Abstract：Abstract Mandarin fish (Siniperca chuatsi) is one of the most important cultured freshwater fish in China. In order to carry out pedigree analysis of selective mandarin fish, compare identification effects of double digest restriction-site associated DNA sequencing (ddRAD-seq) and microsatellites, and establish paternity identification in mandarin fish, 30 pairs of microsatellite primers were selected from GenBank, and 11 of them with good specificity and polymorphism were screened by polyacrylamide gel electrophoresis. Then, these 11 primers were used to amplify 4 full-sib families whose pedigree information was known. Capillary electrophoresis was used to carry out microsatellite genotyping. The result of cluster analysis showed these four full-sib families were divided into four groups using the 11 pairs of microsatellite primers. All 3 283 SNP markers were obtained by ddRAD-seq, and they were used to construct a clustering tree, including 155 samples from the same family and 12 samples from other families. Finally, the 12 samples were effectively distinguished. The above 11 pairs of microsatellite primers were used to detect the 12 samples from other families, eight samples from the same family and their parents, and clustering tree was constructed. The result showed that the 8 samples from the same family and their parents were clustered together, while the 12 samples were clustered in the other cluster. These results illustrated that these 11 pairs of microsatellite primers had good family identification ability. Then, parentage determination of 126 offsprings, which were bred by 7 male and 13 female breeding parents, were carried out by these microsatellite loci. Simulation analysis showed that the non-exclusion probability of each locus ranged from 0.276 to 0.895, and the highest calculate exclusion probability was 99.999%. Among these 126 offsprings, 105 offsprings had been successfully identified for their pedigree relationship by parentage analysis and the success rate was 83.3%. These results illustrated that these 11 pairs of microsatellite primers had high calculate exclusion probability and good identification effect in pedigree identification. The paternity identification method established in this study will be helpful for breeding of mandarin fish.
[1]Anderson E C, Garza J C.The power of single-nucleotide polymorphisms for large-scale parentage inference[J].Genetics, 2006, 172(4):2567-2582[2]Bolger A M, Lohse M, Usadel B.Trimmomatic: a flexible trimmer for Illumina sequence data[J].Bioinformatics, 2014, 30(15):2114-2120[3]Borrell Y J, Alvarez J, Blanco G, et al.A parentage study using microsatellite loci in a pilot project for aquaculture of the European anchovy Engraulis encrasicolus L[J].Aquaculture, 2011, 310(3-4):305-311[4]Brookes A J.The essence of SNPs[J].Gene, 1999, 234(234):177-186[5]Catchen J, Hohenlohe P A, Bassham S, et al.Stacks: an analysis tool set for population genomics[J].Molecular Ecology, 2013, 22(11):3124-3140[6]Gjedrem T, Baranski M.Selective breeding in aquaculture: an introduction (Vol. 10)[M]. Springer Science & Business Media, 2009, New York, USA.[7]Hara K, Watabe H, Sasazaki S, et al.Development of SNP markers for individual identification and parentage test in a Japanese black cattle population[J].Animal Science Journal, 2010, 81(2):152-157[8]Hara M, Sekino M.Efficient detection of parentage in a cultured Japanese flounder Paralichthys olivaceus using microsatellite DNA marker[J].Aquaculture, 2003, 217(2003):107-114[9]Hauser L, Baird M, Hilborn R, et al.An empirical comparison of SNPs and microsatellites for parentage and kinship assignment in a wild sockeye salmon (Oncorhynchus nerka) population[J].Molecular Ecology Resources, 2011, 11(Supplement s1):150-161[10]Heaton M P, Leymaster K A, Kalbfleisch T S, et al.SNPs for parentage testing and traceability in globally diverse breeds of sheep[J].PLoS One, 2014, 9(4):e94851-[11]Jeffreys A J, Wilson V, Thein S L.Idividual-specific 'fingerprints' of human DNA[J].Nature, 1985, 316(6023):76-79[12]Kennedy G C, Matsuzaki H, Dong S, et al.Large-scale genotyping of complex DNA[J].Nature Biotechnology, 2003, 21(10):1233-1237[13]Kerékgyártó M, Kerekes T, Tsai E, et al.Light-emitting diode induced fluorescence (LED-IF) detection design for a pen-shaped cartridge based single capillary electrophoresis system[J].Electrophoresis, 2012, 33(17):2752-2758[14]Lien S, Gidskehaug L, Moen T, et al.A dense SNP-based linkage map for Atlantic salmon (Salmo salar) reveals extended chromosome homeologies and striking differences in sex-specific recombination patterns[J].BMC Genomics, 2011, 12(1):615-[15]Liu S, Palti Y, Gao G, et al.Development and validation of a SNP panel for parentage assignment in rainbow trout[J].Aquaculture, 2016, 452:178-182[16]Liu Z J, Cordes J F.DNA marker technologies and their applications in aquaculture genetics[J].Aquaculture, 2004, 238(1-4):1-37[17]Magdeldin S.Gel Electrophoresis-Principles and Basics[M]. InTech, 2012, Rijeka, Croatia, pp. 1-13.[18]Peterson B K, Weber J N, Kay E H, et al.Double digest RADseq: an inexpensive method for de novo SNP discovery and genotyping in model and non-model species[J].PLoS One, 2012, 7(5):e37135-[19]Rosa A J M, Sardina M T, Mastrangelo S M, et al.Parentage verification of Valle del Belice dairy sheep using multiplex microsatellite panel[J].Small Ruminant Research, 2013, 113(1):62-65[20]Taylor C, Guillen I M, Fernandez J, et al.Electrophoretic techniques applied to the detection and analysis of the human microsatellite DG10s478[J].Journal of Biomolecular Techniques, 2007, 18(5):298-305[21]Vandeputte M, Rossignol M N, Pincent C.From theory to practice: Empirical evaluation of the assignment power of marker sets for pedigree analysis in fish breeding[J].Aquaculture, 2011, 314(1-4):80-86[22]Yue G H, Xia J H, Liu P, et al.Tracing Asian seabass individuals to single fish farms using microsatellites[J].PLoS One, 2012, 7(12):e52721-[23]Yue G H, Xia J H.Practical considerations of molecular parentage analysis in fish[J].Journal of the World Aquaculture Society, 2014, 45(2):89-103[24]Zhang J, Ma W, Wang W, et al.Parentage determination of yellow catfish (Pelteobagrus Fulvidraco) based on microsatellite DNA markers[J].Aquaculture International, 2015, 24(2):567-576[25]陈睿毅, 王桂兴, 刘海金, 等.牙鲆亲本对子代贡献率的实验研究[J].中国水产科学, 2013, 20(4):698-705[26]成为为, 杨凯, 高银爱, 等.基于微卫星标记建立翘嘴鳜亲子鉴定技术[J].淡水渔业, 2016, 46(1):29-45[27]吕德坚, 陆惠玲.DNA亲权鉴定[M]. 暨南大学出版社, 2005, 广东, 广州.(Lv D X, Lu H L. Paternity Identification[M]. Jinan University Press, 2005, Guangzhou, Guangdong.)[28]文萍, 赵建, 李伟, 等.基于微卫星多重技术的黄喉拟水龟亲子鉴定[J].水生生物学报, 2015, 39(6):1134-1141[29]武耀, 贾智英, 李池陶, 等.筛选杂交鲤亲子鉴定的微卫星标记[J].农业生物技术学报, 2012, 20(5):549-559
