收稿日期: 2008-03-27
修回日期: 2008-07-21
网络出版日期: 2010-05-24
基金资助
国家高技术研究发展计划(“863”)项目(2006AA10A409), 国家科技支撑计划项目(2006BAD01A13)和广东省科技计划项目
Isolation and screening of microsatellite markers from the Chinese pearl oyster Pinctada fucata based on FIASCO
Received date: 2008-03-27
Revised date: 2008-07-21
Online published: 2010-05-24
Supported by
国家高技术研究发展计划(“863”)项目(2006AA10A409), 国家科技支撑计划项目(2006BAD01A13)和广东省科技计划项目
用FIASCO(Fast Isolation by AFLP of Sequences Containing Repeats)技术开展了合浦珠母贝Pinctada fucata基因组微卫星标记的分离与筛选研究。合浦珠母贝基因组DNA经限制性内切酶MseI酶切后与接头连接, 用生物素标记的(CA)15探针与其杂交, 然后用磁珠富集、洗脱获得单链目的片段, 经PCR扩增后形成双链, 最后进行克隆转化, 构建微卫星富集文库。挑选克隆用探针引物(CA)15 和载体引物进行第2次筛选, 获得阳性克隆357个, 测序结果表明, 297个克隆(83.2%)含有微卫星序列, 包括479个微卫星DNA结构域。其中完美型微卫星有370个(77.3%), 非完美型95个(19.8%), 复合型14个(2.9%)。合成引物49对, 有31对(63%)扩增出目的产物, 其中9 对在种群中(n=32)具有扩增多态性, 其多态信息含量PIC值在0.375―0.809之间, 平均为0.536; 等位基因数 在2―9个之间, 平均为4.889个; 观测杂合度介于0.200―0.600之间, 平均为0.415; 期望杂合度的变化范围为0.454―0.844, 平均为0.598。表明FIASCO技术适合于合浦珠母贝微卫星标记的分离与筛选。
关键词： FIASCO法; 合浦珠母贝Pinctada fucata; 微卫星标记; 分离与筛选
曲妮妮,龚世园,黄桂菊,童金苟,喻达辉 . 基于FIASCO技术的合浦珠母贝微卫星标记分离与筛选研究[J]. 热带海洋学报, 2010 , 29(3) : 47 -54 . DOI: 10.11978/j.issn.1009-5470.2010.03.047
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.
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