Methods for egg membrane and fertilization membrane removal and karyotype analysis of the sea urchin (Strongylocentrotus intermedius)
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S917

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    Abstract:

    is a commercially important sea urchin species in China and Japan. The chromosomes are difficult to prepare, so the karyotype of is unknown. Egg membrane or fertilization membrane removal is a key step in the preparation of the echinoderm chromosome. To identify the optimal method of egg membrane and fertilization membrane removal, four reagents[triazole solution (2 g/L), dithiothreitol solution (3×10-3 mol/L), p-aminobenzoic acid solution (3×10-3 mol/L), and hydrochloric acid solution (pH 4.75)] were used to treat the eggs and embryos for 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min post-fertilization at the 2-cell, 4-cell, 8-cell, and 16-cell stages, respectively. The chromosomes of the blastocyst embryos whose membranes were removed were prepared by conventional air-drying, and the karyotypes were analyzed. The results showed that all four reagents could remove the egg membrane or fertilization membrane. The eggs treated with 2 g/L of triazole for 30 min had an 85.50% membrane removal, 97.25% fertilization rate, and 1.75% deformity rate, indicating that this was the best method. Sixty-one metaphase cells with well-dispersed and well-preserved chromosomes were successfully prepared from the early blastocysts by the optimal membrane removal method. Karyotype analysis showed that the diploid chromosome number of was 42, and the karyotype was composed of 10 pairs of metacentric chromosomes, 10 pairs of submetacentric chromosomes, one pair of subtelocentric chromosomes, and 84 chromosome arms. Thus, the karyotypic formula is 2n=42=20m+ 20sm+2st and NF=84. The chromosome number is the same as that in previous reports for this species and in other sea urchins, such as . The karyotype is the same as . In conclusion, this study compared the membrane removal efficiencies of four reagents, and the results showed that treating with 2 g/L triazole solution for 30 min was the optimal method. We also obtained the karyotype of . The results provide a technical reference for the preparation of sea urchin chromosomes and chromosome manipulation breeding.

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陈小慧,张伟杰,李雅娟,刘雷,张宝警,丁君,常亚青. 中间球海胆卵膜与受精膜去除方法及染色体核型分析[J]. Jounal of Fishery Sciences of China, 2020,[volume_no](9):1033-1041

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  • Online: September 09,2020
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