| 微卫星标记在长牡蛎三倍体鉴定中的应用 |
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| 引用本文： | 姜群,李琪,于红,孔令锋.微卫星标记在长牡蛎三倍体鉴定中的应用[J].水产学报,2014,38(12):1970-1975. |
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| 作者姓名： | 姜群 李琪 于红 孔令锋 |
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| 作者单位： | 中国海洋大学水产学院, 海水养殖教育部重点实验室, 山东 青岛 266003;中国海洋大学水产学院, 海水养殖教育部重点实验室, 山东 青岛 266003;中国海洋大学水产学院, 海水养殖教育部重点实验室, 山东 青岛 266003;中国海洋大学水产学院, 海水养殖教育部重点实验室, 山东 青岛 266003 |
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| 基金项目： | 国家“八六三”高技术研究发展计划(2012AA10A405-6);国家自然科学基金(31372524);山东省优秀中青年科学家科研奖励基金(BS2012HZ020) |
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| 摘 要： | 为开发一种简单有效的倍性检测方法对长牡蛎三倍体诱导结果进行准确评估,本实验采用细胞松弛素B抑制第二极体释放诱导产生长牡蛎三倍体,选用7个微卫星位点扩增基因组DNA,通过亲子代基因分型进行倍性检测,检测结果采用流式细胞仪加以验证,以评估微卫星标记倍性检测的准确性。另外,本研究探讨了准确鉴定倍性所需的微卫星标记数量与微卫星—着丝粒重组率(y)之间的关系,以期为其他物种采用分子标记进行倍性检测提供理论依据。结果显示,细胞松弛素B诱导产生的115个长牡蛎子代经7个微卫星位点鉴定得到40个三倍体,与流式细胞仪检测结果一致,准确率达到100%,7个微卫星位点的(1-y)的乘积为0.005。随机挑选6个位点,也可鉴定出所有三倍体,(1-y)的乘积为0.005~0.042。研究表明,本研究中开发的微卫星标记可以简单高效地鉴定长牡蛎三倍体,微卫星位点的(1-y)的乘积小于0.005,倍性检测的准确率可达100%,采用微卫星标记进行倍性检测对于加速长牡蛎三倍体育种进程具有十分重要的意义。
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| 关 键 词： | 长牡蛎 微卫星 倍性检测 微卫星—着丝粒重组率 |
| 收稿时间： | 6/7/2014 12:00:00 AM |
| 修稿时间： | 2014/10/10 0:00:00 |
A microsatellite panel for triploid verification in the Pacific oyster(Crassostrea gigas) |
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| JIANG Qun,LI Qi,YU Hong and KONG Lingfeng.A microsatellite panel for triploid verification in the Pacific oyster(Crassostrea gigas)[J].Journal of Fisheries of China,2014,38(12):1970-1975. |
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| Authors: | JIANG Qun LI Qi YU Hong and KONG Lingfeng |
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| Institution: | Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao 266003, China;Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao 266003, China;Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao 266003, China;Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao 266003, China |
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| Abstract: | In the Pacific oyster(Crassostrea gigas),triploidy induction is the most common genetic method to enhance production yield through phenotypic improvement.Production of triploids necessarily requires a proper method to verify the ploidy level after induction.Various methods have been developed such as flow cytometry,chromosome counting by karyotype analysis,counting of nucleoli,particle size analysis and so on.However,each method has some drawbacks considering size of sample,accuracy,complexity in conduction and cost.This study aims to identify a microsatellite panel for triploid validation in the Pacific oyster and further explore the number of microsatellites required to successfully verify ploidy status.Triploid oysters are usually induced by suppressing the extrusion of the second polar body.If the distance between a microsatellite and centromere is great,homologous chromosomes might cross over at prophase I of most meiosis allowing the triploid progenies having both of the two maternal alleles.This study screened the 56 microsatellites located in the centromere mapping and obtained seven successfully amplified loci at which the female parent is heterozygous and does not share any allele with the male parent.Using these seven microsatellites,40 triploid oysters were verified from the 115 individuals treated by cytochalasin B(CB,0.5 mg/L)for 15 min starting from the time when about 50% of the eggs released the first polar body.Ploidy status was then verified by flow cytometry and the correspondence was 100%.The ability of a microsatellite in identifying triploids relies on its frequency of microsatellite-centromere recombination(y).To further explore the number of microsatellites required to successfully identify all the triploids,different combinations of microsatellites were analyzed.The results indicate approximate 100% accuracy when product of(1-y)of all microsatellites is no more than 0.005.This study shows that microsatellite markers can serve as an accurate,fast,cost-effective and technically simple method for triploid verification in the Pacific oyster.The protocol described in this work will help in the application of triploids verification using molecular markers in other species. |
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| Keywords: | Crassostrea gigas microsatellite triploids verification microsatellite-centromere recombination |
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