Communication by: Yi Sun, Wen-Qun Song, Yi-Cheng Zhong, Run-Sheng Zhang, Rui-Yang Chen-1998
We have used Amplified Fragment Length Polymorphism (AFLP) DNA markers to analyze 15 Artemia species and strains for genetic diversity. AFLP markers are extremely sensitive to even a small sequence variation and are very stable. It is more polymorphism than RAPD. Using only 10 pairs of primer combinations, we detected 580 AFLP bands of which were polymorphic. We have obtained clear DNA fingerprinting by using silver staining, which does not need special facilities or protection when using gama32P or gama33P. We also reduced the time and the cost by using the silver staining technique.
Sources of Artemia used for AFLP
Species and Populations; Sources; Chromosome Numbers; Reproduction Mode; Geographical Coordinates
A. franciscana from Great Salt Lake; USA; 42; Bisexual; -
A. franciscana from San Francisco Bay; USA; 42; Bisexual; -
A. persimilis; Argentina; 44; Bisexual; -
A. urmiana from Lake Urmia; Iran; 42; Bisexual; -
A. sinica from Yuncheng Lake; China; 42; Bisexual; N34.9, E110.8
A sinica from Beidachi; China; 42; Bisexual; N37.6, E107.4
A. sinica from Chagannao; China; 42; Bisexual; N39.0, E109.0
A. sinica from Ejinao; China; 42; Bisexual; N45.2, E116.5
A. species from Xizang; China; - ; Bisexual; -
A. species from Aibi Lake; China; 42, 84; Parthenogenetic; N44.9, E82.9
A. species from Balikun Lake; China; - ; Parthenogenetic; N43.7,
E92.7
A. species from Gahai Lake; China; - ; Parthenogenetic; N37.1; E97.5
A. species from Daqinghe; China; - ; Parthenogenetic; N39.2, E118.9
A. species from Yingko; China; 42, 84, 105; Parthenogenetic; N40.7, E122.5
A. species from Tianjin; China; 42, 105; Parthenogenetic; N39.1, E117.7
The hatching of Artemia and DNA extraction:
see Sun Yi et al. ELECTRONICAL LARVICULTURE NEWSLETTER ISSUE 56, 1 May 1998.
The AFLP procedure including Digestion of DNA, adapter legation, pre-amplification and selective amplification was performed following the protocol of Keygene n.v. (Zabeau and Vos, 1993) with minor modifications.
Gel Analysis:
The PCR product was mixed with formamide loading buffer. The resulting mixtures were heated for 10 min at 94, and then quickly cooled on ice. Samples were loaded on a 6% denaturing (sequencing) polyacrylamde gel. 0.5M Na2Ac in 1xTBE were used as positive and negative running buffer. Electrophoresis was performed at constant power 30W for approx. 2h.
Silver Staining:
After electrophoresis, the plates were carefully separated. The gel plate (wiped by binding solution and the other plate was wiped by Sigma Cote solution before pouring the gel) was covered with a fix/stop solution for 60 minutes until the tracking dyes were no longer visible, then it was rinsed with ultrapure water and transferred to staining solution and agitated well for 30 minutes. The gel was dipped briefly into ultrapure water, drained and placed immediately into chilled (10-12) developing solution. The gel was agitated well until all bands became visible. 1L of fix/stop solution was added directly to the developing solution. The gel was rinsed in ultrapure water, and was dried at room temperature.
Data Analysis:
Bands were scored as present or absent of every amplification product and the data were entered into a binary data matrix. Cluster analysis was then performed to create dendrograms using the Unweighted Pair-group Method with an Arithmetic Mean (UPMGA) by NTSYS (version 1.8).
Results:
The AFLP techniques are powerful DNA fingerprinting methods for classification of Artemia species and strains. The polymorphism analysis led us to the following conclusions: 1. The dendrogram shows that the Xizang Salt Lake populations might be genetically different from sibling species from Shanxi and Inner Mongolia. More work needs to be done here.
2. These data suggest that A. parthenogenetica of China might have originated from bisexual populations. But the parthenogenetic populations from inland salt lakes could follow a different evolutionary path unlike that of the coastal parthenogenetic populations.