Molecular based assessment of genetic diversity within Barbary fig (Opuntia ficus indica (L.) Mill.) in Tunisia

In this work, we report for the first time on the analysis of genetic diversity within a set of 36 Tunisian Opuntia ficus indica (L.) Mill. ecotypes using RAPD markers.     

Origin and genetic diversity of Tunisian grapes as revealed by microsatellite markers

Ten SSR loci, previously developed for grapevine, were analyzed to evaluate the genetic variability, cultivar relatedness, and parentage in a collection of 61 autochthonous Vitis vinifera cultivars from Tunisia.The number of alleles per locus ranged from 6 to 11, while the number of genotype patterns varied between 10 and 21. The expected heterozygosity varied between 0.621 and 0.855 and the observed heterozygosity was higher than 0.9 at 4 loci (VVMD28, VVMD5, VVIP31 and VVS2) indicating that the SSRs were highly informative.Cluster analysis using unweighted pair group method with arithmetic averaging (UPGMA) suggested 14 groups among studied cultivars and 53 grapevine denominations out of 61 were unequivocally distinguished, with all accessions showing at least one-specific combination of alleles.On the other hand, in order to overcome the existing confusion in Tunisian grapevine nomenclature, of the analyzed homonymous pairs of cultivars, only ‘Balta 2’ and ‘Balta 3’ have shown identical allelic profiles, consistent with their being the same genotype. Hence, nomenclature distinction is meaningless and only one denomination should be retained.Due to the high overall power of exclusion (Q) (greater than 99.99%) and to the absence of null alleles, the set of microsatellite loci used is appropriate to determine parentage in Tunisian grapevines beyond any reasonable doubt. The analysis of fingerprints indicated that the Tunisian grape vines have evolved through out crossing between five possible parents: Balta 1, Beldi Baddar, Beldi Rafraf, Beldi Local Rafraf and Khedhiri 3.     

Morphological variation in Astragalus hamosus L. and Coronilla scorpioides L. populations of Tunisia

Morphological variation between Astragalus hamosus and Coronillascorpioides populations was studied using local germoplasm collected in northern and central Tunisia. Twenty-one morphological traits were recorded and data were analysed using complementary statistical analysis. Considerable variation based on morphological and agronomical traits was found between populations and for both species. Pod and peduncle lengths as well as flowers number were the most discriminant variables between populations. Differences were also found for variables like seed number per pod, seed yield and dry matter production. Significant correlations were found between plant traits and the environmental parameters of the origin sites. This study revealed enough variation among populations to initiate a selection programme for pasture improvement in arid and semi-arid areas of Tunisia, where the two species are well adapted. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic structure and differentiation among grapevines (<Emphasis Type="Italic">Vitis vinifera</Emphasis>) accessions from Maghreb region

Three gene pools representative of Vitis vinifera L. subsp. vinifera (=subsp. sativa Beger) growing in the Maghreb regions (North Africa) from Tunisia (44), Algeria (31) and Morocco (18) and 16 wild grape accessions (Vitis vinifera L. subsp. sylvestris (Gmelin) Beger) from Tunisia were analysed for genetic diversity and differentiation at twenty nuclear microsatellites markers distributed throughout the 19 grape chromosomes. 203 alleles with a mean number of 10.15 alleles per locus were observed in a total of 109 accessions. Genetic diversities were high in all populations with values ranging from 0.6775 (Moroccan cultivars) to 0.7254 (Tunisian cultivars). F _st pairwise values between cultivated grapevine populations were low but found to be significantly different from zero. High F _st pairwise values were shown between wild and cultivated compartments. Two parent offspring relationships, two synonyms and two clones of the same cultivar were detected. The rate of gene flow caused by vegetative dissemination of cultivated grapevine plants was not sufficient to genetically homogenise the pools of cultivars grown in different regions. The Neighbour Joining cluster analysis showed a clear separation according to geographical origins for the cultivated grapevines gene pools and revealed a high dissimilarity between cultivated and wild grapevine. However, three cultivars (Plant d’Ouchtata 1, Plant de Tabarka 3 and Plant d’Ouchtata 3) are very close to wild accessions and may result from a hybridisation between cultivated and wild accessions. The high level of differentiation between cultivated and wild accessions indicates that the cultivated accessions do not derive directly from local wild populations but could mostly correspond to imported materials introduced from others regions during historical times or derived from crossing between them.     

Arbuscular mycorrhizal fungus and rhizobacteria affect the physiology and performance of Sulla coronaria plants subjected to salt stress by mitigation of ionic imbalance

Salt stress has become a major menace to plant growth and productivity. The main goal of this study was to investigate the effect of inoculation with the arbuscular mycorrhizal fungi (AMF; Rhizophagus intraradices) in combination or not with plant growth‐promoting rhizobacteria (PGPR; Pseudomonas sp. (Ps) and Bacillus subtilis) on the establishment and growth of Sulla coronaria plants under saline conditions. Pot experiments were conducted in a greenhouse and S. coronaria seedlings were stressed with NaCl (100 mM) for 4 weeks. Plant biomass, mineral nutrition of shoots and activities of rhizosphere soil enzymes were assessed. Salt stress significantly reduced plant growth while increasing sodium accumulation and electrolyte leakage from leaves. However, inoculation with AMF, whether alone or combined with the PGPR Pseudomonas sp. alleviated the salt‐induced reduction of dry weight. Inoculation with only AMF increased shoot nutrient concentrations resulting in higher K⁺: Na⁺, Ca²⁺: Na⁺, and Ca²⁺: Mg²⁺ ratios compared to the non‐inoculated plants under saline conditions. The co‐inoculation with AMF and Pseudomonas sp. under saline conditions lowered shoot sodium accumulation, electrolyte leakage and malondialdehyde (MDA) levels compared to non‐inoculated plants and plants inoculated only with AMF. The findings strongly suggest that inoculation with AMF alone or co‐inoculation with AMF and Pseudomonas sp. can alleviate salt stress of plants likely through mitigation of NaCl‐induced ionic imbalance, thereby improving the nutrient profile.     

Characterization of single nucleotide polymorphism in Tunisian grapevine genome and their potential for population genetics and evolutionary studies

In this study, two gene fragments corresponding to the VvMYBA1 and VvMYBA2 loci were sequenced on a sample of grapes including cultivated and wild accessions originating from Tunisia, Germany and France. A total of 42 SNPs were detected in the sequenced fragments giving an average of 1 SNP every 33 bp. High level of polymorphism was observed in the samples either in cultivated or wild accessions. Pattern of nucleotide diversity indicates a non departure from neutrality expectations for wild grapevine sample for gene VvMYBA1 and VvMYBA2 and for cultivated sample for gene VvMYBA1. However, a linkage to a selective sweep was revealed for cultivated grapevine gene pool in gene VvMYBA2. A genetic structure of the studied sample according to accession taxonomic status was revealed by the UPGMA clustering with a considerable overlap. This result was confirmed by significant but low genetic differentiation values between cultivated and wild sample. The number of migrants Nm based on sequence data information between Tunisian cultivars and Tunisian wild accessions showed a low level of gene flow between those germplasms. This finding indicates that Tunisian cultivars do not derive directly from local wild populations but could mostly correspond to imported materials introduced during historical times. However, the possibility that some cultivars derived from ancestral events of local domestication or cross hybridization with native wild plants was not completely excluded for Tunisian grapevine accessions.     

Genetic stability of long-term micropropagated Opuntia ficus-indica (L.) Mill. plantlets as assessed by molecular tools: Perspectives for in vitro conservation

Randomly amplified polymorphic DNA markers (RAPD) were employed to assess the level of genetic stability of long term micropropagated prickly pear (Opuntia ficus-indica) plantlets.Thirteen micropropagated plantlets were chosen from a clonal collection of shoots that originated from a single mother shoot. This clonal collection had been maintained under in vitro culture conditions for at least 5 years, as achieved for the time by axillary branch multiplication in Opuntia ficus-indica.Twenty arbitrary primers were used to compare RAPD patterns between in vitro raised material and the mother plant. Only 11 primers were found to yield distinct and reproducible amplification products resulting in a total of 87 amplified products, out of which 82 bands were monomorphic across all the plantlets and 5 showed polymorphisms.Cluster analysis performed on the basis of similarity indices indicated that all micropropagated plantlets and their mother plant grouped together in one major cluster with a 91% level of similarity.Low level of genetic variation has been detected, as polymorphic bands accounted for just 2.79% of the total genetic variation. This very low level of genetic variation, despite more than 5 years of in vitro culture, demonstrates the genetic stability of Opuntia ficus-indica and indicates that the axillary branch multiplication method is highly reliable for the multiplication of genetically true-to-type plant material.The high degree of clonal fidelity detected here, recommend the use of axillary-branching micropropagation technique for the safe in vitro conservation of prickly pear interesting genetic resources.     

Use of chloroplast microsatellite markers as a tool to elucidate polymorphism,classification and origin of Tunisian grapevines

Chloroplast microsatellite markers were used in this study to genotype 43 grapevines accessions grown in Tunisia. Size variation was observed for the three cpSSR loci, both in the sample of cultivars and in wild accessions. The seven alleles observed in the sample of cultivars for the three loci are present in wild accessions except that their distribution is different. Levels of genetic diversity obtained for the Tunisian grapevines either in wild or cultivated gene pools are high and comparable with values obtained with other studied samples of Vitis vinifera. The distribution of haplotypes within the two samples is differential. Indeed, the chlorotype A is most abundant in the wild sample, whereas the chlorotype C is majority in the sample of cultivars. Haplotypes frequencies for cultivated grapevine distinguish haplotypes B and C as the most frequent (28% and 44% respectively) and haplotypes A and D as the least frequent (16% and 12% respectively). For wild grapevines, the seven alleles combined in three haplotypes, A, C and D. The haplotype A is the most frequent (44%) in the analyzed sample of wild accessions while haplotypes C and D show a frequency of 28%. Chlorotype distribution in Tunisian cultivars is comparable with that of cultivars in the Eastern Region representing the primary centre of domestication of the species. These results agree with the higher relevance of table grape cultivars in Tunisian viticulture and support an oriental origin of a large part of autochthons cultivars. Our results agree with other studies based in nuclear and chloroplast microsatellite markers and suggest independent domestication events for V. vinifera L. species.     

Germination of Astragalus hamosus L. and Coronilla scorpioides (L.) as influenced by temperature

Seed germination of two pasture legumes was tested under dark conditions at three alternating temperatures of 5-20, 15-20, 20-35 degrees C and constant 20 degrees C. Duration of the alternating temperatures was 12 h. The seedling counts were checked every day during four weeks. Species differed significantly in germination response to temperature. A. hamosus was more sensitive to temperature than C. scorpioides. The temperature of 20-35 degrees C was more favourable for the germination of A. hamosus than constant 20 degrees C which markedly reduced its germination. The interaction between accessions and temperatures for total germination was significant in both species. In A. hamosus, almost 50% of germination was achieved by all accessions by the first week at alternating 20-35 degrees C temperature while in C. scorpioides, average total germination was significantly low (4%) even by the 28th day. A total rate of 11 and 9% of germination were recorded by two accessions from Ouesslatia (210) and Agareb (19), respectively. A considerable variation for germination was observed among accessions of both species. Temperature requirements for seed germination of the studied species suggest why they are relatively easy or difficult to establish in the field. A. hamosus, with very small seeds required relatively high alternating temperature for germination than C. scorpioides which was insensitive to temperature and has relatively bigger seeds.