Optimizing pear micropropagation and rooting with light emitting diodes and trans-cinnamic acid

Meta-Topolin riboside was selected as cytokinin for these experiments. The effects of combinations of monochromatic blue, red and far red LED light were compared to fluorescent light during the micropropagation and rooting of a recalcitrant pear (Pyrus communis L.). During the micropropagation phase, red light gave some particular advantages: maximal shoot length and leaf area were obtained. Under blue light, callus weight quadrupled compared to fluorescent light. Although far red was advantageous for shoot number, shoot quality was inferior because of hyperhydricity and chlorosis as indicated by a low Chlorophyll a+b and carotenoid content. The smallest leaf area was detected under fluorescent light. Blue+red showed significant improvements. Shoot cluster weight and shoot weight/callus weight' ratio was maximal, as well as shoot number and shoot length. Leaves were also dark green, showing a maximal Chlorophyll a+b and carotenoids content. Adventitious rooting of in vitro cultured pear plants was highly affected by different light spectra and the addition of a new rooting compound: trans-cinnamic acid. Without trans-cinnamic acid, limited rooting was observed under red, blue and blue+red light. In combination with trans-cinnamic acid, 100% rooting was achieved under red light

Effects of meta-topolin derivatives and temporary immersion on hyperhydricity and in vitro shoot proliferation in Pyrus communis

Although micropropagation in temporary immersion systems might increase plant growth and multiplication, it can also cause specific problems such as hyperhydricity and losses by contamination. A new commercial temporary immersion bioreactor, SETIS™, was used to micropropagate two Tunisian pear cultivars, ‘Arbi’ and ‘Mahdia 6’. The latter cultivar was endogenously contaminated by Sphingomonas. Hyperhydricity was inevitable when 5 µM benzyladenine was applied. However, the symptoms could be reduced by lowering the immersion frequency to 3 times per day. Applying 5 µM meta–Methoxy topolin riboside (MemTR) or meta–Topolin riboside (mTR) completely inhibited hyperhydric shoot formation. Moreover, the addition of Plant Preservative Mixture was effective to control Sphingomonas and allowed the plants to proliferate. For both pear cultivars, the highest number of shoots per explant was induced by 5 µM MemTR, whereas the highest leaf area was obtained with 5 µM mTR. The longest shoots were obtained with 5 µM mTR for ‘Arbi’ and 5 µM MemTR for ‘Mahdia 6’. Key message Methoxy topoline-riboside (MemTR) and meta-topoline-riboside (mTR) were used as alternatives to benzyladenine to prevent hyperhydricity during the micropropagation of pears in a new temporary immersion system. These cytokinins also increased the number of good quality shoots, characterized by large leaves and longer shoots

In vitro mycorrhization of pear (Pyrus communis)

The Mycelium Donor Plant system (MDP) was adapted to study the time course of the colonization of Pyrus communis by Rhizophagus irregularis under in vitro conditions. Isolated germinated spores did not colonize pear roots. Inoculum composed of R. irregularis spores/mycelium associated with chicory root fragments was used to inoculate Medicago truncatula which became thereafter the MDP of pear plantlets. Typical intraradical structures (hyphae, arbuscules, spores/vesicles) and appressoria were observed in the pear roots. During acclimatization, the pear plants formed a densely branched root system. R. irregularis colonization not only altered the root architecture but also changed the nutrient composition of the acclimatized pear plantlets

A Founder Large Deletion Mutation in Xeroderma Pigmentosum-Variant Form in Tunisia: Implication for Molecular Diagnosis and Therapy

Xeroderma pigmentosum Variant (XP-V) form is characterized by a late onset of skin symptoms. Our aim is the clinical and genetic investigations of XP-V Tunisian patients in order to develop a simple tool for early diagnosis. We investigated 16 suspected XP patients belonging to ten consanguineous families. Analysis of the POLH gene was performed by linkage analysis, long range PCR, and sequencing. Genetic analysis showed linkage to the POLH gene with a founder haplotype in all affected patients. Long range PCR of exon 9 to exon 11 showed a 3926 bp deletion compared to control individuals. Sequence analysis demonstrates that this deletion has occurred between two Alu-Sq2 repetitive sequences in the same orientation, respectively, in introns 9 and 10. We suggest that this mutation POLH NG_009252.1: g.36847_40771del3925 is caused by an equal crossover event that occurred between two homologous chromosomes at meiosis. These results allowed us to develop a simple test based on a simple PCR in order to screen suspected XP-V patients. In Tunisia, the prevalence of XP-V group seems to be underestimated and clinical diagnosis is usually later. Cascade screening of this founder mutation by PCR in regions with high frequency of XP provides a rapid and cost-effective tool for early diagnosis of XP-V in Tunisia and North Africa

Aspergillus flavus genetic structure at a turkey farm

Abstract Background The ubiquitous environmental fungus Aspergillus flavus is also a life‐threatening avian pathogen. Objectives This study aimed to assess the genetic diversity and population structure of A. flavus isolated from turkey lung biopsy or environmental samples collected in a poultry farm. Methods A. flavus isolates were identified using both morphological and ITS sequence features. Multilocus microsatellite genotyping was performed by using a panel of six microsatellite markers. Population genetic indices were computed using FSTAT and STRUCTURE. A minimum‐spanning tree (MST) and UPGMA dendrogram were drawn using BioNumerics and NTSYS‐PC, respectively. Results The 63 environmental (air, surfaces, eggshells and food) A. flavus isolates clustered in 36 genotypes (genotypic diversity = 0.57), and the 19 turkey lung biopsies isolates clustered in 17 genotypes (genotypic diversity = 0.89). The genetic structure of environmental and avian A. flavus populations were clearly differentiated, according to both F‐statistics and Bayesian model‐based analysis’ results. The Bayesian approach indicated gene flow between both A. flavus populations. The MST illustrated the genetic structure of this A. flavus population split in nine clusters, including six singletons. Conclusions Our results highlight the distinct genetic structure of environmental and avian A. flavus populations, indicative of a genome‐based adaptation of isolates involved in avian aspergillosis

Molecular characterization of Mycobacterium tuberculosis strains resistant to isoniazid

Objective/background: Tuberculosis is a major public health problem and the emergence of drug resistance complicates the situation even more. It is therefore crucial to implement all conclusions from the studies that aim at a better understanding of the molecular mechanisms which govern the emergence and the evolution of drug resistance. The aim of this study is to assess the degree of involvement of the inhA and katG genes in the acquisition of isoniazid resistance in clinical strains of Mycobacterium tuberculosis. Methods: The inhA and katG genes were sequenced in 21 strains of M. tuberculosis with different resistance profiles and from different regions. Results: Analysis of the sequences obtained by comparison to those of the reference strain H37Rv showed that 95.2% had mutations. KatG S315T was the most common mutation (85.7%). The mutation katG T275A was revealed in two strains (9.5%). Two different point mutations in the inhA gene and its promoter region were identified as C-15T and G56A at a frequency equal to 14% and 10%, respectively. The G56A mutation is a new silent mutation. Our study showed no correlation between found mutations and multidrug resistance. Among the 21 strains studied, only one strain showed no mutations. Conclusion: In terms of this study, we characterized the mutations involved in resistance to isoniazid. katG S315T was by far the most frequent mutation, followed by C-15T. The frequency of these mutations was concordant with those reported in literature including those in intermediate tuberculosis endemic countries

Family specific genetic predisposition to breast cancer: results from Tunisian whole exome sequenced breast cancer cases

Abstract Background A family history of breast cancer has long been thought to indicate the presence of inherited genetic events that predispose to this disease. In North Africa, many specific epidemio-genetic characteristics have been observed in breast cancer families when compared to Western populations. Despite these specificities, the majority of breast cancer genetics studies performed in North Africa remain restricted to the investigation of the BRCA1 and BRCA2 genes. Thus, comprehensive data at a whole exome or whole genome level from local patients are lacking. Methods A whole exome sequencing (WES) of seven breast cancer Tunisian families have been performed using a family-based approach. We focused our analysis on BC-TN-F001 family that included two affected members that have been sequenced using WES. Relevant variants identified in BC-TN-F001 have been confirmed using Sanger sequencing. Then, we conducted an integrative analysis by combining our results with those from other WES studies in order to figure out the genetic transmission model of the newly identified genes. Biological network construction and protein–protein interactions analyses have been performed to decipher the molecular mechanisms likely accounting for the role of these genes in breast cancer risk. Results Sequencing, filtering strategies, and validation analysis have been achieved. For BC-TN-F001, no deleterious mutations have been identified on known breast cancer genes. However, 373 heterozygous, exonic and rare variants have been identified on other candidate genes. After applying several filters, 12 relevant high-risk variants have been selected. Our results showed that these variants seem to be inherited in a family specific model. This hypothesis has been confirmed following a thorough analysis of the reported WES studies. Enriched biological process and protein–protein interaction networks resulted in the identification of four novel breast cancer candidate genes namely MMS19, DNAH3, POLK and KATB6. Conclusions In this first WES application on Tunisian breast cancer patients, we highlighted the impact of next generation sequencing technologies in the identification of novel breast cancer candidate genes which may bring new insights into the biological mechanisms of breast carcinogenesis. Our findings showed that the breast cancer predisposition in non-BRCA families may be ethnic and/or family specific

A genome wide SNP genotyping study in the Tunisian population: specific reporting on a subset of common breast cancer risk loci

International audienceBackground: Breast cancer is the most common cancer in women worldwide. Around 50% of breast cancer familial risk has been so far explained by known susceptibility alleles with variable levels of risk and prevalence. The vast majority of these breast cancer associated variations reported to date are from populations of European ancestry. In spite of its heterogeneity and genetic wealth, North-African populations have not been studied by the HapMap and the 1000Genomes projects. Thus, very little is known about the genetic architecture of these populations.Methods : This study aimed to investigate a subset of common breast cancer loci in the general Tunisian population and to compare their genetic composition to those of other ethnic groups. We undertook a genome-wide haplotype study by genotyping 135 Tunisian subjects using the Affymetrix 6.0-Array. We compared Tunisian allele frequencies and linkage disequilibrium patterns to those of HapMap populations and we performed a comprehensive assessment of the functional effects of several selected variants.Results : Haplotype analyses showed that at risk haplotypes on 2p24, 4q21, 6q25, 9q31, 10q26, 11p15, 11q13 and 14q32 loci are considerably frequent in the Tunisian population (> 20%). Allele frequency comparison showed that the frequency of rs13329835 is significantly different between Tunisian and all other HapMap populations. LD-blocks and Principle Component Analysis revealed that the genetic characteristics of breast cancer variants in the Tunisian, and so probably the North-African populations, are more similar to those of Europeans than Africans

Whole Exome Sequencing allows the identification of two novel groups of Xeroderma pigmentosum in Tunisia, XP-D and XP-E: Impact on molecular diagnosis

International audienceBACKGROUND: Skin cancers (SC) are complex diseases that develop from complex combinations of genetic and environmental risk factors. One of the most severe and rare genetic diseases predisposing to SC is the Xeroderma pigmentosum (XP) syndrome.OBJECTIVES: First, to identify the genetic etiology of XP and to better classify affected patients. Second, to provide early molecular diagnosis for pre-symptomatic patient and finally to offer genetic counseling for related individuals.METHODS: Whole Exome Sequencing (WES) and Run Of Homozygosity (ROH) were performed for two patients belonging to two different multiplex consanguineous families. The identified mutations were confirmed by Sanger sequencing and researched in ten Tunisian families including a total of 25 affected individuals previously suspected as having XP group V (XP-V) form. All patients had mild dermatological manifestations, absence of neurological abnormalities and late onset of skin tumors.RESULTS: Screening for functional variations showed the presence of the ERCC2 p.Arg683Gln in XP14KA-2 patient and a novel mutation, DDB2 p. (Lys381Argfs*2), in XP51-MAH-1 patient. Sanger sequencing and familial segregation showed that the ERCC2 mutation is present at a homozygous state in 10 affected patients belonging to 3 families. The second mutation in DDB2, is present at a homozygous state in 5 affected cases belonging to the same family. These two mutations are absent in the remaining 10 affected patients. The ERCC2 c.2048G > A mutation is present in a medium ROH region (class B) suggesting that it mostly arises from ancient relatedness within individuals. However, the c.1138delG DDB2 mutation is present in a large ROH region (class C) suggesting that it arises from recent relatedness.CONCLUSION: To our knowledge, this is the first study that identifies XP-D and XP-E complementation groups in Tunisia. These two groups are very rare and under-diagnosed in the world and were not reported in North Africa