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.