(correspondence from S. Yi, Z. Yicheng, S. Wenqin, Z. Runsheng-1998)
Genetic variations in 4 species of brine shrimp, Artemia parthenogenetica, A. franciscana, A. sinica, and A. urmiana were detected using randomly amplified polymorphic DNA (RAPD) markers. Seven hundred and fifty-one amplified fragments were generated by RAPD markers with 101 primers.
The hatching of Artemia cysts and DNA extraction
The cysts of 4 species of Artemia hatched in a 35-ppt hatching medium. A temperature of 26 +/- 1 C was maintained, with continuous aeration, light of 1000 lux and a pH of 8.5.
Nauplii were washed by TE (Tris-HCl 10mmol/L, EDTA 1 mmol/L, pH 8.0), 2 ml nauplii with TE and 0.4 ml 10% SDS was placed in an Eppendorf tube, frozen at -20 C, melted at room temperature, then 0.44 ml 5 M Sodium Perchlorate was added. Samples were extracted with phenol/chloroform/isoamyl alcohol (25:24:1) and with chloroform. Then, they were incubated with RNase (10 microgram/ml) at 37 C for 2.5 h and extracted with phenol/chlorofrom/isoamyl alcohol and chloroform once again. The DNA was precipitated with two volumes of 95% ethanol at -20 C and rinsed by 70% ethanol twice. The DNA was dried and resuspended in TE.
Amplification of DNA
DNA from Artemia of 4 species was amplified through PCR (BIO-RAD, Gene CyclerTM). A total of 200 different primers were tested. The reaction in each case consisted of 15-30 ng of genomic DNA, 10mmol/L Tris-HCl, pH 8.3, 50mmol/L KCl, 2.5mM MgCl2, 0.5 microM primer, 0.3 mM each of dATP, dCTP, dGTP, dTTP and 1.5 units of Taq DNA polymerase (GWT) in a total volume of 25 microliter, 97 C pretreatment 10 min then -20 C 7 min before adding Taq DNA polymerase. The thermal cycles used were 5 cycles of 1 min at 94 C, 1 min at 37 C, and 2 min 72 C, then 35 cycles of 0.5 min at 95 C, 1 min at 37 C and 2 min at 72 C. A final extension was carried out at 72 C for 7 min. Amplified products were resolved by electrophoresis on 1.2% agarose gels run for 50 min at 5V/cm, stained with ethidium bromide and photogramhed under UV light.
Data analysis
101 primers gave amplification products and 71 primers had polymorphic amplification. Each species was scored for the presence or absence of every amplification product and the data entered into a binary data matrix. Cluster analysis was then performed to create dendrograms using UPGMA by NTSYS.
(Department of biology, Nankai University, Tianjin, PR China)