PCR reactions were carried out in 25 μL volumes containing 50 ng of template DNA, 12.5 μl of PCR master mix buffer (2×) (Thermo, USA), and 20 pmol for each primer (
For the phylogenetic the internal transcribed spacer ITS1 and ITS2 regions and the 5.8S ribosomal DNA (rDNA) regions were amplified by using universal primers ITS1 (5’-TCCGTA GGTGAACCTTGCGG-3’) and ITS-4 (5’-TCCTCC GCTTATTGATATGC-3’) [
SCoT bands were binary scored; presence (1) or absence (0) characters to assemble the matrix of the SCoT phenotypes. Then, the indices of genetic diversity were calculated using POPGENE 3.2 Software [
Primer name | Primer sequence (5’-3’) | Total no. of bands | No. of polymorphic bands | No. of monomorphic bands | % of Polymorphism (P) |
---|---|---|---|---|---|
S4 | CAACAATGGCTACCACCT | 18 | 17 | 1 | 94.44 |
S6 | CAACAATGGCTACCACGC | 21 | 20 | 1 | 95.23 |
S7 | CAACAATGGCTACCACGG | 19 | 18 | 1 | 94.73 |
S9 | CAACAATGGCTACCACGT | 13 | 13 | 0 | 100 |
S10 | CAACAATGGCTACCAGCC | 19 | 19 | 0 | 100 |
S12 | ACGACATGGCGACCAACG | 15 | 13 | 2 | 86.66 |
S17 | ACCATGGCTACCACCGAG | 16 | 16 | 0 | 100 |
S32 | CCATGGCTACCACCGCAC | 19 | 19 | 0 | 100 |
S34 | ACCATGGCTACCACCGCA | 14 | 14 | 0 | 100 |
S36 | CATGGCTACCACCCGCCC | 23 | 23 | 0 | 100 |
Total | 177 | 172 | 5 | ||
Average | 17.7 | 17.2 | 0.5 | 97.10 |
Genetic similarity was calculated on the basis of genetic distance coefficient using the NTSYS-pc program [
Pairwise and multiple DNA sequence alignment were carried out using CLUSTAL W version 1.81 (http://seqtool.sdsc.edu/CGI/BW.cgi; Thompson et al. [
For analysis of variability of A. halimus, 10 primers were used for studying the SCoT banding patterns across the entire samples. A total of 177 amplification products were scored of which 172 were polymorphic, exhibiting 97.10% polymorphism. The amplification products using 10 primers ranged from 86.66% to 100% in producing polymorphic bands (
Ten primers (
The estimate of genetic structure of populations is significantly different from zero (P < 0.0001). Analysis of AMOVA showed that genetic variation (74.19%) was observed within the populations, whereas the variance among populations was 25.81% (
Populations | Sample size | Polymorphic loci (Np) | Percentage population level (PPL %) | Observed number of alleles (Na) | Number of effective alleles (Ne) | Shannon’s Index of diversity (I) | Nei’s gene diversity (h) | hS ± SD | ht ± SD | GST | Nm | |||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Pop1 | 4 | 98 | 55.37 | 1.553 | 1.368 | 0.323 | 0.218 | |||||||
Pop2 | 3 | 63 | 35.59 | 1.355 | 1.284 | 0.226 | 0.158 | |||||||
Pop3 | 3 | 82 | 46.33 | 1.463 | 1.371 | 0.294 | 0.205 | |||||||
Pop4 | 2 | 73 | 41.24 | 1.412 | 1.412 | 0.285 | 0.206 | |||||||
Pop5 | 3 | 67 | 37.85 | 1.378 | 1.325 | 0.240 | 0.168 | |||||||
Pop6 | 3 | 91 | 51.41 | 1.514 | 1.411 | 0.327 | 0.228 | |||||||
Population level | 3 | 79 | 44.63 | 1.445 | 1.361 | 0.282 | 0.197 | |||||||
Species level | 18 | 169 | 95.48 | 1.954 | 1.594 | 0.512 | 0.355 | 0.342 ± 0.022 | 0.218 ± 0.108 | 0.362 | 0.881 | |||
Np = number of polymorphic loci; PPL% = percentage of polymorphic loci; Na = observed number of alleles; Ne = effective number of alleles; I = Shannon’s information index; h = Nei’s gene diversity; hS = Gene diversity within population; ht = total gene diversity; SD = standard deviation; GST = diversity among populations; Nm = gene flow 0.25 (1 − GST)/GST.
Source of variation | df | Sum of squares | Variance components | Percentage variation | P |
---|---|---|---|---|---|
Among populations | 5 | 253.028 | 8.647 Va | 25.81 | *** |
Within populations | 12 | 298.250 | 24.854 Vb | 74.19 | *** |
Total | 17 | 551.278 | |||
Fixation Index (Fst) | 0.25813 | *** | |||
Among regions | 2 | 149.736 | 7.081 | 20.09 | ** |
Among populations Within regions | 3 | 103.292 | 3.310 | 9.39 | ** |
Within populations | 12 | 298.250 | 24.854 | 70.52 | ** |
***P < 0.0001, **P < 0.005.
flow Nm based on Gst was 0.880 (
To identify the source of the highest genetic variation, AMOVA analysis was also performed among groups; Group (1) includes the erect form and Group (2) includes the Chameophyte form populations. Genetic variation among the two groups was found to be low, that reaching 1.9% of the total variation. The amount of genetic variation among regions and among populations was 20.09% and 9.39%, respectively, with the reminder (70.52%) occurring within populations, suggesting that there is no significant genetic differentiation of populations.
The Mantel test showed a significant correlation between genetic distance and geographic distance (r = 0.673, P = 0.025).
The neighbour-joining dendrogram based on the genetic distance between populations revealed a similar pattern: the genetic distances among the populations showed a spatial pattern that corresponded to their geographic locations (
Based on UPGMA clustering algorithm generated from the obtained SCoT dataset, the populations were grouped into two distinct groups (
The correlation analysis indicated that the genetic diversity indices of different populations showed insignificant (P > 0.05) correlations between the genetic diversity indexes and the soil factors. This indicates that the soil factors had no effect on population’s structure and there were no local adaptation of the studied populations.
The ITS1-5.8S-ITS2 rDNA gene was successfully amplified from the six individuals of the six populations (
The study of genetic diversity of Atriplex halimus from diverse environment of the Mediterranean Basin showed a very high intra-populational diversity [
Results of SCoT analysis showed differences in the genetic diversity among populations of A. halimus from different locations. The total gene diversity (hT) and the genetic parameters (PPL%, I, h, Na, Ne) at population level were lower in Population 2 from Burg El-Arab and population 5 from El-Hammam than in the other
populations. In the present study, the mean value of Nei’s gene diversity index (h) was 0.197, near to the minimum h value (0.174 - 0.328) of nine out-crossing plants summarized by Schoen and Brown [
The analysis by AMOVA implied that 1/4 of genetic variation occurred among population and most of the variation (61.9%) settled within the populations. Our results are compatible with the pattern of species that are primarily outcrossing and long lived-wind pollinated shrub, which retain most of their genetic variability within populations [
Outcrossing species usually have a high within-population diversity and low population differentiation, whereas selfing species often have low within population diversity and high differentiation among populations [
The results (
The low estimates of gene flow (Nm = 0.881) among wind-pollinated A. halimus populations, correspond well with the geographic isolation of the populations, in which genetic differentiation among populations appears to be highly correlated with geographic distance between populations (r = 0.673, P = 0.025), although the detected geographical effect might be associated with differences between the type of isolation between populations (resort facilities, sand transport etc.). For example, Population 1 (bushy habit) and population 2 (erect habit) from Burg El-Arab are geographically close, but separated with a high genetic distance. The two locations are isolated by buildings of resorts, contrary to population 5 (bushy habit) and population 6 (erect habit) which are geographically and genetically close. The Nm would be low, with increased spatial isolation of small populations caused by habitat fragmentation [
Another factor may play an important role in the evolution of A. halimus; morphogenesis of reproductive structures is remarkably plastic in A. halimus, since flowers of both architectural patterns might be both male, female or hermaphroditic and their distribution along the reproductive axes as well as their relative occurrence were dependent on environmental conditions. A. halimus is mainly considered a monoecious species which is occasionally dioecious. However, some authors have found individuals that present unisexual and hermaphrodite flowers so this species could be polygamous or, more precisely, trimonoecious [
The present study used two forms of A. halimus shrubs; erect habit (A. halimus) and bushy habit shrub (A. schweinfurthii). The two morphotypes are used naturally isolated by a considerable distance from each other and occupying the same area to explore the effect of natural isolation on the genetic basis of the two forms using Start Codon Targeted (SCoT) and the phylogenetic relationships of A. halimus by sequencing ITS1-5.8S-ITS2 regions of the ribosomal DNA. Besides, we compared the previous results obtained with isozymes markers and RAPDs from Mediterranean basin to the present marker. According to previous reports, A. halimus includes two quite different groups in terms of habitat and morphology; subspecies halimus and subspecies schweinfurthii. However, these subspecies described by Le Houérou are not accepted taxonomical units. Ortíz-Dorda [
Habitat fragmentation through land use change can limit connectivity between populations and gene flow between A. halimus populations. There is interference between the two forms of A. halimus viz. A. halimus and A. schweinfurthii. We conclude that the two forms do not merit specific rank in presence of intermediate morphotypes between the two forms and absence of a breeding barrier.
The authors would like to express their thanks to Prof. Dr. Sania Ahmed Kamal for her aspiring guidance and advice during the field trips habitat description and samples collection. Her help in analyzing the soil data is greatly indebted.
Asmaa Elframawy,Hisham Deif,Ranya El-Bakatoushi, (2016) Genetic Variation among Fragmented Populations of Atriplex halimus L. Using Start Codon Targeted (SCoT) and ITS1-5.8S-ITS2 Region Markers. American Journal of Molecular Biology,06,101-115. doi: 10.4236/ajmb.2016.62011
The sequences of the fragment ribosomal DNA amplified by PCR of the six studied populations.
El-Hammam (6) CCGGGGAATCGCTTCGCCTTGGCGGGGCGTCCTTCCCGGCACAATAACGAACCCCGGCGC
Burg El-Arab (2) CCGGGGAATCGCTTCGCCTTGGCGGGGCGTCCTTCCCGGCACAATAACGAACCCCGGCGC
El-Gharbaniat (3) CCGGGGAATCGCTTCGCCTTGGCGGGGCGTCCTTCCCGGCACAATAACGAACCCCGGCGC
El-Hammam (4) CCGGGGAATCGCTTCGCCTTGGCGGGGCGTCCTTCCCGGCACAATAACGAACCCCGGCGC
El-Hammam (5) CCGGGGAATCGCTTCCCCTTGGCGGGGCGTCCTTCCCGGCATAATAACCAACCCCGGCGC
Burg El-Arab (1) CCGGGGAATCGCTTCGCCTTGGCGGGGCGTCCTTCCCGGCACAATAACGAACCCCGGCGC
*************** ************************* ****** ***********
El-Hammam (6) GGTCTGCGCCAAGGAACATGAATACAAGCGTGCCCTTCTCCGACCGGTTCGCCGGTCGTG
Burg El-Arab (2) GGTCTGCGCCAAGGAACATGAATACAAGCGTGCCCTTCTCCGACCGGTTCGCCGGTCGTG
El-Gharbaniat (3) GGTCTGCGCCAAGGAACATGAATACAAGCGTGCCCTTCTCCGACCGGTTCGCCGGTCGTG
El-Hammam (4) GGTCTGCGCCAAGGAACATGAATACAAGCGTGCCCTTCTCCGACCGGTTCGCCGGTCGTG
El-Hammam (5) GGTCTGCGCCAAGGAACATGAATACAAGCGTGCCCTTCTCCGACTGGTTCGCCGGTCGTG
Burg El-Arab (1) GGTCTGCGCCAAGGAACATGAATACAAGCGTGCCCTTCTCCGACCGGTTCGCCGGTCGTG
******************************************** ***************
El-Hammam (6) GACGTGGCACCAAGTCGTATATAACATTAAACGACTCTCGGCAACGGATATCTCGGCTCT
Burg El-Arab (2) GACGTGGCACCAAGTCGTATATAACATTAAACGACTCTCGGCAACGGATATCTCGGCTCT
El-Gharbaniat (3) GACGTGGCACCAAGTCGTATATAACATTAAACGACTCTCGGCAACGGATATCTCGGCTCT
El-Hammam (4) GACGTGGCACCAAGTCGTATATAACATTAAACGACTCTCGGCAACGGATATCTCGGCTCT
El-Hammam (5) GACGTGGCACCAAGTCGTATATAACATTAAACGACTCTCGGCAACGGATATCTCGGCTCT
Burg El-Arab (1) GACGTGGCACCAAGTCGTATATAACATTAAACGACTCTCGGCAACGGATATCTCGGCTCT
************************************************************
El-Hammam (6) CGCATCGATGAAGAACGTAGCGAAATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAA
Burg El-Arab (2) CGCATCGATGAAGAACGTAGCGAAATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAA
El-Gharbaniat (3) CGCATCGATGAAGAACGTAGCGAAATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAA
El-Hammam (4) CGCATCGATGAAGAACGTAGCGAAATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAA
El-Hammam (5) CGCATCGATGAAGAACGTAGCGAAATGCGATACTTGGTGTGAATTGCACAATCCCGTGAA
Burg El-Arab (1) CGCATCGATGAAGAACGTAGCGAAATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAA
************************************************ ***********
El-Hammam (6) CCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCTTTAGGTTGAGGGCACGCCTGCCT
Burg El-Arab (2) CCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCTTTAGGTTGAGGGCACGCCTGCCT
El-Gharbaniat (3) CCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCTTTAGGTTGAGGGCACGCCTGCCT
El-Hammam (4) CCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCTTTAGGTTGAGGGCACGCCTGCCT
El-Hammam (5) CCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCTTTAGGTTGAGGGCACGCCTGCCT
Burg El-Arab (1) CCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCTTTAGGTTGAGGGCACGCCTGCCT
************************************************************
El-Hammam (6) GGGCGTCACGCATCGCGTCTCCCCCCACCACCCCGTGTGGATGGGGAGGAGGATGATGGC
Burg El-Arab (2) GGGCGTCACGCATCGCGTCTCCCCCCACCACCCCGTGTGGATGGGGAGGAGGATGATGGC
El-Gharbaniat (3) GGGCGTCACGCATCGCGTCTCCCCCCACCACCCCGTGTGGATGGGGAGGAGGATGATGGC
El-Hammam (4) GGGCGTCACGCATCGCGTCTCCCCCCACCACCCCGTGTGGATGGGGAGGAGGATGATGGC
El-Hammam (5) GGGCGTCACGCATCGCGTCTCCCCCCACCACCCCGTGTGGATGGGGAGGAGGATGATGGC
Burg El-Arab (1) GGGCGTCACGCATCGCGTCTCCCCCCACCACCCCGTGTGGATGGGGAGGAGGATGATGGC
************************************************************
El-Hammam (6) CTCCCATGCCTCACCGGGCGTGGATGGCCTAAATATGGAGCCCCCGGTTACGAAGTGCCG
Burg El-Arab (2) CTCCCATGCCTCACCGGGCGTGGATGGCCTAAATATGGAGCCCCCGGTTACGAAGTGCCG
El-Gharbaniat (3) CTCCCATGCCTCACCGGGCGTGGATGGCCTAAATATGGAGCCCCCGGTTACGAAGTGCCG
El-Hammam (4) CTCCCATGCCTCACCGGGCGTGGATGGCCTAAATATGGAGCCCCCGGTTACGAAGTGCCG
El-Hammam (5) CTCCCATGCCTCACCGGGCGTGGATGGCCTAAATATGGAGCCCCCGGTTACNAANTGCCG
Burg El-Arab (1) CTCCCATGCCTCACCGGGCGTGGATGGCCTAAATATGGAGCCCCCGGTTACGAAGTGCCG
*************************************************** ** *****
El-Hammam (6) CGGCAATTGGTGGAATACAAGGCCACGCCTAGGATGAAACGGTAGTCGCGCACATCGTGG
Burg El-Arab (2) CGGCAATTGGTGGAATACAAGGCCACGCCTAGGATGAAACGGTAGTCGCGCACATCGTGG
El-Gharbaniat (3) CGGCAATTGGTGGAATACAAGGCCACGCCTAGGATGAAACGGTAGTCGCGCACATCGTGG
El-Hammam (4) CGGCAATTGGTGGAATACAAGGCCACGCCTANGATGAAACGGTAGTCGCGCACATCGTGG
El-Hammam (5) CGGNNNTTGGTGGAATACAAGGNCACCCCTANGATGAAACGGTANTCGCGCACATCNTGG
Burg El-Arab (1) CGGCAATTGGTGGAATACAAGGCCACGCCTAGGATGAAACGGTAGTCGCGCACATCGTGG
*** **************** *** **** ************ *********** ***
El-Hammam (6) CTCTTGAGGACTCGCAGGACCCTTACTTGTTTGCCCCTAGGGGCGGCAAAACCGTTGCGA
Burg El-Arab (2) CTCTTGAGGACTCGCAGGACCCTTACTTGTTTGCCCCTAGGGGCGGCAAAACCGTTGCGA
El-Gharbaniat (3) CTCTTGAGGACTCGCAGGACCCTTACTTGTTTGCCCCTAGGGGCGGCAAAACCGTTGCGA
El-Hammam (4) CTCTTGAGGACTCGCAGGACCCTTACTTGTTTGCCCCTAGGGGCGGCAAAACCGTTGCGA
El-Hammam (5) CTCTTGANGACTNNNNNGACCCTTACTTGTTTGCCCCTANGGGCGGCAAAACCGTTGCGA
Burg El-Arab (1) CTCTTGAGGACTCGCAGGACCCTTACTTGTTTGCCCCTAGGGGCGGCAAAACCGTTGCGA
******* **** ********************** ********************
El-Hammam (6) CCCC-AGGTCAGGCGGGGCTACCCGCTGAGTTTAAGCATAT
Burg El-Arab (2) CCCC-AGGTCAGGCGGGGCTACCCGCTGANTTTAAGCATAT
El-Gharbaniat (3) CCCC-AGGTCAGGCGGGGCTACCCGCTGAGTTTAAGCATAT
El-Hammam (4) CCCC-AGGTCAGGCGGGGCTACCCGCTGAGTTTAANCATAT
El-Hammam (5) CCCCCAGGTCAGGCGGNGCTACCCGCTGAGTTTAAGCATAT
Burg El-Arab (1) CCCC-AGGTCAGGCGGGGCTACCCGCTGAGTTTAAGCATAT
**** *********** ************ ***** *****