11.817.04.19F: Functional and genetic-molecular aspects of sunflower genome (Helianthus annuus L.)
Project type: institutional (registration no. 590.INST)
Project duration: 2011 – 2014
Project director: DUCA Maria, professor, doctor habilitated, academician

Executors:

Duca Maria, director, chief researcher; Clapco Steliana, senior researcher; scientific secretary; Port Angela, leading researcher; Glijin Aliona, leading researcher; Budeanu Oleg, leading researcher; Leviţchi Alexei, senior researcher; Şestacova Tatiana, researcher; Rotarenco Victoria, senior researcher; Dencicov-Cristea Lidia, researcher; Munteanu Viorel, researcher; Martea Rodica, researcher; Chilari Rodica, junior researcher; Calmîş Ana, researcher; Nechifor Victoria, researcher; Abduşa Daniela, researcher; Acciu Adriana, junior researcher; Gorceag Maria, junior researcher; Tabără Olesea, junior researcher; Gordeev Victor, junior researcher; Doroş Irina, technician; Manole Alexandru, technician.

Research methods:

Analytical approach by integrating experimental data and knowledge obtained by a complex of bioinformatic tools (databases, programming tools), laboratory techniques (electrophoresis, spectrophotometry, RT-PCR, AFLP, SCAR, etc.) will reveal the level of spatial and temporal expression of some genes involved in certain intracellular signaling pathways and will contribute to the explication of the possibilities of physiological processes regulation.

Project objectives:

The aim for the project is to reveal some functional and genetic-molecular aspects of gene expression and signal transduction at sunflower.
Research objectives include:

• identification of gene networks, which participate in the morphogenesis and signal transduction at plants;
• relevation of the physiological processes of the nucleus- cytoplasm interaction by the model of Cytoplasmic Male Sterility - Fertility Restoration (CMS-Rf);
• the investigation of the physiological, biochemical and genetic aspects in the frame of the host (sunflower) - parasite (Orobanche Cumana) system and the resistance potential of sunflower genotypes.

Results:

The role of gibberellins in the mechanisms of reproductive development has been determined and gene networks have been created for genes involved in regulation of the redox homeostasis, in mitochondrial activity and biogenesis, in energetic metabolism, microsporogenesis, signaling, in processes of DNA repair and recombination, etc., in key processes linked to the manifestation of male sterility in sunflower.
Differentially expressed genes have been identified depending on the development stage and the action of different factors – important components of metabolic networks, which can serve as models for the study of similar processes in other vegetal species.

09.817.04.001A: Plant functional genomics
Project type: institutional (registration no. 383.INST)
Project duration: 2009 – 2010
Project director: TODERAS Lidia, habilitated doctor

Executors:

Maria Duca, chief researcher, habilitated doctor, professor; Lidia Toderas, chief researcher, habilitated doctor; Oleg Budeanu, chief of laboratory, PhD; Veaceslav Reva, chief of laboratory, habilitated doctor; Dan Zgardan – chief of laboratory, PhD; Vitalii Minciuna, chief of laboratory, habilitated doctor; Nicolae Barbacar, managing researcher, habilitated doctor; Sergiu Port,  managing researcher, PhD; Alexei Levitchi, managing researcher, PhD; Irina Kahovscaia,  managing researcher, PhD; Victor Lupascu,  senior researcher, PhD; Victoria Rotarenco, senior researcher, PhD; Viorel Munteanu,  researcher; Tatiana Sestacova,  researcher, doctoral student; Andrei Midoni,  researcher; Svetlana Rezinciuc,  researcher, doctoral student; Iulia Lazareva,  researcher; Elena Mita, researcher; Ana Calmis, researcher; Ana-Maria Cosovan, technician; Lucia Tapu, technician, doctoral student; Marina Cordun, technician; Iulia Ticu, technician; Aurelia Ciobanu, technician; Igor Beletchi, technician; Victoria Nechifor, technician; Victoria Balan, technician; Rodica Martea, technician; Ana Babii, technician.

Research methods:

In the implementation of the project the following databases were exploited: GenBank, GEO, Regs Database of Plant Regulatory Elements, AGRIS, PlantPromoter DB, and TAIR. The applications used at different stages of processing of retrieved information were the following: BLAST, ORF-Finder; Scan Prosite, NCTS NCTS-PL-PL under Molquest2, and Antheprot; Multiexperiment viewer; PLACE; Predotar and Mitoprot.

Project objectives:

Identification of target genes involved in flower development (especially the stamens) and mtDNA recombination for selected transcription factors

Results:

Data on the model plant Arabidopsis thaliana was analyzed to identify genes susceptible to GA3, their promoters, transcription factors and nuclear proteins involved in mtDNA recombination. The production of ASC forms will be accelerated. The results demonstrate the possibility of using data obtained from the model plant (Arabidopsis) for less studied species (in particular those whose genome is not yet sequenced, as in sunflower). For this purpose, exploratory data analysis offers the possibility of developing hypotheses for the practical application of fundamental knowledge of biological processes such as ASC.

15.817.05.03F: Resistance of sunflower (Helianthus annuus L.) to broomrape (Orobanche cumana Wallr.): genetic-molecular and physiological mechanisms
Project type: institutional
Project duration: 2015 – 2018
Project director: DUCA Maria, professor, doctor habilitated, academician

Executors:

Maria Duca, director, chief researcher; Steliana Clapco, scientific secretary, researcher; Angela Port, leading researcher; Oleg Budeanu, leading researcher; Tatiana Sestacova, researcher; Victoria Rotarenco, researcher; Viorel Munteanu, researcher; Rodica Martea, researcher; Lucia Tapu, researcher; Ana Mutu, researcher; Victoria Nechifor, researcher; Daniela Abdusa, researcher; Adriana Acciu, junior researcher; Olesea Tabără, junior researcher; Victor Gordeev, junior researcher; Irina Doros, technician; Alexandru Manole, technician; Ion Zara, technician.

Research methods:

In order to achieve the objectives the following methods will be used: cytochemical (highlighting various biological compounds); anatomical and morphological; biochemical (spectrometry, electrophoresis); molecular biological (PCR with RAPD, SCAR, SSR, ISSR, RTime-PCR primers); bioinformatics (R programming language, RefSeq data analysis, ShortRead); microbiological (isolation, cultivation, identification of microorganisms); sociological (surveys, observation sheet); statistics.

Project objectives:

• Assessment of genetic, morphological and physiological variability of different geographical populations of O. cumana (study of genetic intrapopulational variation; determination of distribution, identification of subspecies and characterization of virulence of O. cumana populations; study of the evolution, morphology and biology of the parasite).
• Molecular and phenotypic screening of different sunflower genotypes (identification of molecular markers associated with resistance, sunflower germoplasm testing; assessment of the phenotypic response reaction of sunflower genotypes to natural and artificial infestation with O. cumana).
• Study of the mechanisms of host – parasite interactions (assessment of the level of expression of genes associated with resistance and development of gene networks involved in biotic stress; determination of key enzymes activity in the host plant response to the action of the phytopathogen; qualitative study of proteins involved in the system of sunflower – O. cumana interaction; highlighting host – parasite anatomical particularities and specific locating of various biochemical compounds).

Expected results:

The expected research outcome is the determination of intra-population variability, genetic structure and origin of mono- and polyphyletic origin of the population of O. cumana existing in the area of cultivation of sunflower in the Republic of Moldova; the presence of gene Or5 will be identified and sunflower genotypes will be assessed under artificial infestation with O. cumana , that will further be classified into resistant, tolerant and susceptible; molecular and physiological mechanisms of host-parasite interaction will be determined, and the main factors that determine the impact of the parasite on the host plant will be identified.

13.820.15.10 GA: Digital holographic microscopy in the study of biological tissues using LabVIEW
Project type: bilateral, Moldova – Germany 
Project duration: 2013 – 2015
Project director: DUCA Maria, professor, doctor habilitated, academician

Executors:

Maria Duca, project director, chief researcher; Aliona Glijin, senior researcher; Ludmila Batir, senior researcher; Adriana Acciu, junior researcher; Maria Gorceag, junior researcher

Project objectives:

• The development of the concept of optical configuration for the digital holographic microscope using state-of-the-art technologies
• The development of software based on LabVIEW image processing of biological preparations for optical configuration by using the digital holographic microscope
• The use of digital holographic microscope in the study of biological objects

Expected results:

The concept of using the digital holographic microscope by state-of-the-art technologies in microscopic analysis of biological preparations. The first digital images of biological objects obtained by using the digital holographic microscope. The possibility of using the holographic microscope in the analysis of biological preparations. 3D digital images of biological preparations. Database of images of biological objects obtained and stored in LabVIEW.

13.820.18.06/RoA: Intraspecific genetic polymorphism analysis in the development of chemotype molecular markers in medicinal and aromatic plants
Project type: bilateral, Moldova – Romania 
Project duration: 2013 – 2014
Project director: BUDEANU Oleg, PhD in Biology (Moldova)/ GILLE Elvira, dr. prof. (Romania)

Executors:

Oleg Budeanu, project director; Maria Duca, leading researcher; Steliana Clapco, senior researcher; Alexei Levitchi, senior researcher; Rodica Martea, researcher; Mutu Ana, researcher; Catalin-Alexandru Grosu, researcher.

Project objectives:

• Prelevation from the wild flora and assessment of biodiversity by studying natural populations and their morphological and phytochemical characterizations.
• Extracting vegetal raw materials due to the principles of the European Pharmacopeia and analysis of biosynthetic capacity of medicinal plants.
• Qualitative and quantitative phytochemical analysis: thin layer chromatography (TLC), high performance liquid chromatography (HPLC), gas chromatography coupled with mass spectrometry (GC-MS).
• Identification, characterization and multiplication of genotypes/ecotypes with biosynthetic potential and chemotyping by the aforementioned methods.
• Extraction of nuclear DNA and proteins from fresh and dried plant tissue.
• Electrophoretic separation of nucleic acids and proteins.
• Performing polymerase chain reactions with RAPD random primers.
• RAPD profile analysis and association with other data (morphological, biochemical).

Results:

The morphological, phytochemical, genetic analysis of 90 genotypes of Origanum vulgare and Hyssopus officinalis collected from the spontaneous flora of the Republic of Moldova and Romania have highlighted the intra- and inter-population diversity of the species. Chemotypes have been established for Origanum vulgare and chemovarieties for Hyssopus officinalis which correspond to the pharmacological requirements and can be used for drugs and cosmetics. The chemotypes of Origanum vulgare collected from the spontaneous flora of the Republic of Moldova have a decreased level of thymol and carvacrol which allows their utilization for fitotherapeutic purposes.

10.820.04.14BF: Imprinting of sunflower genotypes and determining the degree of seed hybridization using molecular markers
Project type: bilateral, Moldova – Belarus (registration no. 315.BA)
Project duration: 2010 – 2011 
Project director: PORT Angela, PhD in Biology

Executors:

Maria Duca, doctor habilitated, professor, leading researcher; Angela Port, PhD, associate professor, project director; Alexei Levitchi, PhD in Biology, senior researcher; Tatiana Sestacova, doctoral student, researcher; Maia Popa, accountant.

Research methods:

CTAB DNA extraction (Hettich centrifuge, 2008, Sanyo freezer, 2008, photo documentation system, 2009); PCR analysis (Corbet amplifier, 2008); denaturing polyacrylamide gel electrophoresis (electrophoresis system, 2008); agarose gel electrophoresis.

Project objectives:

• Analysis of helianthinin profile in different genotypes in order to highlight the specific spectra.
• Selection of SSR primers with a high degree of polymorphism.
• SSR analysis of different sunflower genotypes (lines, varieties and hybrids) with subsequent selection of specific loci (polymorphs) as an indicator in genotypic authenticity assessment.
• Comparative study of specific traits in genetic profiles and identifying SSR markers and protein markers based on helianthinin.
• Establishing of quality assessment criteria of breeding material in sunflower. Testing based on genotyping can be applied on new or recognized varieties for copyright protection or standard analysis aiming the certification of new sunflower lines and hybrids offered by physical persons and commercial seed companies.

Results:

The genetic profile of 21 sunflower genotypes, homo- and heterozygous with 28 ORS primers that highlighted 163 alleles was studied. The number of alleles per locus ranged from 1 (ORS795) and 15 (ORS70), demonstrating the polymorphism of investigated markers with values of the PIC (Polymorphic Content Information) index ranging from 0.5 to 0.92. To assess the genotype authenticity the following primers can be recommended: ORS78, ORS261, ORS349, ORS366, ORS509, ORS795, ORS811, ORS836, and for those that manifest codominance the following: ORS78, ORS509, ORS811, ORS815, being potential markers in the estimation of the degree of hybridization.
Five hundred and forty helianthinin polypeptide profiles of genotypes native to Belarus were analyzed. Among the nine genotypes native to Belarus, eight shared a single profile, demonstrating that these lines possess a high genetic purity level. Genotype no.4 native to Belarus revealed four profiles with different frequencies, while two paternal forms – LC Raus and LC 39 indicated 3 and, respectively, 2 atypical profiles. The data will be further tested in order to determine the cause of atypical spectra (genetic impurity, contamination, heterogeneous population, etc.).

10.820.09.09GA: Assessment of pharmaceutical potential of medicinal plants in the spontaneous flora of Moldova
Project type: bilateral, Moldova – Germany (registration no. 300.GA)
Project duration: 2010
Project director: Maria Duca, associate professor, PhD in Biology (Moldova)/ Frieder Müller-Uri, dr. (Germany)

Executors:

Maria Duca – managing researcher, PhD; Angela Port – researcher, PhD; Veaceslav Reva – senior researcher, PhD; Mihail Mirza, senior researcher, associate professor; Alexei Levitchi – senior researcher, PhD; Oleg Budeanu,  researcher, PhD; Maia Popa, accountant.

Research methods:

Biochemical (TLC, HPLC), bioinformatics (databases), botanical (plant determination).

Project objectives:

• Identification of medicinal plant species in the spontaneous flora of Moldova.
• Prelevation of biological material and obtaining of experimental samples.
• Biochemical analysis of secondary metabolites from endemic medicinal plants.
• Dissemination of obtained data.

Results:

Synthesis and analysis of current information on the diversity of medicinal plants and potential uses in the diseases prevention, phytotherapy and cosmetology were performed. Global databases were studied on the accessibility and degree of available information on medicinal plants from the spontaneous flora of Moldova. The obtained results reveal the presence of cardenolide compounds in three plants collected from the spontaneous flora of Moldova. The cardenolides in the quantities sufficient for pharmaceutical needs have been revealed in the examined samples. A database with references to taxonomy, habitat, therapeutic actions, etc., available at www.plante.asm.md was created.

13.819.14.12A: Gene expression studies of sunflower defensive response to downy mildew (Plasmopara halstedii F. Berl. et de Toni)

Type of project: Projects for young researchers (registration no. 456 IND_TC)
Project duration: 2013 – 2014
Project director: ŞESTACOVA Tatiana, PhD

Executors:

Şestacova Tatiana, project director, researcher; Rotarenco Victoria, senior researcher;   Nechifor Victoria, researcher; Calmîş Ana, researcher; Tabără Olesea, researcher; Darii Mariana, technician.

Research methods:

The planned research will be realized by applying a set of bioinformatics tools and databases: NCBI Gene Expression Atlas, Gene Ontology, PRGdb, BLAST, Clustal W, etc. Also, special software for experimental primer design will be used: Primer3, Oligo Analyzer, etc.
Analysis techniques will include the following: RNA extraction and purification with TRI Reagent, spectrophotometric determination of sample concentration and quality, RNA sample electrophoresis under denaturing conditions, DNAse treatment of samples, cDNA production by reverse transcriptase, real-time polymerase chain reaction, DNA electrophoresis, etc.

Project objectives:

• Analysis of genomic database (NCBI, EMBL, PRGdb, etc.) relating to genes involved in specific and nonspecific resistance for the purpose of experimental primer design.
• Assessment of the presence of Pl specific resistance genes by screening, using molecular markers.
• Obtaining of RNA samples from downy mildew infested plants and the control group.
• Obtaining cDNA samples by reverse transcription.
• Estimating the degree of gene expression by quantitative PCR.

Results:

The molecular screening of the genes Pl1 and Pl6 in 117 genotypes of sunflower have revealed 46 genotypes resistant to 6 races of Plasmopara. 5 maternal and 23 paternal lines are recommended for use in obtaining hybrid combinations resistant to the pathogen. Infection with Plasmopara has differentially modified the expression of certain genes encoding antioxidative enzymes and essential components of the Systemic Acquired Resistance system as a function of the attack rate. It has been proved that an average to a high level of infection is positively correlated with the expression of the non-pathogen related 1 (NPR1) gene, while a weak level of infection is determined by the rapid response of the plant to the attack of the pathogen, ensured by an increase in the activity of the gene encoding the transcription factor Why1 (Whirly 1).

11.819.09.15A: UDaCoT – Data Collecting Tool
Type of project: Projects for young researchers (registration no. 379)
Project duration: 2011 – 2012
Project director: LEVITCHI Alexei, PhD in Biology

Executors:

Alexei Levitchi, project director, PhD; Rodica Martea, junior researcher, doctoral student; Daniela Abdusa, junior researcher, doctoral student; Tatiana Sestacova, junior researcher, doctoral student; Lidia Dragomir, junior researcher; Ana Babii, junior researcher; Igor Beletchii, junior researcher; Andrei Cibotari, junior researcher; Alina Antoci, junior researcher; Alexandru Levitchi, junior researcher; Filipp Gincota, senior software engineer; Dan Schiopu, technician.

Project objectives:

• Identification of the scientific domains with major interest in RM;
• Determination of the databases to extract information;
• Elaboration of the methodology of extraction and display of the data;
• Ontological study of the extracted results;
• Elaboration of the primary report regarding selected scientific fields.

Results:

The UDaCoT tool was created as an interdisciplinary elaboration which offers various options of the information search by proposed keywords for several biologic and medical domains through general and specialized bioinformatic databases. The data are identified in major databases of the NCBI portal and the results are represented in a graphical way. Each user must register by filling registration form in order to explore the tool.

11.220.10.05A: The investigation of different sunflower genotypes (Helianthus annuus L.) to biotic stress
Type of project: equipment procurement (registration no. 384)
Project duration: 2011
Project director: GLIJIN Aliona, PhD in biology, associate professor

Executors:

Aliona Glijin, PhD, chief of laboratory, project director; Alexei Levitchi, PhD, chief of laboratory; Oleg Budeanu, PhD, leading researcher; Angela Port, PhD, chief of laboratory; Victoria Rotarenco, PhD, senior researcher; Lucia Tapu, doctoral student, junior researcher; Rodica Martea, doctoral student, junior researcher; Tatiana Sestacova, PhD, researcher intern, Victoria Nechifor, PhD, researcher intern; Victoria Balan, doctoral student, junior researcher; Elena Mita, doctoral student, junior researcher; Ana Calmis, master, junior researcher; Daniela Abdusa, master, junior researcher; Lidia Dragomir, master, junior researcher; Adriana Acciu, master, technician; Maia Popa, chief accountant.

Project objectives:

Purchasing additional equipment necessary for conducting molecular researches will allow the realization of the following objectives:

• The study of the mechanisms of genetic and physiological resistance to phytopathogenic agents in plants and analysis of database relating to resistance genes.
• Assessment of the frequency of infestation of sunflower with rust and Orobanche under natural conditions and collection of experimental samples.
• Obtaining of DNA samples.
• Screening of genotypes based on molecular markers associated with resistance to rust and Orobanche.

Results:

The results relating to the frequency of sunflower infestation with rust were obtained. The intensity and the attack degree were determined for five differentiating sunflower lines resistant to races D, E, F and G.

The genes have been identified and the primers suitable for performing molecular screening for determining the resistance to rust and Orobanche were developed.

Equipment purchased:

• Vertical protein electrophoresis system (with electrical source), CONSORT.
• Grow rooms with adjustable temperature and lighting systems, FRIOCELL.
• Gel photo documentation system, UVITEK UVISAVE HD2 HOOD.

10.817.05.02AE: Education and research laboratories equipped with virtual tools
Type of project: equipment procurement, Institute of Applied Physics of the Republic of Moldova – Academy of Sciences of the Republic of Moldova (registration no. 381.INST)
Project duration: 2010
Project director: CULIUC Leonid, professor, doctor habilitated in physics and mathematics

Executors:

Elena Achimov, chief of laboratory, PhD in physics, Corneliu Gherman, leading researcher, PhD in physics, Vladimir Abaskin, leading researcher, PhD in physics, Andrei Colev, researcher, Sergei Levchenco, senior researcher, PhD in physics, Galina Guriev, researcher, Sergiu Anghel, doctoral student.

Research methods:

LabVIEW basic package; measuring module set; collection, storage and loading of experimental data files; real action with the support and guidance of external sensors using motors, video cameras, relays, valves, taps; virtual simulation experiments and computer-based technological processes.

Project objectives:

• Equipment for the training and experimentation processes.
• Adapting the educational process to the research process using LabVIEW virtual instruments.

Results:

Obtained equipment allows us to create an entirely new platform for the modernization of training and conducting various scientific law-cost experiments: detailed analysis of the investigated problems, researched rationale of problems, etc. Using Virtual Instruments in LabVIEW will help us to make closer the educational process with that of scientific research.

Equipment purchased:

Computer lab: CASE: 400w, front: 2 USB, IEEE-1392 + audio, CPU: 2 nuclee, 2,7 GHz, RAM:3 Gb DDRII, HDD: 250 Gb, VIDEO: 512 M DDR3, 256 bit, UNITATE OPTICĂ: DVD + -RW-, DL KEYBOARD/MOUSE: standard + mouse pad, Monitor 19" TFT

Computerized laboratory equipment for practical training of physics using LabVIEW tools network.