Isolation and characterization of microsatellite loci from orange-spotted grouper, Epinephelus coioides
Date Issued
2010
Date
2010
Author(s)
Lin, Bou-Yun
Abstract
In recent years, microsatellites have become one of the most popular molecular markers used with applications in many fields. The objective of this study is to isolate the microsatellite loci from orange-spotted grouper, Epinephelus coioides and characterize those microsatellite loci.
The approach to achieve the target is based on the characteristics of the microsatellite loci. First, the genomic DNA extracted from the orange-spotted grouper had to be cut into smaller fragments by RsaI. Then, 6 sets of specific di-nucleotide probe (CG15, GA15, AC15, AT15, TG15 and CT15) were used to select the microsatellite-enriched fragments by the selective hybridization with two different hybridization buffers. Select totally 470 colonies from the microsatellite-enrich library. There were 201 positive colonies confirmed by the digestion check. Among all these positive colonies, 28 microsatellite loci have been successfully isolated. Before the samples were analyzed, finding the optimized annealing temperature was essential.
Total 54 samples E. coioides were collected from Cha-Dong, then the allelic distribution and the allele frequency of those microsatellite loci were analyzed. The number of the allele range from 0 to 8 alleles was found. Locus Ec-21 has the most PIC value of 0.85 and consider as the highest polymorphic locus. In the result from the analysis of cross-species amplification, locus Ec-19 can distinguish different species of Serranidae. Base on the distribution of separating alleles, the approach of mutiloci PCR amplification can be used in the further research.
Subjects
Epinephelus coioides
Serranidae
microsatellite DNA
polymorphic information content (PIC)
Type
thesis
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