Chloroplast DNA Diversity of Tunisian Barley Landraces as Revealed by cpSSRs Molecular Markers and Implication for Conservation Strategies.

In Tunisia, barley local landraces are still cropped for human and animal consumption in some subsistence farming systems under marginal and stressed conditions. These high-value genetic resources present a potential source of resistance genes to biotic and abiotic stresses useful for both national and international breeders. Actually, they are represented by threatened small populations, which face a high risk of genetic erosion and progressive substitution by modern varieties. In this study, the genetic diversity of 60 Tunisian barley landraces was assessed using six chloroplast microsatellites. All loci were found polymorphic, with 2 or 3 alleles per locus. Thirteen alleles were detected across the studied sample, which were combined into 8 haplotypes, giving a haplotype diversity (Hd) of 0.847. High punctual and haplotype genetic diversity was observed for Tunisian barley landraces when compared to other germplasms from other regions of the world. The genetic structure analysis revealed two major clusters of Tunisian barley landraces, which confirms their multiorigin. This result was corroborated by the median-joining network showing the genetic relationships among the eight detected haplotypes. The AMOVA analysis revealed that 83% of the genetic variation is between populations, which requires the in situ and ex situ conservation of plant material for all Tunisian populations of barley landraces. Information on genetic variation within the chloroplast genome is of great interest to ensure an efficient conservation strategy that takes into account the preservation of the various maternal lineages of Tunisian barley.

New insights into the intraspecific cytoplasmic DNA diversity, maternal lineages classification and conservation issues of Tunisian pearl millet landraces.

Tunisian pearl millet (Pennisetum glaucum L.) landraces are still growing in contrasting agro-ecological environments and are considered potentially useful for national and international breeders. Despite its genetic potential, the cropping areas of this species are still limited and scattered which increases the risk of genetic erosion. The chloroplast DNA polymorphism and maternal lineages classification of forty nine pearl millet landraces representing seven populations covering the main distribution area of this crop in Tunisia were undertaken based on informative cpSSR molecular markers. A total of 21 alleles combining to 9 haplotypes were detected with a mean value of 3.5 alleles per locus and a haplotype genetic diversity (Hd) of 0.82. The number of chloroplast haplotypes per population ranged from 1 to 4 with an average of 1.28. The haplotypes median-joining network and UPGMA analyses revealed two probable ancestral maternal lineages with a differential pearl millet seed-exchange rate between the investigated areas. Northern and Central populations presented unique genetic backgrounds while historical farmers' practices in the South-East area resulted in the isolation of their own local landraces. The genetic evidences strongly support at least two introduction origins of pearl millet in Tunisia, one in the North and the other in the South followed by distinct local dispersal histories. Complementary in-situ and ex-situ conservation strategies taking into account the conservation of the maternal lineage cytoplasmic diversity are required. The investigated chloroplast SSRs provide useful molecular markers which could be used in further genetic studies and breeding surveys of pearl millet genetic resources.

Conservation priorities for endangered coastal North African Pennisetum glaucum L. landrace populations as inferred from phylogenetic considerations and population structure analysis.

The increasing anthropologic pressure and the modernization of agriculture have led to a forsaking of pearl millet traditional cultivars, inducing a progressive loss of the genetic variability encompassed in this locally adapted germplasm. Imperatively, national efforts based on robust data gleaned from genetic surveys have to be undertaken in order to set up suitable conservation priorities. In this study, in addition to the assessment of the genetic diversity and population structure among and within a set of seven pearl millet landrace populations from coastal North Africa, demographic and phylogenetic data, conservation priority scores were calculated according to Vane-Wright et al. (1991). To date, genetic diversity of pearl millet in North Africa is still poorly documented. The present survey reports for the first time the use of highly informative nSSR markers (PIC = 0.74) on Pennisetum glaucum landraces representative of the Mediterranean coastline of North Africa. A high level of genetic diversity was obtained within the investigated landraces (He = 0.80) at the population level. FST, AFC-3D, and Bayesian clustering underlined significant differentiation and an apparent genetic structure, according to geographical origin. Phylogenetic considerations integrated with demographic and genetic information enabled conclusive inferences of highly prioritized populations for conservation. Populations Haouaria, Hammem Laghzez, Mahdia, and Medenine, representatives of the main pearl millet growing areas in Tunisia and cultivated in the North African littoral, should be strongly recommended for an ex situ conservation program. Dynamic on-farm conservation method is also required as it allows the local landraces to evolve in different environments, while maintaining their adaptation potentials.

Metabolomic fingerprint of Mentha rotundifolia L. Leaf tissues promotes this species as a potential candidate for sustainable production of biologically active molecules.

Background The Mentha rotundifolia L. (Lamiaceae family), is a medicinal herb used since the ancient times as an antiseptic, analgesic and anti-inflammatory agent. In the present work, metabolomic profiling of two Mentha rotundifolia L. ecotypes leaf tissues spontaneously growing in the North of Tunisia was achieved. Methods Phenolic contents (TPC, TFC and TTC) were assessed using colorimetric methods. Metabolomic profiling of leaf tissues extracts was assessed based on Gas Chromatography-Mass Spectrometry (GC/MS) analysis. The antioxidant ability of M. rotundifolia extracts was achieved based on two test systems namely DPPH and FRAP assays. Antimicrobial activity against a set of Gram negative and Gram positive bacteria was estimated by measuring ID, MIC and MBC values. Results Fifty metabolites were identified as belonging mainly to phenolics, fatty acids, terpenes, steroids and aldehydes classes with qualitative and quantitative variability. Most of the identified compounds are reputed bioactive with potent antioxidant, antimicrobial and anti-inflammatory among others effects. To confirm these findings common in vitro biological activities were achieved. The investigated extracts showed significant antioxidant abilities based on both 1,1-diphenyl-2-picrylhydrazyl (DPPH) and Ferric reducing antioxidant potential (FRAP) assays. Furthermore, the extracts revealed promising antimicrobial ability against tested Gram+ and Gram- bacterial strains (ID: 12.5-14.5 mm, MIC: 3.125-25 (µg/mL), MBC: 6.25-100 (µg/mL)). Conclusions Based on our findings Mentha rotundifolia L. leaves extracts present a potential source of natural antioxidants and diverse bioactive compounds which could be used in green pharmacy, food preservation, alternative medicine and natural therapies.

Fingerprinting and genetic purity assessment of F1 barley hybrids and their salt-tolerant parental lines using nSSR molecular markers.

Hybridity and the genuineness of hybrids are prominent characteristics for quality control of seeds and thereby for varietal improvement. In the current study, the cross between two local barley genotypes (Ardhaoui: female; Testour: male) previously identified as susceptible/tolerant to salt stress in Tunisia was achieved. The hybrid genetic purity of the generated F1 putative hybrids and the fingerprinting of the parents along with their offspring were assessed using a set of 17 nuclear SSR markers. Among the analyzed loci, 11 nSSR were shown polymorphic among the parents and their offspring. Based on the applied 11 polymorphic SSR loci, a total of 28 alleles were detected with an average of 2.54 alleles per locus. The locus HVM33 presented the highest number of alleles. The highest polymorphism information content value was detected for the locus HVM33 (0.6713) whereas the lowest PIC value (0.368) was revealed by the loci BMAC0156, EBMAC0970 and BMAG0013 with a mean value of 0.4619. The probabilities of identical genotypes PI for the 11 microsatellite markers were 8.63 × 10-7. Banding patterns among parents and hybrids showed polymorphic fragments. The 11 SSR loci had produced unique fingerprints for each analyzed genotype and segregate between the two parental lines and their four hybrids. Parentage analysis confirms the hybrid purity of the four analyzed genotypes. Six Tunisian barley accessions were used as an outgroup in the multivariate analysis to confirm the efficiency of the employed 11 nSSR markers in genetic differentiation among various barley germplasms. Thus, neighbor joining and factorial analysis revealed clearly the discrimination among the parental lines, the four hybrids and the outgroup accessions. Out of the detected polymorphic 11 nuclear SSR markers, a set of five markers (HVM33, WMC1E8, BMAC0154, BMAC0040 and BMAG0007) were shown to be sufficient and informative enough to discriminate among the six genotypes representing the two parental lines and the four hybrids from each others. These five nSSR markers presented the highest number of alleles per locus (An), expected heterozygosity (He), PIC values and the lowest probabilities of identity (PI). These nSSR loci may be used as referral SSR markers for unambiguous discrimination and genetic purity assessment in barley breeding programs.

Patterns of genetic structure and evidence of gene flow among Tunisian Citrus species based on informative nSSR markers.

This study investigates the extent of genetic diversity, phylogenetic relationships and the amount of gene flow among Tunisian Citrus species based on a set of 15 informative nuclear SSR molecular markers. Genotyping data highlighted an allelic richness among Tunisian Citrus species and has allowed the detection of 168 alleles among them 104.19 were effective. The partition of the total genetic diversity (HT=0.832) showed that the highest amount of variation within the Citrus species is HS=0.550, while the relative amount of the between-species genetic diversity GST does not exceed 0.338. This pattern of genetic structure was supported by low-to-moderate FST pairwise values and the presence of a gene flow (Nm) among the eight Citrus species. The lowest genetic differentiation was revealed between the species C. sinensis and C. insitorum (FST=0.111, Nm=1.99), while the highest genetic differentiation was recorded between the species C. aurantifolia and C. paradisi (FST=0.367, Nm=0.43). The established Neighbor Joining analysis showed that all genotypes were widely discriminated and clearly pooled according to their species of origin, with minor exceptions.

Identification of barley landrace genotypes with contrasting salinity tolerance at vegetative growth stage.

In the present study, we evaluated the behavior of 21 Tunisian barley landraces under salt stress. The evaluation was performed using 14 morphological and physiological traits at vegetative growth stage under severe salt stress (200 and 250 mM). A multivariate analysis was used in order to select the genotypes with contrasting behavior towards salinity and to identify the major traits conferring salinity tolerance. According to the PCA analyses the genotypes exhibited diverse behavior with the salt stress concentration, indeed 3 different clustering profiles were obtained. Eleven quantitative characters were considered the most pertinent for the ranking of genotypes for salt tolerance. Among them the total fresh weight and the net CO2 assimilation rate were the most discriminating descriptors at 250 mM NaCl. These parameters allowed as the identification of the contrasting pair genotypes toward salinity. "Testour" was classified as the most sensitive and "Enfidha" the most tolerant toward salinity stress. These findings would be of great relevance in breeding programs.

Pathogenicity Spectra and Screening for Resistance in Barley Against Tunisian Pyrenophora teres f. teres.

This work aimed to determine patterns of pathogenicity in Pyrenophora teres f. teres and to identify potentially effective resistance sources that could be used as breeding material to control net blotch in Tunisia. Extensive pathogenic variability was detected in 85 isolates of P. teres causing net blotch of barley in Tunisia. Based on unweighted pair-group method with arithmetic averaging clustering and mean disease rating scores, three distinct virulence groups were identified. The isolates were classified into 23 pathotypes. Pathogenic variability within the groups was higher than that between the groups, a finding that can guide a rational choice of isolates for screening lines as part of a breeding program. Conversely, studying the relationship between geographic and pathotypic structure allowed us to detect a significant isolation by distance pattern, suggesting a regular and gradual dispersal of the pathogen over this spatial scale. Using specific resistance properties of individual barley genotypes as virulence markers, all the differential barley genotypes were shown to be distinct, and no single source of resistance was totally effective against all isolates.

Pathotype and microsatellite analyses reveal new sources of resistance to barley scald in Tunisia.

We examined the variation and relationships between pathogenicity and a microsatellite-based haplotype in 79 Tunisian Rhynchosporium secalis isolates that were collected from the most commonly cultivated barley populations in Tunisia, Rihane cv. and local landraces, with the goal of finding genes that might be used to monitor resistance to scald. Isolates could be classified into three distinct virulence groups based on artificial inoculation of 19 differential cultivars with known scald resistance genes. The resistance gene BRR2 carried by the Astrix differential cultivar appeared to be the most effective in Tunisia. Pathotypes sampled from the Rihane host were more virulent than those sampled from local barley landraces. Because some differential cultivars that carried the same resistance genes showed different reaction patterns to 48 of the isolates, we postulated that other unknown resistance gene(s) specific to Tunisian isolates may be prevalent and could be used in Tunisian barley breeding programs. Microsatellite fingerprinting allowed the detection of 11 alleles linked to the virulence and pathogenic identification of 52% of the tested isolates. Thus, microsatellite analysis may provide a rapid tool for pathogen detection, without an inoculation step that requires long incubation periods before ultimate disease assessment.