INTRODUCTION
Alfalfa (Medicago sativa L.) is the most cultivated forage legume.
It is an autotetraploid with 2n = 4x = 32; (Dermaly, 1954) and allogamous
species.
Alfalfa (Medicago sativa L.) is a cross pollinated forage legume
(Julier et al., 2000), a traditional forage crop, widely used for
animal feeding. Lucerne (alfalfa) is the most important sown perennial
fodder in Tunisia. It is widely adapted and can be found throughout the
country. Lucerne originates from Asia Minor, Transcaucasia, Iran and Turkmenistan
and is diversified in the Mediterranean area.
Variability for agronomic and morphological traits of alfalfa is frequently
used in breeding programs for developing cultivars with a high forage
production and quality but seed-yielding ability has rarely been an important
criterion in the early stages of selection programs (Julier et al.,
2000). Alfalfa is able to fix atmospheric nitrogen in symbiosis with Rhizobium
meliloti, (Kiss et al., 1992).
Several methods were used to study the level of differentiation among
a set of cultivars or populations using biochemical, isozymes and molecular
markers, DNA polymorphisms, such as Restriction Fragment Length Polymorphisms
(RFLPs) or Random Amplified Polymorphic DNAs (RAPDs), Simple Sequence
Repeat (SSR), it is possible to efficiently mark any portion of the genome
for inheritance studies. RFLP are generally detected using low copy nuclear
DNA sequences as hybridation probes on Southern blots of restriction genomic
DNA (Botstein et al., 1980), RAPDs are identified by the Polymerase
Chain Reaction (PCR) using arbitrary primers, making them simpler to assay
than RFLPs and can detect polymorphism in both low copy and repetitive
DNA sequences (William et al., 1990). While, most RFLP markers
are codominants and may detect many alleles at a locus, most RAPD markers
are dominants and can detect only two alleles for a locus (presence or
absence of the marker). Dominant RAPD markers thus provide less genetic
information than RFLP markers in certain mating and may segregate in only
a limited number of populations (Echt et al., 1992). The use of
SSR loci as polymorphic DNA markers has expanded considerably over the
past decade both in the number of studies and in the number of organisms,
primarily due to their facility and power for population genetic analyses
(Estoup and Angers, 1998). To date, few studies have been conducted using
SSR markers to assess the level of variation among perennial Medicago
species and populations. Diwan et al. (1997) have been the first
to develop SSR markers in Medicago. They have shown how SSR can
be used to describe genetic diversity and to analyze the genetic relationships
among genotypes in alfalfa. Recently a set of 107 SSRs identified in the
EST data base of Medicago truncatula was mapped in Medicago
sativa (Julier et al., 2003) and can be used to perform genetic
diversity analysis. Among these markers, some were easy to score allele
doses with tetraploid genotypic information.
Inter Simple Sequence Repeat (ISSR) is a dominant molecular marker revealed
in mass. Under conditions of adapted amplifications, the DNA fragments
were separated in agarose or acrylamide gel.
The genetic resources presently available are the register varieties
and the wild populations and the landraces in which most of the genetic
diversity in most of the cultivated species underlines the need to describe
the genetic diversity available in the material under collection which
could be stored as genetic resources or used in breeding programs.
In the present study, we report the use of molecular markers such as
ISSR for the assessment of genetic diversity and relationships among different
populations of cultivated alfalfa collected from three different sites
of Tunisian south, Gabes, Tozeur and Kebilli; Italy; France; Morocco and
Austerely.
MATERIALS AND METHODS
Plant material: Twenty nine populations of the cultivated alfalfa
(Medicago sativa) were involved in this study, including 15 local
originating in the Tunisian South and 12 introduced. They are shown in
Table 1. This study was carried out in experiment field
of Institute Arid Area of Medenine. Tunisia Seeds of different genotype
were sowed in April 2005. The young leaflets were carried two month after
this date. There are dried and conserved at-40 °C for other use.
| Table 1: |
List of various studied populations of cultivated alfalfa |
 |
DNA extraction
Experimental protocol: Genomic DNA was extracted from young
leaves of each plant following the method described by Pallotta et
al. (2003) with minor modifications relating to the treatment of the
AND with two enzymes: proteinase K and RNase.
Quality and quantity of the DNA
Optical density: OD260 nm: DNA concentration was determined
by spectrophotometer at 260 nm. The measurement of the OD 280 nm is used
to detect the contaminants. The ratio OD260/OD280 must situate between
1.8 and 2. A value lower than 1.8 testifies a contaminations by proteins,
whereas a value higher than 2 testifies to a contamination by salts.
Electrophoresis on agarose gel: DNA concentration was determined
by 2% agarose gel. The absence of smear testifies the purity to the DNA.
Primers and ISSR-PCR assays: The detection of genetic polymorphism
inter populations has been performed using 19 primers. These were based
on either di- or- multinucleotide repeats that were complementary to microsatellite
(Table 2). The dinucléotides repeats were anchored
at 3` ends.
DNA samples of the 29 individuals plants were adjusted to 80 ng μL-1
and used in the amplification reactions with a final volume of 20 μL
containing: 80 ng DNA; 1.6 μL of primer (10 μM); 1.5 μL
of dNTP (2 mM); 0.2 μL Taq DNA polymerase (5 u μL-1);
2 μL of PCR Buffer (1 X); 2 μL of MgCl2 (25 mM),
adjusted at 20 μL by deionised water. DNA amplification was carried
out using a Gene Amp PCR System 9700 thermal cycler programmed with 6
min at 94 °C for initial denaturation, followed by 35 cycles of 30
sec at 94 °C; 45 sec at 72 °C. After DNA amplification, the DNA
fragments were separated by electrophoresis for about 2 h under constant
voltage (60 V) in 3% agarose gel submersed in 1X TBE buffer. The gels
were stained with ethidium bromide solution and observed under ultraviolet
light. Each gel was photo documented using the image capturing system
bio print. The Jules DNA ladder (Q Bio gene) was used standard molecular
weight marker. The gels obtained by the two primers A12 and UBC 818 were
represented, respectively by the Fig. 1 and 2.
| Table 2: |
Various primers tested |
 |
 |
| Fig. 1: |
ISSR PCR fingerprints of 29 populations of cultivated
alfalfa (Medicago sativa L.) using the 3` anchored primer (GA)
6CC. M: Molecular size (pb); 2-35: DNA templates |
 |
| Fig. 2: |
ISSR PCR fingerprints of some populations of cultivated
alfalfa (Medicago sativa L.) using the 3` anchored primer (CA)
7G. M: Molecular size (pb); 2-35: DNA templates |
Data analysis: The amplification bands were scored as 1 and 0
based on band (allele) presence and absence, respectively. Sizes amplification
bands were estimated using Gel Pro analyser software. The similarity of
all samples for at scored bands was assessed using Rogers and Tanimoto
(1960) similarity coefficient. The matrix generated were analysed with
SPSS version 12 software to group the different populations by hierarchical
classification.
RESULTS
Molecular polymorphism: The 19 primers were screen for their ability
to generate ISSR polymorphic DNA bands using the accessions total DNA.
The Number of Total Bands and their polymorphism and Percentage of Polymorphism
(%) were present in Table 3. According to this table
one notes that: The total number of bands varies between the various populations
from 9-16; the number of polymorphic bands lies between 4 and 11; the
percentage of polymorphic bands is located between 44 and 67% and the
percentage of total polymorphism is about 60%.
The data exhibit that the dinucléotides repeat: A12 [(GA) 6CC]
and UBC-818 [(CA) 7G] are more informative than tetra nucleotide and three
nucleotide ones.
Figure 1 shows typical examples of the amplified ISSR
banding patterns with DNA stretches ranging from 1200-1900 pb. In this
case A12 oligonucleotid was applied using all the samples DNA.
Figure 2 shows typical examples of the amplified ISSR
banding patterns by UBC-818 oligonucleotid.
A total of 355 ISSR bands were amplified using the two available primers
with 29 populations of cultivated alfalfa. Among these bands 210 bands
are unambiguously reproducible and polymorphic. The percentage of polymorphic
bands and the percentage of polymorphism inter populations were estimated.
| Table 3: |
A number of polymorphic bands and their percentage of
polymorphism for the different populations, by combining the two starters:
A12 and UBC - 818 |
 |
Relationships among populations of alfalfa: Hierarchical classification:
Cluster analysis (Fig. 3) devised the 29 populations
into four large groups:
Group 1 (G1): It is formed by the majority of the introduced populations:
Melissa, Mamuntanas, Demnat, Rich2, Siriver, Coussouls, Ecotiposiciliano,
African ABT805, Ameristand and Erfoud which have got 12 at 16 bands with
a molecular weight between 1300 and 1900 pb.
Group 2 (G2): It is included several locals` populations: Tboulbou,
Metwia, Essdada, Ghannouch, Zerkine and Nouael and a single introduced
population, Sardi, which have got 12 at 17 bands with a molecular weight
between 1550 and 1950 pb.
Group 3 (G3): It is included only one local population, Dgach,
which have got 12 bands, with molecular weight between 1300 and 1800 pb.
Group 4 (G4): It is included several locals populations: Chenchou,
Limaguess, Elgolaa, Douz, Zaafarane, Jersine, Bouhlel, Hammajerid, Chenenni
1 and Chenenni 3, which have got 9 at 13 bands, which a molecular weight
between 1500 and 1800 pb.
 |
| Fig. 3: |
Dendrogram grouped the various populations after combining
the two primers: A12 and UBC-818 by hierarchical classification |
DISCUSSION
The cultivated alfalfa (Medicago sativa L.) is characterized by
a height genetic variability (Mauriès, 2003). The study of genetic
diversity, in this research is based on mineral composition, some morphological
traits and ISSR markers.
The clustering grouped the studied populations into three clusters with
no correlation to geographical origins.
In this study we conclude that the dinucléotides repeat: A12 [(GA)
6CC] and UBC-818 [(CA) 7G] are more informative than tetra nucleotide
and three nucleotide ones. By combining the two primers, the total number
of bands varied between 9-16 and the polymorphic bands from 4-11. These
markers were successfully for the polymorphism genetic study of maize,
rice, potato, to study the genetic relations between the various coffee
species. These markers are also used to identify and analyze genetic variability
of date palm. In addition to the ISSR markers, several molecular markers
are used to identify and to study of genetic diversity of cultivated alfalfa,
as the RAPD markers witch are very much used for the Medicago genus,
primarily, to estimate the genetic relations of cultivated alfalfa (Yu
and Pauls, 1993a) the development of the genetic carts (Echt et al.,
1993) analysis of the genetic variability of the diploids annual species,
for the characterization of the Medicago species and to target
the genes (Yu and Pauls, 1993b). SSR markers were used by Julier et
al. (2005) to study the genetic diversity of the cultivated alfalfa
(Medicago sativa L.).
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