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(draft Last up-dated December 12th, 2011 - by Dr. Liliana TÖRÖK)

" FISHGENETICS LABORATORY"

TEAM IN 2007-2011 PERIOD

Specialists of DDNIRD constantly involved in activities of the "Fishgenetics Laboratory" team as main research team are:

Scientific researcher gr.I
Dr. SUCIU Radu - chromosome preparation, molecular biology

Scientific researcher gr.III
Dr. ONĂRĂ Dalia-Florentina - molecular biology, fish immuno-genesics

Scientific Researcher Assistent
MSc HOLOŞTENCU Daniela - molecular biology and biotelemetry

Note: the "Fishgenetics Laboratory" has as support staff  one biologist (technician) involved in chromosome preparation and molecular biology


HISTORY / TRADITION

The interest in fish genetics of Danube Delta National Institute for Research and Development (DDNIRD) started 25 years ago by establishing in 1986 the Cytogenetic Laboratory that focused on investigating fish karyotypes. Later on, keeping up with the advances in molecular biology, in 2000 was established at DDNIRD the molecular biology laboratory, investigating genetic variability of sturgeon species from the Lower Danube River, as part of Sturgeon Research Group (SRG).

A scholarship awarded by the British Royal Society to Prof. Andrew Ferguson of the Queen’s University of Belfast and Radu Suciu in 1998 resulted in two training and working stages (1998 and 1999) at the Molecular Biology Laboratory of the Medical Biology Centre in Belfast, which conducted to the first attempts to describe genetic variability of "Lower Danube River" sturgeons using 6 microsatellites and RFLP of mtDNA region ND5/6 and eventually lead to the development of the Fish Genetics Team.  Boyd Kynard also advised us in conducting experiments on behaviour of early life stages (ELS) of  "Lower Danube River" sturgeons in endless oval tanks.

During the last decades there was a great development worldwide of molecular biology research on sturgeons.
The knowledge concerning the genetic variability of the sturgeons from Danube River basin, the last wild self-sustaining sturgeon population of European Union gradually enlarged every year. The preliminary studies on genetic structure of sturgeon populations of Danube River have been conducted in collaboration with the Laboratory of Molecular Genetics of the Queen's University of Belfast.

It is widely recognized that conservation of biological diversity is essential for preserving existing biological systems. Identifying patterns of hierarchical population structure and diversity are critical steps toward setting conservation priorities and understanding processes of evolution and speciation. Information gained during the last 11 years has been used to augment endangered populations still spawning in the Lower Danube River . Both understanding of the genetic structure of sturgeon populations and the anthropic influence on the genetic diversity of these fish populations will have a major impact on the protection and recovery measures that should be established for sustainable management of the natural fish resources.

ORGANIZATION

Methodology used in the molecular biology laboratory of DDNIRD:

1. DNA extraction from fin clips, larvae and caviar according to a standard protocol.
2. DNA quantification – using a spectrophotometer UV/VIS Lambda 10, at 260 nm.
3. PCR– Polymerase Chain Reaction using primers able to amplify the target genes.
Several pairs of primers were designed based on DNA sequences available in GenBank database, analyzed using BioEdit software. Primers characteristics and compatibility were estimated using software OligoExplorer1.2 and OligoAnalyzer1.2.
4. RFLP – Restriction Fragment Length Polymorphism, a molecular biology based on the presence in genome of restriction sites specifically reacting with the restriction enzymes.
5. Gene cloning procedure, an advanced molecular biology technique applied for the first time in analysis of ribosomal gene 28S of anadromous sturgeon species of Danube River . Positive clones were sequenced by Macrogen Inc. Korea.
6. Bioinformatics - Primer design (OligoExplorer1.2 and OligoAnalyzer1.2), sequence alignment (Clustal W, BioEdit), standard online databases (GenBank, EMBL-Bank), statistic and phylogenetic analysis (Mega 3.1, Genepop, Dispan program suite, Haplotype Analysis).
 

PROJECTS IN 2007-2011 PERIOD

In the last 5 years the team-members were involved in 8 research projects dealing with identification of species, populations and interspecies hybrids, study of fish genetic diveristy and population structure, cloning, isolation and sequencing of new genes for phylogenetic studies as follow:

Genetic diversity

2

2

2

1

2

Molecular biology     1 1 1
Impact     1 1 1

The team-members involved in the above mentioned projects contributed to:
- describing of the consistent variability in the case of stellate sturgeon
- design of a new pair of primers for amplifying the region D-loop from mtDNA to assess the genetic variability of beluga sturgeon from Lower Danube River
- identification of four maternal genealogies of beluga sturgeon
- the first amplification, cloning and sequencing of 28S ribosomal gene of anadromous sturgeon species of Lower Danube River
- recomandation for gene pool conservation of sturgeon populations in Lower Danube River
- identification and certification of yong Rossian sturgeons used in the supportive stocking of the Lower Danube
- DNA analysis of caviar from the Black Market in Romania and Bulgaria
- identification of sturgeon species using RFLP of mtDNA cytochrome b region
- comparation of production traits and genetic parameters of the pike perch population of the Balaton Lake and the Danube Delta

Our further studies will include:

a) estimation of supportive stocking program of LDR using aquaculture fingerlings from wild parents by determining their survival rate and distribution along the Black Sea coasts, in relation to their genetic features (project proposal submitted to Romanian Fishery Operational Program in June 2011),

b) developing of genetic markers for migration studies and increase our understanding of the spawning behaviour of sturgeon populations of Danube River (Life + project proposal LINKSTUR - submitted in July 2011),

c) identifying populations best suited for genetic improvement / breeding programme by crossing brood-fish from genetically differentiated lines (using separated spawning grounds) captured from the wild  (project proposal to be submitted to Romanian Fishery Operational Program).


CONSULTANCY

Guidance of MSc Students: The "Fishgenetics Laboratory" provide support and supervising the PCR-RFLP work for one of the team member to develope the MSc thesis: Conservation of genetic diversity in populations of stellate sturgeon (Acipenser stellatus) of the NW Black Sea and Lower Danube River carried out at NTNU, Trondheim, 2011.  The Best Combat project  has offered supporting evidence for the existence of a population sub-structuring within the LDR basin stellate sturgeon.

DISSEMINATION


Scientific publication:

Dudu A., Suciu R., Paraschiv M., Georgescu S. E., Costache M. and Berrebi P., 2011, Nuclear Markers of Danube Sturgeons Hybridization International Journal of Molecular Sciences, vol. 12, no. 10, pp. 6796-6809
Onara DF, Forlenza M, Gonzalez SF, Rakus K, Pilarczyk A., Irnazarow I., Wiegertjes GF., 2008, Differential transcription of multiple forms of alpha2-macroglobulin in carp (Cyprinus carpio) infected with parasites.  Developmental and Comparative Immunology vol. 32, no. 4, pp. 339-347
Dudu A, Georgescu S.E., Suciu R., Dinischiotu A., Costache M., 2008, Microsatelitte DNA variation in the black sea beluga sturgeon (Huso huso), Romanian Biotechnological Letters vol. 13, no.3, pp. 3779-3783
Toană A., Luca C., Georgescu S. E., Suciu R., Costache M. 2008, Characterization of seven microsatellites in the Black Sea sturgeon (Huso huso), Scientific Annals of the Danube Delta Institute. vol 14. pp. 75-78

Presentation at international conferences:

- ECI XII 2007, Dubrovnik,
- International Conference on Conservation, Recovery and Sustainable Use of Danube River Sturgeons (29th of March  - 2nd of April 2011, Tulcea, Romania)
- Sixth International Symposium on Sturgeon - Wuhan / China 26-29 Oc. 2009
- Annual Symposium of Danube Delta Institute for Research and Development - 2011 _DELTNET International Conference & "Deltas and Wetlands "

 
     

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