1. Population Structure and Genetic Diversity in South Tunisian Hedysarum
Mohamed Neji, Sondes Rahmouni, Malek Smida, Wael Taamalli, Mhemmed Gandour et Chedly Abdelly
Corresponding author: Mhemmed Gandour
Laboratory of Extremophiles Plants, Centre of Biotechnology of Borj-Cédria
Abstract:
Hedysarum carnosum is diploid (2n=16), allogamous, seed-propagated species and has a vegetative shoot system characterized by an orthotropic main stem bearing some lateral plageotropic shoots. Leaves are symmetrical on the first one and non symmetrical on the second ones. Its agronomical interest is based on its high protein content, suitable feeding value and favorable environmental impact. It is of interest for rangeland revegetation and production. This study characterize the genetic relationships of H. carnosum populations, so plant breeders and geneticists will have the necessary information to maintain a broad genetic base within selected germplasm populations. Random Amplified Polymorphic DNA markers were used on 5 populations. Fourteen percent of the total genetic variation was found between populations, in addition each population showed significant isolation by distance with a rate of 3.1 of gene flow between populations and by generation. Cluster analysis reveals three groups of metapopulations: the first group is in the extreme south of Tunisia (Douiret), the second is in the west southern of Tunisia (Metlaoui) and the last one contains Sidi Bouzid, Kairouan and Karkar populations.
Table 3: Pairwise Nei's genetic distances matrix between H. carnosum populations
Material and methods
1-DNA extraction
DNA was extracted from fresh leaves of 45 days aged plants according to Geuna et al
2- PCR Amplification
10 primers for Random Amplified Polymorphic DNA (RAPD) were used. PCR products were separated by electrophoresis in 1.5% TBE agarose gel, stained with ethidium bromide and photographed under UV in a gel-doc system.
3-Statistical analysis
Based on the binary matrix obtained, genetic diversity indexes (the percentage of polymorphic bands (PPB), the observed number of alleles (Na), the expected number of alleles (Ne), Nei’s gene diversity (He), and Shannon’s information index of diversity (I)), were calculated by POPGENE 32 program.
Nei’s genetic differentiation index (Gst = [(Ht − Hs)/Ht]), gene flow (Nm = 0.5 (1–Gst) / Gst) were calculated using the population mean diversity and the total diversity indexes.
A dendrogram based on Nei’s genetic distance coefficient matrix was generated using the same program
pop
Douiret
Kairouan
Karkar
Sidi Bouzid
Metlaoui
0.1215
0.1137
0.0507
0.0945
0.0604
0.0567
0.0813
0.0934
0.1182
0.1047
Fig 2: Percentages of polymorphism for the five studied Hedysarum carnosum populations
Table 2: Genetic structure of Hedysarum carnosum populations
Expected number of alleles per locus (Ne) within populations
1.191
gene diversity within populations (Hs)
0.31
Gene diversity at the species level (Ht)
0.36
Shannon’s index (I)
0.48
Nei’s genetic differentiation index (Gst )
0.14
gene flow (Nm )
3.05
Fig 3: UPGMA dendrogram based on Nei’s genetic distance matrix clustered the fife-studied populations into two groups.
Results
Table 1: Nucleotide sequences of the primers used in RAPD analysis, total number of bands per primer and the percentages of polymorphism.
Conclusions
Results were coherent to show a moderate genetic differentiation among the studied populations
The values of genetic differentiation detected among populations (Gst = 0.14) were comparable to the average for outcrossing plant species (Gst = 0.22)
The high level of gene flow may be from the concerned populations being in close proximity and the outcrossing nature of H. carnosum.
the populations of H. carnosum showed a pattern of genetic differentiation related to the regional ecogeography and environmental conditions with a proportion of the variation explained by the geographical distance.
primer
Séquence (5’- 3’)
Total Nre B
% of polymorphism
A04
AGTCAGCCAC 10
100
D05
TGAGCGGACA 4
Q11
GTAGCACTCC 12
R18
TCTCCGCAAC 11
S20
TCGCCCAGTC 13
S19
TCCTGGTCCC 8
S12
CTGGGTGAGT
S02
GAGTCAGCAG 9
S09
TCTGGACGGA
T12
GGGTGTGTAG
Total 96
The Gst value was 0.14, indicating that genetic diversity in S.carnosawas moderately low.
14% of the total genetic variability was distributed among populations
The overall level of inferred gene flow (Nm) was estimated at 3.05 individuals among populations and per generation, indicating a striking gene flow and notable pollen and seed dispersal between populations.
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Fig 1: localization of studied populations