Genetic analysis of drought stress response in Arabidopsis thaliana and Brassica rapa

Drought is the major abiotic stress affecting plant growth and limiting crop productivity worldwide. Plants have evolved three adaptive strategies, drought escape, drought avoidance and drought tolerance, to cope with drought. Knowledge on how Quantitative Trait Loci (QTL), or genes underlying these strategies interact with their environments will significantly increase our understanding and the success of breeding for drought tolerance. This thesis focused on phenotyping shoot and root traits ofA. thaliana and B. rapa grown on sand and in greenhouses,to further understand how plants can adapt to natural drought stress. In chapter 2, an already existing ArabidopsisRIL population was selected based on the differential root drought response of the two parental lines, Sha and Col, to be evaluated under different water regimes. Chapter 3 illustrated the use of GWAS in identifying candidate genes that are associated with pant response to drought.. In order to apply the same methodology in crop breeding, chapter 4 introduces a contribution to the genetic mapping of a new B. rapa RIL population, consisting of 160 lines and genotyped with 270 different markers was achieved. The morphological and physiological responses of this population to drought was evaluated in chapter 5. The results presented in the present thesis demonstrate that QxE is an important component of the genetic variance and can play a great role in improving drought tolerance in future breeding programs. In general, several QTL and SNPs were mapped either with main effect or with interaction with environments QxE. Many of the mapped QTL showed conditional neutrality and antagonestic pleiotropy.</p

A framework for gene mapping in wheat demonstrated using the Yr7 yellow rust resistance gene

We used three approaches to map the yellow rust resistance gene Yr7 and identify associated SNPs in wheat. First, we used a traditional QTL mapping approach using a double haploid (DH) population and mapped Yr7 to a low-recombination region of chromosome 2B. To fine map the QTL, we then used an association mapping panel. Both populations were SNP array genotyped allowing alignment of QTL and genome-wide association scans based on common segregating SNPs. Analysis of the association panel spanning the QTL interval, narrowed the interval down to a single haplotype block. Finally, we used mapping-by-sequencing of resistant and susceptible DH bulks to identify a candidate gene in the interval showing high homology to a previously suggested Yr7 candidate and to populate the Yr7 interval with a higher density of polymorphisms. We highlight the power of combining mapping-by-sequencing, delivering a complete list of gene-based segregating polymorphisms in the interval with the high recombination, low LD precision of the association mapping panel. Our mapping-by-sequencing methodology is applicable to any trait and our results validate the approach in wheat, where with a near complete reference genome sequence, we are able to define a small interval containing the causative gene

Evaluation of the probiotic potential of lactic acid bacteria isolated from faeces of breast-fed infants in Egypt

The probiotic-related characteristics of 55 strains of lactic acid bacteria isolated from the faeces of 3 - 6 months old breast-fed infants were determined. The API 50 CH and SDS-PAGE techniques wereemployed to ascertain the identity of the isolated strains. The predominant species among the isolated strains were Lactobacillus (Lb.) acidophilus, Lb. plantarum, Enterococcus (E.) faecium, and E. faecalis. Probiotic properties such as bile resistance, acid tolerance, and adhesion to intestinal mucous were assessed. In vitro results obtained showed that five strains, Lb. plantarum (P1 and P164), Lb. pentosus (P191), and Lb. fermentum (P10, P193) were able to meet the basic requirements for probiotic functions as they demonstrated probiotic characteristics such as tolerance to pH 3, growth in 0.4% oxgall and adhesion to intestinal mucous. The results obtained in this investigation will be used to selectpotentially probiotic strains for in vivo study

Enumeration and identification of lactic microflora in Algerian goats’ milk

A total of 153 strains of lactic acid bacteria were isolated from Algerian goats’ milk. The strains were identified according to morphological, biochemical and physiological criteria, as well as the use of theAPI system and SDS-PAGE technique. Identification of the isolates revealed the presence of six genera: Enterococcus (41.82%), Lactobacillus (29.40), Lactococcus (19.60%), Leuconostoc (4.57%),Streptococcus thermophilus (3.26%) and Pediococcus (1.30%). The predominant strains belong to Enterococcus faecium (24 isolates), Enterococcus durans (22 isolates), Lactococcus lactis subsp. lactis(25 isolates), Lactobacillus rhamnosus (9 isolates) and Lactobacillus delbrueckii subsp. bulgaricus (7 isolates)

High-density SNP-based association mapping of seed traits in fenugreek reveals homology with clover

Fenugreek as a self-pollinated plant is ideal for genome-wide association mapping where traits can be marked by their association with natural mutations. However, fenugreek is poorly investigated at the genomic level due to the lack of information regarding its genome. To fill this gap, we genotyped a collection of 112 genotypes with 153,881 SNPs using double digest restriction site-associated DNA sequencing. We used 38,142 polymorphic SNPs to prove the suitability of the population for association mapping. One significant SNP was associated with both seed length and seed width, and another SNP was associated with seed color. Due to the lack of a comprehensive genetic map, it is neither possible to align the newly developed markers to chromosomes nor to predict the underlying genes. Therefore, systematic targeting of those markers to homologous genomes of other legumes can overcome those problems. A BLAST search using the genomic fenugreek sequence flanking the identified SNPs showed high homology with several members of the Trifolieae tribe indicating the potential of translational approaches to improving our understanding of the fenugreek genome. Using such a comprehensively-genotyped fenugreek population is the first step towards identifying genes underlying complex traits and to underpin fenugreek marker-assisted breeding programs

Preparation, Structure, and Surface Chemistry of Ni-Au Single Atom Alloys

Ni/Au is an alloy combination that while, immiscible in the bulk, exhibits a rich array of surface geometries that may offer improved catalytic properties. It has been demonstrated that the addition of small amounts of Au to Ni tempers its reactivity and reduces coking during the steam reforming of methane. Herein, we report the first successful preparation of dilute Ni-Au alloys (up to 0.04 ML) in which small amounts of Ni are deposited on, and alloyed into, Au(111) using physical vapor deposition. We find that the surface structure can be tuned during deposition via control of the substrate temperature. By adjusting the surface temperature in the 300-650 K range, we are able to produce first Ni islands, then mixtures of Ni islands and Ni-Au surface alloys, and finally, when above 550 K, predominantly island-free Ni-Au single atom alloys (SAAs). Low-temperature scanning tunneling microscopy (STM) combined with density functional theory calculations confirm that the Ni-Au SAAs formed at high temperature correspond to Ni atoms exchanged with surface Au atoms. Ni-Au SAAs form preferentially at the elbow regions of the Au(111) herringbone reconstruction, but at high coverage also appear over the whole surface. To investigate the adsorption properties of Ni-Au SAAs, we studied the adsorption and desorption of CO using STM which allowed us to determine at which atomic sites the CO adsorbs on these heterogeneous alloys. We find that small amounts of Ni in the form of single atoms increases the reactivity of the substrate by creating single Ni sites in the Au surface to which CO binds significantly more strongly than Au. These results serve as a guide in the design of surface architectures that combine Au's weak binding and selective chemistry with localized, strong binding Ni atom sites that serve to increase reactivity

KLC1-ALK: A Novel Fusion in Lung Cancer Identified Using a Formalin-Fixed Paraffin-Embedded Tissue Only

The promising results of anaplastic lymphoma kinase (ALK) inhibitors have changed the significance of ALK fusions in several types of cancer. These fusions are no longer mere research targets or diagnostic markers, but they are now directly linked to the therapeutic benefit of patients. However, most available tumor tissues in clinical settings are formalin-fixed and paraffin-embedded (FFPE), and this significantly limits detailed genetic studies in many clinical cases. Although recent technical improvements have allowed the analysis of some known mutations in FFPE tissues, identifying unknown fusion genes by using only FFPE tissues remains difficult. We developed a 5′-rapid amplification of cDNA ends-based system optimized for FFPE tissues and evaluated this system on a lung cancer tissue with ALK rearrangement and without the 2 known ALK fusions EML4-ALK and KIF5B-ALK. With this system, we successfully identified a novel ALK fusion, KLC1-ALK. The result was confirmed by reverse transcription-polymerase chain reaction and fluorescence in situ hybridization. Then, we synthesized the putative full-length cDNA of KLC1-ALK and demonstrated the transforming potential of the fusion kinase with assays using mouse 3T3 cells. To the best of our knowledge, KLC1-ALK is the first novel oncogenic fusion identified using only FFPE tissues. This finding will broaden the potential value of archival FFPE tissues and provide further biological and clinical insights into ALK-positive lung cancer

Identification of a novel polyfluorinated compound as a lead to inhibit human enzymes aldose reductase and AKR1B10 : structure determination of both ternary complexes and implications for drug design

Aldo-keto reductases (AKRs) are mostly monomeric enzymes which fold into a highly conserved ([alpha]/[beta])8 barrel, while their substrate specificity and inhibitor selectivity are determined by interaction with residues located in three highly variable external loops. The closely related human enzymes aldose reductase (AR or AKR1B1) and AKR1B10 are of biomedical interest because of their involvement in secondary diabetic complications (AR) and in cancer, e.g. hepatocellular carcinoma and smoking-related lung cancer (AKR1B10). After characterization of the IC50 values of both AKRs with a series of polyhalogenated compounds, 2,2',3,3',5,5',6,6'-octafluoro-4,4'-biphenyldiol (JF0064) was identified as a lead inhibitor of both enzymes with a new scaffold (a 1,1'-biphenyl-4,4'-diol). An ultrahigh-resolution X-ray structure of the AR-­NADP+-JF0064 complex has been determined at 0.85 Å resolution, allowing it to be observed that JF0064 interacts with the catalytic residue Tyr48 through a negatively charged hydroxyl group (i.e. the acidic phenol). The non-competitive inhibition pattern observed for JF0064 with both enzymes suggests that this acidic hydroxyl group is also present in the case of AKR1B10. Moreover, the combination of surface lysine methylation and the introduction of K125R and V301L mutations enabled the determination of the X-ray crystallo­graphic structure of the corresponding AKR1B10-NADP+-JF0064 complex. Comparison of the two structures has unveiled some important hints for subsequent structure-based drug-design efforts