The genus Opuntia belongs to the Cactaceae family (subfamily Opuntioideae)
and includes several species that originated in North and South America, some
of which were relatively recently distributed throughout the world. The number
of species included in the genus is unknown (Chavez-Moreno
et al., 2009). The cactus was introduced to the Mediterranean during
the 16th century (Barbera, 1995). In Morocco, Opuntia
ficus represented particularly in arid and semi-arid areas, from Oriental
(Oujda, Figuig) to South (Sidi Ifni) through the arid plateaus of Haouz. Cactus
pear constitutes, in these areas a resource to control water and wind erosion
in eroded soils. The taxonomy of cacti has been traditionally based on comparative
observation of morphological and biogeographic data (Metzing
and Kiesling, 2008). The knowledge of the detailed descriptions are needed
in the use of germplasm, because in this germplasm may have valuable genes for
use in breeding programs.
Due to the multiple uses and the ability of cacti to thrive in arid and semiarid
environments, it has become increasingly important to describe and characterize
these valuable resources. The latter is a challenging goal since up to now;
knowledge regarding the amount of genetic variation and genetic relationship
by means of molecular tools is missing in Moroccan Opuntia ficus indica. In
fact, though this crop is widely cultivated in the country, the majority of
research works were especially oriented towards the characterization of the
nutritional value of the cladodes as an important fodder crop in arid areas,
independently from their genetic potential (Boujghagh and
Chajia, 2001). At present, molecular markers have been proved to be valuable
tools in the characterization and evaluation of genetic diversity within and
between species and populations. It has been shown that different markers might
reveal different classes of variation (Russell et al.,
1997). The advent of the Polymerase Chain Reaction (PCR) favored the development
of different molecular techniques (Saiki et al.,
1988). These molecular markers had been successfully used in Opuntia genus
for detecting genetic diversity and relationships (Arnholdt-Schmitt
et al., 2001; Labra et al., 2003).
Of these techniques, RAPD has several advantages, such as simplicity of use,
low cost and the use of small amount of plant material, etc.
The success of cactus plantations in arid and sub-arid environments insists
on leading researches on the management of Moroccan genetic resources, which
will permit a well knowledge of the planted cacti. At the present, no study
is yet available based on genetic variation and relationship by molecular tools
in Opuntia ficus indica of Morocco.
The objectives of the present study were to assess the usefulness of RAPD to
differentiate Opuntia ecotypes and to investigate genetic relationships
among different ecotypes and to set up rational decisions concerning the establishment
of a national reference collection. Indeed, though this crop is widely cultivated
in the country, collection repositories are missing.
MATERIALS AND METHODS
The Cladodes of Opuntia ficus indica, representing 13 provenances were
collected from 13 localities in Morocco (Table 1) and planted
in Faculty of Sciences Agadir, this material were used for molecular polymorphism
(RAPD) research. For each accession, an external slice of the cladode was taken
for analysis. A piece of about 1 g of the chlorenchyma was cut using a scalpel
and taking care not to include areole. The protocol of DNA extraction used here
is that of (Saghai-Maroof et al., 1984) modified.
DNA was quantified by visual comparison with lambda DNA molecular marker on
ethidim bromide stained agarose gels.
The amplification is performed according to the protocol (Arnholdt-Schmitt
et al., 2001
), 14 oligonucleotide (decamer) were used for the amplification
of random DNA (OPA-03, OPA-08, OPA-09, OPA-10, OPA-11, OPA-12, OPA-13, OPA-14,
OPA-15, OPA-16, OPA-17, OPA-18, OPA-19, OPA-20). PCR reactions were performed
in a 25 mL reaction mixture containing: 2.5 ng of template DNA, 2.5 mL of Go Taq
buffer (Promega), 3.5 mL of dNTPs, 2 mL of 25 mM MgCl2
, 25 pmol of
primer and 0.5 U of Go Taq DNA polymerase (Promega). The PCR was performed in
a Thermoblock thermocycler (Techne).
PCR amplification was performed using the following profile: 5 min at 95°C;
45 cycles of denaturation (1 min at 95°C), annealing (1 min at 36°C)
and extension (5 min at 72°C); then a final step for 5 min at 72°C.
Products of the PCR were separated by electrophoresis in 2% agarose gels using
a volt range of 2 Vcm-1 during 3 h. The gel was finally fixed with
ethidium bromide and photographed under UV light.
||Geographic localization of several provenances of Opuntia
ficus indica under study
Size of alleles was defined using the molecular weight marker 100 bp DNA leaders.
Different RAPD amplified bands were scored for their presence (1) or absence
(0) and the resulting data matrices were computed with Nei coefficient to provide
a similarity matrix. Different parameters have been estimated and the ability
of primers to distinguish between individuals was calculated by evaluating the
resolving power, POPGENE32 software was used to treat the binary matrices obtained
to estimate genetic parameters intra-provenances (%P: percentage of loci polymorphic,
h: Nei's genetic diversity (Nei, 1973) and inter-provenance
(Gst: coefficient of genetic differentiation and genetic distance (Nei,
RESULTS AND DISCUSSION
Photometric measurements at 260 and 280 nm of the nucleic acid extracts of
the various accessions of Barbary fig indicated a quotient (260/280) between
1.82 and 2.02. Thus, the obtained DNA is of high quality.
Among the 14 primers used to assess polymorphism in the tested ecotypes, 13
have revealed unambiguously scorable bands, these mentioned primers generated
multiple banding each produced two to twenty readable tapes and are reproducible
(Table 2). OPA-13 primer was abandoned because of the difficulty
of reading the gel. A total of 105 bands were obtained which 55 were monomorphic
and 50 were polymorphic. The average number of bands per primer was 8.07. All
bands reported 73 different electrophoretic profiles. The resolving power of
primers varied from 0 for the primer OPA-18 to 7.68 for primer OPA-14 with an
average value of 1.55. The primer OPA-14, followed by primer OPA-11 and OPA-12,
primers seem to be the most efficient to assess the genetic diversity, since
they presented relatively high resolving power rates among the prickly pear
Genetic diversity (h) has a nil value for the accessions analyzed of Meknes
Mohammedia and Safi and a value ranges from 0.01 to 0.11 for other provenances
(Fig. 1). Thus, the ecotypes tested from Meknes, Mohammedia
and Safi are monomorphic, while other ecotypes have different levels of polymorphism.
The ecotype of Chaoun is the most highly polymorphic (h = 0.11).
Gst coefficient has a value of 0.29 if we takes into account all the loci studied
and 0.75 if we limit only to polymorphic loci. It seems that in both cases,
studied provenances have a significant structuring and gene flows are very limited.
Gst furthermore the Gst coefficient has the greatest value for loci OPA-11-01-03
and OPA-04 (Gst = 1).
|| Neis Genetic diversity (h) within provenances
|| Dendrogramme of 13 provenances of Opuntia ficus indica
throughout Neis genetic distance
Thus, these loci characterize provenances of Ait Baha and Tafraout. The genetic
distance of (Nei, 1978) in terms of dendrogram (Fig.
2, 3) shows different levels of similarities and dissimilarities
between studied provenances.
Therefore, apart from provenances of Safi and Mohamedia which are similar,
all others provenances shows different cases, including the isolation of Ait
Baha and Tafraout on the one hand and Berkane and Meknes on the other hand.
These results showed a disparity between the varieties named Moussa (late) and
Aissa (early) from Sidi Ifni. Also, the geographical distribution of the studied
provenances is partially respected throughout the genetic similarities.
The results presented in this study are the first contributions to the application
of molecular markers to assess the genetic diversity of prickly pear in Morocco.
Exploring RAPD showed that provenances studied are genetically quite divergent.
The loci analyzed participated differently to the description of this divergence.
Thus, some loci were polymorphic and easily characterized some provenances.
These results may contribute to the establishment of a conservation program
and management of the genetic diversity of prickly pear in Morocco. They can
be developed by analyzing other markers with a larger sample. This work could
also be combined to agro morphological traits to start a breeding program and
a genetic selection.