| That experiment used burbot (Lota Lota) as subject. Microsatellite enrichment by magnetic beads is a fast and efficient method for isolating this molecular genetic marker. Burbot is similar to its marine relatives in that its distribution is circumpolar. The burbot is a unique member of freshwater codfish, and it has high commercial value in China.Microsatellite markers of burbot were obtained by using the method. The genomic DNA was cut by the enzyme of MboI, and targeted segments were collected with the size of 500～1000bp base pairs by centrifugation of sucrose density gradient .Then the segments were ligated to created Brown adaptor and were filtrated to remove superfluous adaptor sequences by Centifugal Devices, from which, the"genomic PCR library"was created. These genomic DNA fragments hybridized to STREPTAVIDIN-coated magnetic bead capture using biotinylated capture molecules. We used the enriched fractions as the templates for the second round of PCR, and then the products were cloned. The plasmid library was transformed into competent Escherichia coli DH5αusing the CaCl2 method, using colony PCR to test the condition of insertion being 70.0%. The replicative recombinant colonies onto nitrocellulose membranes were second hybridized by oligonucleotide 32P-labelled probes to screen positive clones at the second times. 702 from screened 1650 colonies gave a positive signal, and 145 microsatellite loci were confirmed form selected 150 positive clones. The proportion of the CA/GT, AG/TC and ACA/TGT taken up in the microsatellites is 82.46%, 12.41% and 5.60%, respectively.This allowed us to designed 120 pairs of primers with the softwarePrimer5.0 and Primer 3.0. 40 pairs of primers were composted and screened. 22 pairs amplified special PCR products in the population of Jingbo Lake burbot and used to analyze genetic diversity of three populations of Heilong River region: Jingbo Lake, Wusuli River and Heilong River. Fifteen microsatellite loci were isolated from the burbot populations, two microsatellite loci were used to identify populations and eleven microsatellite loci are polymorphism using PCR and 8% polypropylene electrophoretic analysis.Using software of POPGENE3.2, GENEPOP and PHYLIP3.63 analyzed populations. Every microsatellite locus of three populations, the mean number of effective alleles (Ne) was 2.103, 2.779 and 2.583, the mean observed heterozygosity (Ho) was 0.284, 0.310 and 0.372, the mean expected heterozygosity (He) was 0.347, 0.378 and 0.427, and the mean polymorphism information contents (PIC) was 0.310, 0.340 and 0.370. The cluster analysis was constructed by UPGMA method based on the pairwise Nei's standard distances using PHYLIP. The mean genetic similarity index (I) of the three populations was 0.913 and the genetic distance (Ds) of the populations was 0.087. Cluster analysis showed the populations of JBL and WSL were closer than HLJ in genetics. And the result of cluster analysis was relate to geographic distribution.Up to now, there's no report about the application of relative microsatellite markers of burbot in China. The microsatellite markers isolated here will become very useful genetic tools in the population diversities analyze, conservation biology and strains identification. |
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