Isolation And Characterization Of Microsatellite Loci In Three Cephalopods

Octopus minor, Octopus vulgaris and Sepioteuthis lessoniana, have becomecommercial cephalopods in the world. In recent years, the sharp decline of naturalresources was due to environmental destruction and overfishing. It is necessary forspecies conservation to detect genetic variation and population structure.Microsatellite DNA marker, with the advantage of codominant, multiallelic andhighly polymorphic nature, is an available tool in genetics and evolution researches.The microsatellite loci in the three cephalopods were isolated based on magneticspheres and microsatellite enriched genomic library, and the polymorphism of theseprimers in each population were also analyzed by6%denaturing polyacrylamide gelusing silver staining. Total53microsatellite sequence were submitted to GenBank。Many biologists focus their attention on the study of population geneticstructure and population diversity in cephalopods. However, the development andapplication of microsatellite loci in cephalopods are still initial and seldom. Up to now,no microsatellite loci from O. minor and S. lessoniana were reported, and Greatorexet al.(2000) developed only6microsatellite loci from O.vulgaris. As for O. minor,625clones were selected for amplification, and115clones that generated two or morebands were selected and sequenced.50sequences were anaylized to be suitable forprimer design.12microsatellite loci obviously showed polymorphism in thepopulation sampled from Rongcheng, Shandong Province (37.16°N,122.41°E).Meanwhile,250clones for O.vulgaris were selected for amplification.70clones wereisolated for sequencing, and55PCR primer pairs for each microsatellite locus weredesigned. At last,17polymorphic microsatellite loci were screened and characterizedin the population of O. vulgaris sampled from Xiapu, Fujian Province (26.89°N,120.00°E).360clones for amplification from S. lessoniana were selected and97clones of them were isolated for sequencing with52PCR primer pairs being designed.The polymorphism was showed at24loci in the population of S. lessoniana from Lingshui, Hainan Province (18.48°N,110.02°E).The number of alleles，observed (HO) and expected (HE) heterozygosities at eachlocus were evaluated. Firstly,12microsatellite loci obviously showed polymorphismin the population of O. minor. The number of alleles at each locus ranged from2to11with an average of5.4, and the observed (HO) and expected (HE) heterozygositiesvaried from0.200to1.000and0.235to0.869with an average of0.635and0.630,respectively. Secondly, the levels of variability at17polymorphic microsatellite lociin O.vulgaris were evaluated. The number of alleles at each locus ranged from3to17with an average of8.5, and observed (HO) and expected (HE) heterozygosities rangedfrom0.154to1.000and from0.214to0.925with an average of0.809and0.750,respectively. Thirdly,24microsatellite loci showed polymorphism in S. lessoniana.The number of alleles at each locus ranged from4to26with an average of13.25, andthe observed (HO) and expected (HE) heterozygosities varied from0.188to0.828and0.392to0.921with an average of0.675and0.852, respectively. Genetic variability ofthese loci in three cephalopods indicated their practicability as appropriate populationgenetic markers of the investigation of the population genetic diversity.