基于FIASCO技术的合浦珠母贝微卫星标记分离与筛选研究

doi:10.11978/j.issn.1009-5470.2010.03.047

Abstract

Abstract:

Isolation and screening of microsatellite markers from Pinctada fucata were performed using the method of Fast Isolation by AFLP of Sequences Containing Repeats (FIASCO). After restriction of genomic DNA with MseI, the digested fragments were ligated with adaptors and then hybridized with biotinylated (CA)15 probes. The tentative microsatellite DNA was isolated by streptavidin-coated magnetic beads from the hybridized mixture. After purification, the isolated microsatellite DNA was amplified using degenerated primer MseⅠ-N and then cloned into T-vector. After transforming, the microsatellite-enriched library was constructed. The second PCR screening was performed using the primer of (CA)15 probe and T vector’s primers, and 357 positive clones were obtained. Sequencing analysis showed that 297 clones (83.2%) contained microsatellite DNA, including 479 microsatellite domain. Among them, 370 microsatellites (77.3%) were perfect type, 95 (19.8%) imperfect type and 14 (2.9%) compound type. Forty nine pairs of primers were designed and 31 (63%) pairs were effective for PCR amplification. Nine loci showed polymorphism as tested by a population (n=32). PIC values for the nine loci ranged from 0.375 to 0.809 with an average of 0.536. The number of alleles ranged from 2 to 9 with an average of 4.889. The observed heterozygosity ranged from 0.200 to 0.600 with an average of 0.415, and the expected heterozygosity ranged from 0.454 to 0.844 with an average of 0.598. This study demonstrated that FIASCO is a useful technique of microsatellite isolation for Pinctada fucata.

Key words: FIASCO method, Pinctada fucata, microsatellite marker, isolation and screening

QU Ni-ni ,GONG Shi-yuan ,HUANG Gui-ju ,TONG Jin-gou ,YU Da-hui . Isolation and screening of microsatellite markers from the Chinese pearl oyster Pinctada fucata based on FIASCO[J].Journal of Tropical Oceanography, 2010, 29(3): 47-54.

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Isolation and screening of microsatellite markers from the Chinese pearl oyster Pinctada fucata based on FIASCO

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