摘要摘 要 微卫星多重PCR技术在生物的亲子鉴定、群体遗传分析以及家系管理等方面均得到了广泛的应用。长鳍吻鮈(Rhinogobio ventralis)是我国长江上游特有珍稀鱼类，目前已达3级急切保护状，被列为低危鱼类。为了提高长鳍吻鮈微卫星分型效率，本研究从已开发的微卫星中筛选出15个多态性较高的微卫星位点进行组合扩增，并对位点组合的引物浓度、退火温度和反应体系等条件进行优化，建立了5组各含3个微卫星位点多重PCR体系。利用5组微卫星多重PCR，通过ABI 3730测序仪和Cervus v.3.0软件对136尾长鳍吻鮈进行个体基因型检测以及遗传多样性分析，结果显示，群体的平均等位基因数(number of allele, Na)为15.600，平均期望杂合度(expected heterozygosity, He)为0.825，平均观测杂合度(observed heterozygosity, Ho)为0.813，平均多态信息含量(polymorphism information content, PIC)为0.801。对40尾候选亲本和96尾子代进行亲子鉴定分析，结果显示，第一亲本的累积排除概率(combined exclusion probability of the first parent, CE-1P)、第二亲本累积排除概率(combined exclusion probability of the second parent, CE-2P)和双亲累积排除概率(combined exclusion probability of a parent pair, CE-PP)分别为0.999 986 07、0.999 999 97和1.000 000 00。使用该5组微卫星多重PCR体系进行亲子鉴定准确率为100%。长鳍吻鮈微卫星多重PCR技术的建立为亲子鉴定、分析群体遗传多样性和辅助家系管理提供了一种高效的技术手段。
Abstract：Abstract Multiplex PCR technique of microsatellites has been wildly used in the field of parent assignment, population genetic analysis and family management. Rhinogobio ventralis is endemic and rare fish in the upper Yangtze River, China. From the species vale and threatened degreed evaluation, it has reached three-level desperate protection sate and has been classified as low risk fish. In order to develop a rapid, economically efficient and robust approach for genetic studies of R. ventralis, a technique for the parentage assignment in R. ventralis was established based on 5 multiplex PCR panels using previously reported microsatellites loci with high polymorphism. The annealing temperatures, the primer ratio and the concentrations of reaction system were optimized in this technique. With the multiplex PCR tool, the genotyping and the genetic diversity analysis on 136 individuals of R. ventralis were performed using ABI 3730 genetic analyzer and Cervus v.3.0 software. The results showed that the average number of alleles (Na) was 15.600; the average expected heterozygosity (He) was 0.825; the average observed heterozygosity (Ho) was 0.813; the average polymorphism information content (PIC) was 0.801. The parentage assignment was performed on 40 candidate parents and 96 offspring. The combined exclusion probability of the first parent (CE-1P), the second parent (CE-2P), and a parent pair (CE-PP) were shown to be 0.999 986 07, 0.999 999 97 and 1.000 000 00, respectively. When using multiplex PCR group A, B and C, the CE-1P was 0.998 348 13 and CE-2P was 0.999 960 49. All of offspring could correctly find their real parents. The accuracy rate of parentage assignment was 100% using 5 multiplex PCR panels. These results indicated that 15 polymorphic loci and microsatellite multiplex PCR technique were suitable for paternity test of R. ventralis. The development of microsatellite multiplex PCR system provides a high efficiency technical method for parentage assignment, genetic diversity analysis, and marker-assisted family management in R. ventralis.
参考文献Bao X G, Xie W X, Huang D M and Zhu B K, 2011. Analysis of the ratio of flesh content and nutrient and evaluation of nutritional value for Rhinogobio ventralis. Journal of Hydroecology, 32(5):142–148（鲍新国, 谢文星, 黄道明, 朱邦科，2011. 金沙江长鳍吻鮈的含肉率及营养成分分析. 水生态学杂志, 32(5):142–148）Cao W X, 2000. Consideration on the construction of special fish nature reserve in the upper reaches of the Yangtze River. Resources and environment in the Yangtze River, 9(2):131—132（曹文宣. 2000. 长江上游特有鱼类自然保护区的建设及相关问题的思考. 长江流域资源与环境, 9(2):131–132）Chamberlain J S, Gibbs R A Rainer J E, et al. 1988. Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res, 16(23): 11141–11156．Chen M J, Fang C, Ke T, Xiong Z Y and He G Y, 2005. Multiplex PCR—A molecular biotechnique of high efficiency and speed. Journal of Wuhan University of Technology，27(10) : 33—36（陈明洁, 方倜, 柯涛, 熊志勇, 何光源, 2005. 多重PCR—一种高效快速的分子生物学技术. 武汉理工大学学报, 27(10) : 33–36）Chen Y Y，1998. The Osteichthyes Cypriniformes of Fauna Sinica. Beijing: Science Press, 200—208（陈宜瑜, 1998. 中国动物志硬骨鱼纲鲤形目(中卷). 北京：科学出版社，200–208）Chen X F，2013. Genetic Structure of Rhinogobio ventralis Endemic to the Upper Yangtze River.Chongqing: Southwest University（程晓凤. 2013. 长江上游特有鱼长鳍吻鮈(Rhinogobio ventralis)遗传结构分析. 重庆：西南大学）Ding R H，1994. The Ichthyography of Sichuan.The science and technology of Sichuan Press（丁瑞华, 1994. 四川鱼类志. 成都：四川科学技术出版社）Fu H T, Qiao H, Li F J, Wu Y, Gong Y S, Jiang S F, Xiong Y W and Wang N, 2010. Genetic diversity of Macrobrachium nipponense on a regional scale in the Yangtze River. Journal of Fisheries of China, 34(2):204-212（傅洪拓，乔慧，李法君，吴滟，龚永生，蒋速飞，熊贻伟，王宁，2010. 长江不同江段青虾的遗传多样性．水产学报，34(2)：204—212）Guan M, Qu H T, Hu M H, Liu Y, Lu X B, Ni Y, Xiao K, Yang Y J, Li S and Guo W T, 2015a. Artificial propagation of Rhinogobio ventralis. Fisheries Scince (5)，294—299（管敏, 曲焕韬, 胡美洪, 刘勇, 鲁雪报, 倪勇, 肖衎, 杨元金, 李莎, 郭文韬, 2015a. 长鳍吻鮈人工繁育的初步研究. 水产科学(5), 294–299）Guan M, Hu M H, Liu Y, Qu H T, Xiao K, Li S and Guo W T, 2015b. MS-222 study of Rhinogobio ventralis juvenile anesthesia. Fisheries Science & Technology Information, 42(2)72-77（管敏, 胡美洪, 刘勇, 曲焕韬, 鲁雪报, 肖衎, 李莎, 郭文韬, 2015b. MS-222对长鳍吻鮈幼鱼麻醉效果的研究. 水产科技情报, 42(2)72-77）Guo J T, Zhao J N, Yan B, Tang S J, Li S F, Ye W, Fu Y and Chen H C, 2013. Inheritance and segregation of microsatellite loci in the hybrid of Oreochromis niloticus♀×Sarotherodon melanotheron♂. Journal of Fishery Sciences of China(3), 528-535（郭金涛, 赵金良, 颜标, 唐首杰, 李思发, 叶卫, 符云, 陈辉崇, 2013. 尼罗罗非鱼♀×萨罗罗非鱼♂杂交后代的遗传分离分析. 中国水产科学(3), 528-535）Holland M M, Parson W. 2011. GeneMarker (R) HID: A reliable software tool for the analysis of forensic STR data. Journal of Forensic Sciences, 56(1): 29–35.Huang Y H, Hu X X, Xu W Z, Gao Y, Feng J D, Sun H and Li N, 2003. The factors affecting the efficiency of multiplex PCR. Hereditas, 25(1):65—68（黄银花, 胡晓湘, 徐慰倬, 高宇, 冯继东, 孙汉, 李宁, 2003. 影响多重PCR扩增效果的因素. 遗传, 25(1): 65–68）Ji W Z, Su B, 1999. Principles and Methodologies of Genetic Diversity Studies. Zhejiang: Zhejiang Science and Technology Press（季维智, 宿兵, 1999. 遗传多样性的研究原理与方法. 浙江: 浙江科学技术出版社）Jones A G, Ardren W R. 2003. Methods of parentage analysis in natural populations. Molecular Ecology, 12: 2511–2523.Kalinowski S T, Taper M L, Marshall T C. 2007. Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment. Molecular Ecology, 16(5): 1099–1106.Kong F, Gilbe G L. 2006. Multiplex PCR-based reverse line blothybridization assay (mPCR/RLB)-a practical eqidemiological and diagnostic tool．Nat Protoc，1(6)：2668–2680.Li B, Pan W T, Li G Q and Li H G, 2013. A Comparison among three softwares of parentage analysis (CERVUS,COLONY and PAPA)—A case study on parentage analysis of control pollination progenies in Liriodendron. Molecular Plant Breeding, 11(2): 241-248（李博, 潘文婷, 李康琴, 李火根. 2013. 三种常用亲本分析软件效率的比较—以鹅掌楸属树种控制授粉子代亲本分析为例. 分子植物育种, 11(2): 241–248）Li D Y, Kong J, Meng X H, Luan S, Luo K, Lu X and Cao B X, 2016. Development of multiplex PCR systems of microsatellite markers for pacific white shrimp(Litopenaeus vannamei) and its application for parentage identification. Progress In Fishery Sciences, 37(3):58—67（李东宇，孔杰，孟宪红，栾生，罗坤，卢霞，曹宝祥， 2016. 凡纳滨对虾(Litopenaeus vannamei)微卫星多重PCR体系的建立及其在家系亲权鉴定中的应用. 渔业科学进展，37(3):58–67）Li X F, Chen X F, Wang S, Duan X B, Wang D Q and Chen D Q, 2012. Optimization and preliminary application of fluorescence multiplex microsatellite PCR for Silver Carp (Hypophthalmichthys molitrix). Journal of Agricultural Biotechnology, 20(7):822-830（李小芳, 程晓凤, 王生, 段辛兵, 汪登强, 陈大庆, 2012. 鲢微卫星标记的荧光多重PCR体系建立及其应用. 农业生物技术学报, 20(7): 822–830）Li Y T, Wong K P, Shekhar M, et al. 2010. Development of two microsatellite multiplex systems for black tiger shrimp Penaeus monodon and its application in genetic diversity study for two populations. Aquaculture, 266(1–4): 279–288.Liu J, 2004. A quantitative analysis on threat and priority of conservation order of the endemic fishes in upper reaches of the Yangtze River. China Environmental Science, 24(4):395—399（刘军, 2004. 长江上游特有鱼类受威胁及优先保护顺序的定量分析. 中国环境科学, 24(4):395–399）Liu ZH B, Gan Q R, Wang R X, et al 2005. Application of multiplex PCR to studies on plant biology. Molecular Plant Breeding, 3(2) : 261–268．Luo W, Gao Z X, Zeng C, Deng W, Yi S K, Qian X Q and Wang W M, 2013. Establishment and application of microsatellite multiplex PCR system for bluntsnout black bream Megalobrama amblycephala. Journal of Dalian Ocean University, 28(5):418-423（罗伟, 高泽霞, 曾聪, 邓伟, 易少奎, 钱雪桥, 王卫民, 2013. 团头鲂微卫星多重PCR体系的建立及应用. 大连海洋大学学报, 28(5):418-423）Miao G D, Du M, Yang J F, Xu Y, Fan T J and Chen S L,2011. Development of two microsatellite multiplex PCR systems for turbot (Scophthalmu smaximus) and its application for family management. Periodical of Ocean University of China,2011, 41(1-2): 97—106（苗贵东, 杜民, 杨景峰, 徐营, 樊廷俊, 陈松林, 2011. 大菱鲆亲子鉴定的微卫星多重PCR技术建立及应用.中国海洋大学学报, 41(1–2): 97–106）Ren X Y, Li J, Liu P, Li X P, Han Z K and Liu L, 2011．Establishment and optimization of a microsatellite-labeling multiplex PCR system in Portunus trituberculatus. Progress In Fishery Sciences, 32(3):76—83（任宪云, 李建, 刘萍, 李晓萍, 韩智科, 刘磊, 2011. 三疣梭子蟹微卫星多重PCR技术建立及条件的优化.渔业科学进展, 32(3) : 76–83）Rooney A P, Honeycutt R L, Davis S K. 1999. Evaluating a Putative Bottleneck in a Population of Bowhead Whales from Patterns of Microsatellite Diversity and Genetic Disequilibria. Journal of Molecular Evolution, 49(5): 682–690.Shao K, Yan S, Xiong M, et al. 2015. Development of sixteen novel polymorphic microsatellite markers in Rhinogobio ventralis (Teleostei, Cyprinidae, Gobioninae). Conservation Genetics Resources, 7(1):225–227.Wang Y, Wang X X, Wang A, et al. 2010. A 16-microsatellite multiplex assay for parentage assignment in the easter noyster (Crassostrea virginica Gmelin). Aquaculture, 308(S1): 28–33.Wang M, 2011. Construction of a genetic linkage map on 11th Chromosome in Cashmere Goats. Huhhot: Inner Mongolia Agricultural University（王敏, 2011. 绒山羊11号染色体遗传连锁图谱的构建. 呼和浩特: 内蒙古农业大学）Weissenbach J. 1993. Microsatellite polymorphisms and the genetic linkage map of the human genome. Current Opinion in Genetics & Development, 3(3): 414–417.Wen P, Zhao J, Wei L I, Li W, H X Y and Zhu X P, 2015. The parentage assignment of Mauremys Mutica using multiplex PCR of microsatellites. Acta Hydrobiologica Sinica, 39(6):1134-1141（文萍, 赵建, 李伟, 洪孝友, 朱新平, 2015. 基于微卫星多重PCR技术的黄喉拟水龟亲子鉴定. 水生生物学报, 39(6):1134-1141）Wu X B, Guo W, Zhu Y J, Yang D G, Zhang M, 2015. Study on the embryonic development of Rhinogobio ventralis. Sichuan Journal of Zoology, 34(6):889-894（吴兴兵, 郭威, 朱永久, 杨德国, 张敏, 2015. 长鳍吻鮈胚胎发育特征观察. 四川动物, 34(6):889–894）Xiong L W, Liu Z Q, Yan Y L and Qiu G F, 2000. The segregation patterns of 37 polymorphism tri-nucleotide microsatellite markers in an F1 family of the Chinese mitten crab(Eriocheir sinensis). Journal of Fisheries of China , 39(2):174-181（熊良伟, 刘志强, 严银龙, 邱高峰, 2000. 中华绒螯蟹37个多态性微卫星标记在亲子遗传中的分离方式分析. 水产学报, 39(2):174-181）Xu N, Shi F, Xiong M H, Que Y F, Yang Z, Zhu B, HuJ X and Chang J B, 2009. Preliminary Studies on Genetic Diversity of Rhinogobio ventralis in Three Gorges Reservoir.Journal of Hydroecology, 2(2):113—116（徐念, 史方, 熊美华, 阙延福, 杨钟, 朱滨, 胡菊香, 常剑波, 2009. 三峡库区长鳍吻鮈种群遗传多样性的初步研究. 水生态学杂志, 2(2):113–116）Zhang Y P, Ryder O A, Zhao Q G, et al. 1994. Non-invasive giant panda paternity exclusion. Zoo Biology, 13(6): 569–573.Zhang Y, Sun D X, Yu Y, Zhang Y, 2008. Optimized multiplex PCR sets and genetic polymorphism of 30 microsatellite loci in domestic buffalo. Hereditas, 30(1) : 59—64（张毅, 孙东晓, 俞英, 张沅, 2008. 家养水牛30个微卫星标记的多重PCR体系建立及其多态性检测. 遗传，30(1) : 59–64）
