Mohamed Marrakchi

Diversity of pomegranate (Punica granatum L.) germplasm in Tunisia

Thirty pomegranate (Punica granatum L.) accessions were studied to determine the overall degree of polymorphism and to detect similarities among genotypes. PCA and cluster analysis showed a considerable phenotypic and genetic diversity in the local pomegranate germplasm. Some polyclonal varieties were identified and cases of homonymy were detected. The geographic origin was not a determinant criterion for cultivars clustering. Parameters with high discriminating values were those related to fruit size and color and juice characteristics. Factors affecting pomegranate germplasm diversity are discussed.

Genetic diversity in Tunisian perennial ryegrass revealed by ISSR markers

The genetic diversity in Tunisian perennial ryegrass (Lolium perenne) was examined by the help of inter-simple sequence repeats (ISSR). Starting from eighteen accessions, a large number of polymorphic ISSR markers were currently generated using appropriate primers (a total of 136, which average of 12.6 polymorphic bands/primer). These markers were considered to estimate the genetic distance among accessions and to draw phylogenetic trees. Our data provide evidence of a high degree of genetic diversity in Tunisian ryegrass. In addition, both cultivars and wild types present a high degree of divergence suggesting a complex domestication process in this crop. Moreover, spontaneous populations of Tunisian ryegrass have been identified as important ecotypes that are suitable in selection programs to improve grasslands.

Genetic inheritance analysis of four enzymes in date-palm (Phoenix dactylifera L.)

The genetic inheritance of four date-palm enzymes hasbeen carried out in 29 Tunisian cultivars. Our data provide evidenceof five polymorphic genes involving 12 alleles. It has been assumedthat either Pgm or Shdh are specified by three different alleles,while Got-1, Got-2 and Pgi-2 loci are biallelicgenes. On the other hand, analysis throughout progenies of twocontrolled crosses support significantly these hypothesis. In thewhole, results are in agreement with reports that describeddate-palm isozymes. Nevertheless, we assume that other alleleare involved in Pgm gene. Also, isozymes controlled by Got-2locus are of a dimeric structure. Opportunely, it can be establishedthat the resultant isoenzymes would constitute polymorphic markerssuitable to analyze the genetic diversity in date-palm.

Isozymic polymorphism and phylogeny of 10 Lathyrus species

Isozyme variation and phylogenetic relationships between ten annual andperennial Lathyrus species: L.aphaca, L.articulatus, L.cicera, L.hirsutus, L.latifolius, L.nissolia, L.odoratus, L.ochrus, L.sativus and L.sylvestris were studied. Four enzyme systems,leucine-amino-peptidase (LAP), 6-phosphogluconatedeshydrogenase (6-PGD), glutamateoxalo-acetate transaminase (GOT) and phosphoglucoisomerase(PGI) were analyzed, using gel electrophoresis. Fivepolymorphic loci were detected and eleven alleles were identified at these loci.Zymogram data revealed that almost all-present species studied exhibited isozymepolymorphism. L. latifolius andL. sylvestris maintain high levels ofisozyme diversity, which is probably associated with the perenniality of thesetwo species and their predominantly outcrossing reproduction system. Incontrast, the low level of genetic diversity observed in other species isattributed to their breeding systems. These species are annuals and have higherproportions of selfing. The distribution of genetic variation within and amongspecies showed large genetic differences between the analysed species.PGD-1 andPGD-2 loci contributed the most to thedistinction between species. GOT-2,LAP-1 and PGI-1loci contributed to the distinction within species. The low level of gene flowrevealed could be partly related to the high level of autogamy in the majorityof species. The regroupement of species revealed by Neis genetic similarityagrees only in parte with Kupichas classification based on morphologicalcharacters. Thus, these isozymic markers are important in germplasm collectionand conservation.

Use of Microsatellite Polymorphisms to Develop an Identification Key for Tunisian Apricots

Microsatellite polymorphisms in 54 apricot landrace cultivars were identified by using 26 Prunus microsatellite primers. Samples of apricot cultivars were collected in eight growing regions in Tunisia ranging from the north to the south of the country and from the sub-humid to the saharian areas. The primers revealed 103 alleles and 155 different genotypes among the 54 apricot accessions. The most polymorphic microsatellite loci were used to develop an identification key. Five microsatellite primers and the 28 resulting alleles were sufficient to discriminate among all 54 cultivars. These results are discussed in the context of cultivar nomenclature, geographic origins, and the comprehensive fingerprinting of Tunisian apricot collections.

Genetic diversity in ecotypes of Tunisian date-palm (Phoenix dactylifera L.) assessed by AFLP markers

Summary Amplified fragment length polymorphisms (AFLP) were used successfully to survey genetic diversity in 40 ecotypes of date-palm (Phoenix dactylifera L.) collected from oases in Tunisia. Six primer pairs were screened to assess their ability to detect polymorphism in this tree crop. As a result, a total of 428 AFLPs have been generated and used to estimate genetic distances which ranged from 0.07 – 0.63. A large, and typically continuous, range of genetic diversity characterises Tunisian date-palm germplasm. In addition, the UPGMA dendrogram derived from these data exhibited two clusterings of ecotypes independent of their geographic origin or the sex of the trees. These data corroborate the hypothesis of the origin of date-palm domestication being in Mesopotamia. Moreover, taking into account the high percentage of polymorphic bands, together with their resolving power (Rp), all the primer pairs tested contributed to the discrimination of date-palm genotypes, suggesting that the AFLP method is efficient in assessing genetic diversity in this crop. The data are discussed in relation to the use of AFLP molecular markers in the management and improvement of date-palm.

Phylogenetic relationships in the North African genus Hedysarum as inferred from ITS sequences of nuclear ribosomal DNA

Sequences of the internal transcribed spacers (ITS) of nuclear ribosomal DNA were analysed to precise their length (637–643 bp) and resolve phylogenetic relationships among eight Mediterranean species of the genus Hedysarum (Fabaceae). The infra-specific variability levels of the ITS sequences of spontaneous population of H. coronarium proved a lack of polymorphism both in the length and in the sequences examined in this species. Hence, a consensus ITS sequence characterising each Hedysarum species has been investigated for analysis of inter-specific polymorphisms. The level of variation of ITS sequence was high enough to make the ITS1 and ITS2 a useful tool for phylogenetic reconstruction. However, ITS2 seems to be relatively more polymorphic and more informative than ITS1 regarding length or GC percent. The phylogenic relationships in the genus Hedysarum based on ITS1 and ITS2 sequences taken independently or together, are discussed in the context of current work in molecular biosystematics. Results exhibited the distinctiveness of the two H. spinosissimum subspecies (i.e. H. spinosissimum ssp. capitatum and H. spinosissimum ssp. spinosissimum). In addition, the great similarity of the ITS sequences between H. coronarium (the only cultivated species of the genus) and H. carnosum suggests the usefulness of the latter in selection programmes to improve pastoral production in semi-arid areas.

Exploration of intra- and inter-population genetic diversity in Hedysarum coronarium L. by AFLP markers

Amplified fragment length polymorphism (AFLP) markers were used to characterize the genetic diversity within and among natural populations and cultivars of Hedysarum coronarium. Twelve populations within Tunisia were evaluated with three AFLP primer combinations. A total of 207 reproducible bands was detected of which 178 (86%) were polymorphic. The great discriminative power of AFLP markers and their ability to represent genetic relationships among Hedysarum plants was demonstrated. Genetic diversity within and among populations was assessed through Principal Component Analysis (PCA) and cluster analysis by using the Neighbor-joining clustering algorithm. AFLP technology has provided evidence of a high degree of intra- and inter-population genetic diversity in H. coronarium. AFLP banding patterns provided molecular markers correlated with the plants’ geotropism. In addition, AFLP markers can differentiate wild accessions from cultivars. Moreover, geographical origins did not correspond to population clustering.

Molecular characterization of Mauritanian date palm cultivars using plasmid-like DNAs markers

Mauritanian date palm cultivars and progenies of two controlled crosses were analyzed according to the identity of mitochondrial plasmid-like DNAs. Starting from total genomic DNA and appropriate primers, polymerase chain reaction was designed to amplify either a 373-bp or a 265-bp fragments corresponding to the S and the R-plasmid respectively. Data proved that 5 cultivars out of 10 studied have exhibited the R-plasmid suggesting their resistance to the fusariosis. The existence of intra-cultivar variability has also been revealed in the cv. Ahmar. In addition, analysis throughout progenies of two controlled crosses suggested the strict maternal transmission of the date palms’ mitochondrial genome.

Genetic variation analysis in the genus Lathyrus using RAPD markers

RAPD analysis was applied to five species belonging to the genusLathyrus (Fabaceae): L.sativus, L.cicera, L.ochrus, L.sylvestris and L.latifolius. All the species under study belong to thesection Lathyrus except L.ochrus which is in section Clymenum.Nine populations representing these species were used and ten random10-mer primers were sampled. A total of 129 amplification products,ranging in size from 0.3 to 3 Kb, were generated. Partitioning ofvariation was studied using the AMOVA technique. The genetic variation proved tobe nearly equally distributed among species and among populations withinspecies. A similar approach was carried out to distinguish populations, whichproved to be efficient. The between population dissimilarities were calculatedand a dendrogram of genetic relationships was drawn. The RAPDs obtained weresufficient to distinguish between the two accessions of a species and toseparate these accessions by clustering them according to species. A highgenetic similarity between the populations of L.sylvestris and L.latifolius was established. A similar result is also shownfor the populations of L. sativus andL. cicera.