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1 The Center of Molecular Genetic Identification at the Russian Federation CITES Scientific Authority on the Sturgeon Fishes. V.Krasnoselskaya, 17, VNIRO, Moscow, Russian Fderedation Phone/fax+ + (095) , molgen@molgen.vniro.ru www. vniro.ru

2 Machinery park

3 One of the workplaces under laminar conditions

4 Amplification Unit Tetrad Thermal Cycler (MJ Research PTC-225, USA)

5 Multifunctional gel scanner Variable mode Imager- Typhoon 8600 (Molecular Dynamics, USA) Multiplex PCR 1 2

6 Sequncer 3100 Genetic Analyzer Manufactured by ABI (now Aplera), USA

7 96-capillary electrophoretic Genetic Analysis System SpectruMedix SCE 9610 (USA)

8 Liquid Handling a part of the Robotic Workstation Genesis, designed by Tecan, Switzerland

9 General View of the LabNet

10 Sturgeon fish and products identification

11 Layout of the Fragment-DNA Analysis 3 Пр 5 DNA Пр Пр Пр 3 30 циклов = 5 3 n х 2 30 копий искомого фрагмента ДНК

12 A row of events for identification of single sturgeon egg cells species specific origin

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16 Samples 5 and 7 belong to Bairii-like haplotype of Acipenser gueldenstaedtii mt DNA 427 bp 214 bp 139 bp 1-4 Samples belong to Azove sea population of Acipenser gueldenstaedtii, ); 5-9 Samples belong to Kaspian sea population of A.gueldenstaedtii (A.gueldenstaedtii, 0008, 0009, 0014, 0016, 0020); Samples belong to Lena river population of A. baerii, и , соответственно); 15 DNA ladder (100 bp «SibEnzyme», Russia).

17 RAPD analysis

18 Fragment Analysis of RAPD- fingerprints Data (6% PAAG Gel)

19 Cluster analysis of Acipenser baerii (red) and A. gueldenstaedtii (yellow) sturgeons RAPD fingerprints.

20 RAPD- fingerprints UPGMA-dendrogram of following species: A. ruthenus (1), Huso huso (2) and Bester (A. ruthenus x H. huso hybrids)(3).

21 PER 1 PER 2 PER 3 PER 4 PER 5 GUE - BL 4 GUE Azov 2 GUE Azov 3 GUE - BL 1 GUE Volga 3 GUE Volga 4 GUE Azov 1 GUE Azov 4 GUE Volga 2 GUE - BL 2 GUE Volga 5 GUE - BL 3 GUE Volga 1 GUE - BL 5 BAE Lena 4 BAE Lena 6 BAE Lena 2 BAE Lena 5 BAE Lena 3 BAE Lena 1 Nuclear DNA fingerprints NJ-dendrogram of Acipenser gueldenstaedtii (GUE), A. persicus (PER), A. baerii (BAE) and A. gueldenstaedtii which has Bairii-like haplotype of mt DNA (GUE BL)

22 RAPD fingerprints Ward s Dendrogramm of Oncorhyncus nerka populations from Kamchatka peninsula west coast. -BolshayaRiver - Palana River - Ozernaya River

23 Multi-Dimensional Scaling of Oncorhyncus nerka populations

24 Molecular genetic identification of Oncorhynchus keta based upon RAPD-PCR data 1. Identification of brood stock 2. The genetic flow Ado-Tymovsky Pobedinsky Buyuklovsky Kalininsky Pobedinsky Buyuklovsky Kalininsky Ado-Tymovsky Okhotsky Okhotsky

25 Molecular genetic identification of Fish strains based upon RAPD-PCR data Cherepetsk Common Carps (Cyprinus carpio) strains Rainbow Trout (O. mykiss) strains Koi Carassius Cherepetsky Ramchaty Cherepetsky zerkalny Ramchaty razbrosany Cherepetsky cheshuychaty Steelhead Rostal Donaldson Adler Salmo salar

26 Microsatellite analysis

27 Microsatellite Multiplex OMM1082-One111-Ots253 Forward primers in each pair were labeled on their 5 end with fluorescent dyes as follows: OMM1082 JOE, One111 FAM, Ots253 TAMRA.

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30 Adaptation of PCR conditions for microsatellite analysis mfw 1 mfw 4 mfw 7 mfw 9 mfw 16 mfw 28 mfw 31 mfw 31 mfw 1 After adjustment of the PCR conditions mfw 4 mfw 7 mfw 9 mfw 16 mfw 31 Strictly following the protocol in original publication Grooijmans et. al Adjusted: Touchdown Annealing t o Mg 2+ etc.

31 Sizing of microsatellite PCR product (green) performed on capillary electrophoresis apparatus. Red internal size standard.

32 Sequencing

33 Sequence alignment of A. baerii (BAE) and A. gueldenstaedtii (GUE) species FSH intron polymorphisms. BAE1 BAE2 BAE3 BAE4 BAE5 GUE1 GUE2 GUE3 GUE4 GUE5 A. baerii A. gueldenstaetdii

34 Phylogenetic reconstruction of sturgeon group based on COI sequence

35 Phylogenetic reconstruction of Sturgeon species based on complete mitochondrial DNA (15780 bp) Sequencing of A. baerii and A. gueldenstaedtii complete mtdna was perfomed in the Center of Molecular Genetic Identification, VNIRO, Russia 100 Psephurus gladius Polyodon spathula Scaphirhynchus albus 100 A. transmontanus A. dabryanus 100 Huso huso Мt DNA A. stellatus A baerii A. gueldenstaedtii