Identification of Acipenseriformes species in trade Prepared by Arne Ludwig IUCN/ SSC Sturgeon Specialist Group (Leibniz-Institute for Zoo and Wildlife Research Berlin, Germany) and IUCN Species Programme 1 Contents Abbreviations 3 Glossary 4 Summary 7 1 Introduction 10 2 Species/caviar identification methods 19 2.1 Morphological methods 19 2.2 Genetic methods 20 2.2.1 DNA-sequencing 21 2.2.2 Genetic identification techniques based on mtDNA 21 2.2.2.1 PCR-Single Strand Confirmation Polymorphism (SSCP) 21 2.2.2.2 Species specific PCR 24 2.2.2.3 PCR-Restriction Fragment Length Polymorphism (RFLP) 26 2.2.3 Nuclear DNA techniques 31 2.2.3.1 Random Amplified Polymorphic DNA (RAPD) 31 2.2.3.2 Amplified Fragment Length Polymorphism (AFLP) 33 2.2.3.3 Microsatellites 33 2.2.3.4 Future developments – DNA barcoding 34 3 Population (stock) identification 37 3.1 Genetic methods 37 3.1.1 Microsatellites 37 3.2 Biochemical methods 39 3.2.1 Allozyme electrophoresis 39 4 Source identification (wild vs. aquaculture) 41 4.1 Gas-liquid chromatography 41 5 Age determination of caviar 43 6 Overview of identification techniques 45 7 Development of a reference collection 47 8 Concluding remarks and next steps 50 Acknowledgements (list of contributions) 53 Bibliography 54 Appendix 61 2 Abbreviations AFLP Amplified Fragment Length Polymorphism bp base pairs (value for the length of a DNA fragment) CITES Convention on International Trade in Endangered Species of Wild Fauna and Flora cytb cytochrome b (gene of the mitochondrial genome) d-loop displacement-loop (part of the mitochondrial DNA) DNA Deoxyribonucleic acid EMBL European Molecular Biology Laboratory (DNA database) IEF Isoelectric Focusing IUCN The World Conservation Union MDH Malate Dehydrogenase mt mitochondrial PCR Polymerase Chain Reaction RAPD Random Amplified Polymorphic DNA RFLP Restriction Fragment Length Polymorphism SEM Scanning Electron Microscope ss single-stranded SSCP Single Strand Confirmation Polymorphism SSR Simple Sequence Repeats STR Short Tandem Repeats Taq Thermus aquaticus VNTR Variable Number Tandem Repeats 3 Glossary allele – Alternative states of a gene, generated by mutations. autosomal DNA – All nuclear DNA which is located on an autosomal (non-sex determining) chromosome. In general, one allele is inherited from each of the parents. bp (base pairs) - Unit for the number of nucleotides currently used to characterize the length of DNA fragments or strands (pairs because DNA is a double stranded molecule and each nucleotide is base paired to its complementary nucleotide). cytochrome b – Gene of the mitochondrial genome coding for cytochrome b and very commonly used for phylogenetic analyses of vertebrates. diagnostic nucleotide or diagnostic substitution – Nucleotide or substitution which is only present in a single species (species-specific), population (population-specific) or stock (stock-specific). All specimens of this species, population or stock have this nucleotide or substitution and all specimens of other species, populations or stocks do not share it. DNA – DNA (deoxyribonucleic acid) is a nucleic acid that contains the genetic instructions. In complex eukaryotic cells such as those from plants, animals, fungi and protists, most of the DNA is located in the cell nucleus (nuclear DNA). Additionally, cellular organelles known as chloroplasts (plants) and mitochondria (animals - mitochondrial DNA) also carry DNA. The mitochondrial (mt) genome of animals is a small genome containing about 16000 bp in length. In contrast, the human nuclear DNA contains 24 distinct chromosomes with a total of about 3 billion DNA bp. The number of chromosomes range between about 120 and >500 in sturgeons and paddlefishes. The number of bp is unknown for these species. DNA sequencing – The process of reading the succession of nucleotides representing the genetic code by a machine called DNA sequencer. EMBL - Nucleotide Sequence Database of the European Molecular Biology Laboratory in Germany. endonuclease - An enzyme that breaks strands of DNA at internal positions; these enzymes are important tools in recombinant DNA technology. gene – A region of the DNA which controls for a specific characteristic and is consequently regarded as the basic unit of heredity. genetic drift - Genetic drift describes the statistical drift of the frequencies of neutral selected alleles over time. genotype - The genotype will contain both member (diploid) or all member (tetraploid or higher ploidy levels) alleles of the nuclear DNA. haplotype - A haplotype is the genetic constitution of an individual chromosome. In the case of diploid organisms, the haplotype will contain one member of the pair of alleles for each site. A 4 haplotype can refer to only one locus, to several loci or to an entire genome. A genome-wide haplotype would comprise half of a diploid genome, including one allele from each allelic gene pair. In this review, the term haplotypes refers to a set of nucleotide polymorphisms found to be in one or more genes. Furthermore, in the case of mitochondrial DNA haplotypes = genotype. heterosomal DNA – Nuclear DNA which is located on an heterosomal (sex determining – X/Y or Z/W) chromosome. A small part of this DNA from the Y or W chromosome is inherited in a clonal (non-recombinant, non-Mendelian) fashion. hybrid – A hybrid is a cross-breed between two (or sometimes more) species. In breeding biology, the term hybridization is also used for crosses between distantly (formerly separated) populations or hatchery strain of the same species. interspecific variability – Genetic differences between species. intraspecific variability – Genetic variability within one species. introgression – An introgression is based on a hybridization event followed by back-crossing with one paternal species/population. This back-crossing with one paternal species results in the loss of nuclear DNA diagnostics for the other paternal species. The first hybrid generation (F1) has an allelic ratio of 50:50 from both species, the second generation resulting from a cross between a F1-hybrid and one paternal species has a ratio of 75:25, third 87.5:12.5 and so on. After a few more generations, no nuclear signs of the second paternal species are detectable. Based on their nuclear profiles and on their morphology this offspring is genetically identical to the first paternal species. However, mitochondrial DNA is passed on maternally and therefore, offspring can still have nuclear signs from one parent and at the same time their mitochondrial DNA can be from another paternal species. lineage sorting - Lineage studies can be done based on the fact that the mitochondrial DNA only comes from the mother (maternal lineage), and the male Y chromosome only comes from the father (paternal lineage). Often these lineages are reflecting population (species) specific- selection resulting in interspecific or intraspecific variability. Mendelian inheritance (fashion) – Inheritance following the rules discovered by Gregor Mendel (1822-1884). microsatellite – A region of DNA that evolves very rapidly allowing the detection of recent evolutionary changes. Microsatellite regions form the basis of DNA profiling (genotyping) which is used in forensic applications. mitochondrial DNA - DNA which is located in the mitochondrial genome. In vertebrates, mitochondrial DNA (mtDNA) is only inherited from the mother. This means that mutations of mtDNA can be passed from mother to all offspring independent of their sex. 5 morphological analysis - The form and structure of an organism or one of its parts (for example oocytes) is analysed. mutation – A change in the DNA sequence that results in an alteration of this sequence. Reasons are non-correct replication or the presence of mutagenic factors, for example. Fixation of a mutation by selection or genetic drift can result in a diagnostic character state (diagnostic substitution). nuclear DNA – DNA which is stored in the nuclear genome, unifying all autosomal and heterosomal DNA. n-1 molecules – Normally primers are stored forzen. The successive thawing/freezing of primers, when they are used for PCR, cause their partial degeneration. This degeneration results in the cut of one (n-1) or more (n-x) nucleotides resulting in an shorting of primers. oocyte - An oocyte or ovocyte is a female gametocyte or germ cell involved in reproduction. Unfertilised egg cell. organoleptic analyses - Relating to perception by a sensory organ. In the case of caviar, colour and taste are proven mainly. PCR – PCR stands for Polymerase Chain Reaction. This is a biochemical (enzymatic) reaction geneticists use to artificially synthesise millions of copies of a target region of DNA. It is the method by which microscopic quantities of tissue can be investigated in forensic and conservation genetic analysis. population – In this paper the term is used for all specimens from the same river basin or water body (including tributaries). phylogeny – The pattern of ancestral relationships between species or taxonomic groups. primer - A short segment of DNA or RNA that is complementary to a given DNA sequence and that is needed to initiate replication by DNA polymerase (used for PCR amplification). RFLP - Restriction Fragment Length Polymorphism is a fingerprinting technique based on sequence- specific cleavages of DNA by restriction enzymes. Because of the diagnostic differences between sequences (species), species-specific restriction patterns can be used for identification. strain – Within this report this term is used for hatchery