preliminary rflp analysis of mtdna from southern african artemia populations horst kaiser,...
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Preliminary RFLP analysis of mtDNA from southern African
Artemia populations
Horst Kaiser, Department of Ichthyology and Fisheries Science Rhodes University, South Africa
and
Alexander Triantafyllidis, Athanasios Baxevanis, and Theodore J. Abatzopoulos; Department of Genetics, Development and Molecular Biology, Aristotle University of Thessaloniki, Greece
• Bisexual– Several species with a wide distribution range
• Parthenogenetic– Different ploidy levels – Genetics is poorly described
• Mixed populations
Artemia life histories
Potentially interesting southern African Artemia populations
Namibia:Smallest recorded cysts
Coega: Undescribed
Madagascar
The current project:Thessaloniki, Greece
• Main topic: Artemia biodiversity– Sub-topic: Genetic structure of southern
African populations– Technique development for Artemia
Project objectives
• To develop a data base of genetic markers for comparison with other populations.
• To find genetic markers for African Artemia populations.• Getting accustomed to molecular techniques
Problems with morphometrics as a tool to differentiate species
• Different species may be very similar morphometrically.
• Results are sensitive to rearing conditions.
• Requires strict standardisation.
Molecular techniques
• Using the16s sub-unit of the mitochondrial rDNA– Maternal inheritance– Small size (540 base pairs)– Stable gene arrangement– Fairly well conserved
Individuals from five populations were used for genetic analysis
• Three parthenogenetic populations from Greece
• Namibia (Swartkopmund)
• Madagascar (near Toliara)
• South Africa (Coega; Cerebos salt works)
RFLP
• Restriction Fragment Length Polymorphism
The molecular geneticist’s version of a
jigsaw puzzle - without knowing the
number of pieces.
The RFLP recipe
• Use only the best Artemia: gently squashed and homogenised.
• For best results use one shrimp at time.• Prepare with a liberal dose of proteinase
(keep in the oven overnight).• Wash in phenol and chloroform, then spin.• Add a few picograms of primers and a
touch of Taq enzyme….
continued
The RFLP recipe
• “Cook” while varying the temperature (PCR)
• Add assorted enzymes to cut the PCR product into smaller units.
• Add the rDNA pieces to a bed of gel.
• Let run on gel until well separated.
• Serve results in a table.
PCR product from 17 different individuals1.5% Agarose gel, ethidium bromide stain,
~ 540 base pairs (bp)
Reference ladder: 100 bpIndividuals
Restriction of PCR product using the DpnII enzyme
120 bp
180 bp
240 bp
100 bp Ladder
Restriction of PCR product using the TaqI enzyme
Artemia parthenogenetica(Greek population)
Artemia parthenogenetica(Greek population)
Restriction of PCR product using the HaeIII enzyme
Six individuals from the Coega population(s) showdifferent restriction patterns:
An indication of a mixed population?
Alu A C D3352802602201951551156055
Examples of different restriction patterns for two enzymes
Taq A C D E420319300261251171161101
54382119
Haplotypes of different populations
Greek (1) P* DBAAABABC 7
Greek (2) P CCCAABABD 7
Greek (2) P CBCAABABD 2
Greek (3) P DBAAABABE 9
Namibia (P) DBAAABABC 8
Madagascar (P) DBAAABABC 10
Coega CBCAAAABD 7
Coega AAAACAABC 1
Coega ABAACAABC 9
A. franciscana AAAAAAAAA -
*P = A. parthenogenetica; numbers are frequencies.
Artemia franciscana
Bisexual component from Coega
A. parthenogenetica (Greece)
A. parthenogenetica (Madagascar)
A. parthenogenetica (Greece)
A. parthenogenetica (Namibia)
Parthenogenetic componentfrom Coega
A. parthenogentica (Greece)
Phylogram based on restriction site patterns
Interesting preliminary results
• There is a considerable genetic distance between the two populations from the Coega salt works (South Africa)
• Populations with different life histories appear to co-exist.
Questions and tasks for next year
• Do the environmental requirements differ between the populations?
• Which species represents the bisexual component?
• Survey and study bird migration routes.