基于微卫星标记的洞庭青鲫倍性鉴定
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摘要
洞庭青鲫产于湖南省澧县北民湖,因个体大且背部青灰而得名,其快速生长的特性和异于普通鲫的体型令作者怀疑前人报道的"二倍体群体"结果。为此,从银鲫微卫星文库中筛选9个位点构建微卫星鲫倍性鉴定体系,同时采用流式细胞术和染色体核型分析完成了洞庭青鲫倍性鉴定。结果显示,建立的微卫星鲫倍性鉴定体系能够100%区分二倍体、三倍体和四倍体鲫,微卫星基因分型结果显示136尾洞庭青鲫均为三倍体,各位点的等位基因数为1～3,样本基因型包括AAA、AAB/ABB、ABC 3种类型;洞庭青鲫血细胞DNA平均含量为159.42±5.64,与二倍体红鲫的血细胞DNA平均含量(102.43±3.54)比值(DI)为1.56∶1;流式细胞术与微卫星体系判定结果匹配度100%;染色体核型分析显示洞庭青鲫染色体数为3n=156±,核型公式为3n=39m+36sm+81sta,NF=231。建立了适用于鲫倍性鉴定的微卫星标记体系,且系统地证实了洞庭青鲫为三倍体鲫品种。
Carassius auratus var. dongting originates from Beimin Lake, Hunan. Compared to other Carassius spp., C. auratus var. dongting had a higher growth rate and larger body size, and we therefore hypothesized that these growth advantages were related to polyploidization. To verify this hypothesis, a 9 microsatellite ploidy identification method was developed to examine the ploidy level of C. auratus var. dongting, and flow cytometry assays and chromosome karyotype assays were used to further confirm the findings. The results showed that 9 microsatellites could completely distinguish 37 ploidy-known specimens. The genotyping results showed that all the 136 ploidy-unknown individuals were triploid, all three genotypes(AAA, AAB/ABB, and ABC) were observed in this population. The mean DNA content of blood cells in C. auratus var. dongting(159.42±5.64) was 1.5 times that of blood cells in C. auratus var. red(102.43±3.54), and the results from microsatellite markers were totally concordant with those from flow cytometry analysis. The karyotype analysis revealed that the number of chromosomes was 3n=156± and the karyotypic formula was 3n=39m+36sm+81sta, NF=231. This study not only established a microsatellite-based system for ploidy identification of C. auratus, but also systematically confirmed that C. auratus var. dongting is triploid.
Carassius auratus var. dongting originates from Beimin Lake, Hunan. Compared to other Carassius spp., C. auratus var. dongting had a higher growth rate and larger body size, and we therefore hypothesized that these growth advantages were related to polyploidization. To verify this hypothesis, a 9 microsatellite ploidy identification method was developed to examine the ploidy level of C. auratus var. dongting, and flow cytometry assays and chromosome karyotype assays were used to further confirm the findings. The results showed that 9 microsatellites could completely distinguish 37 ploidy-known specimens. The genotyping results showed that all the 136 ploidy-unknown individuals were triploid, all three genotypes(AAA, AAB/ABB, and ABC) were observed in this population. The mean DNA content of blood cells in C. auratus var. dongting(159.42±5.64) was 1.5 times that of blood cells in C. auratus var. red(102.43±3.54), and the results from microsatellite markers were totally concordant with those from flow cytometry analysis. The karyotype analysis revealed that the number of chromosomes was 3n=156± and the karyotypic formula was 3n=39m+36sm+81sta, NF=231. This study not only established a microsatellite-based system for ploidy identification of C. auratus, but also systematically confirmed that C. auratus var. dongting is triploid.
引文
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[2]杨品红,张倩,谢春华,等.洞庭青鲫肌肉营养成分分析及营养价值评定[J].动物学杂志,2008,43(1):102-108.Yang P H,Zhang Q,Xie C H,et al.Analysis of nutritional composition and evaluation of nutritional quality in muscle of Carassius auratus var.dongtingking[J].Chinese Journal of Zoology,2008,43(1):102-108(in Chinese).
[3]吴珊,吴维新.洞庭青鲫形态性状遗传分析[J].激光生物学报,2006,15(1):90-93.Wu S,Wu W X.The genetics analysis of morphological trait on Carassius auratus Tungting[J].Acta Laser Biology Sinica,2006,15(1):90-93(in Chinese).
[4]刘良国,杨品红,谢春华,等.洞庭青鲫(Carassius auratus var.Dongtingking)与三个鲫品系群体遗传多样性的ISSR分析[J].海洋与湖沼,2015,446(2):426-431.Liu L G,Yang P H,Xie C H,et al.Genetic diversity of Dongtingking crucian carp and other three strains of crucian carp revealed by ISSR analysis[J].Oceanologia et Limnologia Sinica,2015,446(2):426-431(in Chinese).
[5]张纯,刘少军,李涛,等.红鲫(♀)×鲤(♂)杂交鱼的胚胎染色体组倍性研究[J].水产学报,2011,35(9):1369-1373.Zhang C,Liu S J,Li T,et al.Studies of chromosome sets in embryonic cell of hybrid fish of red crucian crap(♀)×common crap(♂)[J].Journal of Fisheries of China,2011,35(9):1369-1373(in Chinese).
[6]Luo J,Gao Y,Ma W,et al.Tempo and mode of recurrent polyploidization in the Carassius auratus species complex(Cypriniformes,Cyprinidae)[J].Heredity,2014,112(4):415-427.
[7]张纯,刘少军,伍艳红,等.二倍体雌核发育鲫鲤卵子发生的DNA含量和细胞学分析[J].水产学报,2015,39(1):1-7.Zhang C,Liu S J,Wu Y H,et al.DNA contents and cytological analyses of oogenesis of the dipoid gynogenetic progeny of allotetraploid hybrids[J].Journal of Fisheries of China,2015,39(1):1-7(in Chinese).
[8]姜群,李琪,于红,等.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.Jiang Q,Li Q,Yu H,et al.A microsatellite panel for triploid verification in the Pacific oyster(Crassostrea gigas)[J].Journal of Fisheries of China,2014,38(12):1970-1975(in Chinese).
[9]耿波,孙效文.流式细胞术在水生生物DNA含量和倍性分析中的应用[J].水产学杂志,2008,21(2):21-24.Geng B,Sun X W.The application of flow cytometry on the DNA content and ploidy analysis of hydrophily organisms[J].Chinese Journal of Fisheries,2008,21(2):21-24(in Chinese).
[10]Hernández-Urcera J,Vera M,Magadán S,et al.Development and validation of a molecular tool for assessing triploidy in turbot(Scophthalmus maximus)[J].Aquaculture,2012,330-333:179-184.
[11]Leggatt R A,Iwama G K.Occurrence of polyploidy in the fishes[J].Reviews in Fish Biology and Fisheries,2003,13(3):237-246.
[12]杨睿姣,李冰霞,冯浩,等.彭泽鲫染色体数目及倍性的细胞遗传学分析[J].动物学报,2003,49(1):104-109.Yang R J,Li B X,Feng H,et al.Cytogenetic analysis of chromosome number and ploidy of Carassius auratus variety pengze[J].Acta Zoologica Sinica,2003,49(1):104-109(in Chinese).
[13]李学军,王阅雯,高丽霞,等.淇河鲫与野生鲫形态及染色体差异研究[J].水产科学,2010,29(11):674-676.Li X J,Wang Y W,Gao L X,et al.The morphometrical and chromosome differences in crucian carp Carassius auratus in Qihe River and in other waters[J].Fisheries Science,2010,29(11):674-676(in Chinese).
[14]张克俭,万全,李公行,等.滁州鲫染色体组型的研究[J].中国水产科学,1995,2(4):8-15.Zhang K J,Wan Q,Li G X,et al.Studies on karyotypes of Chuzhou crucian carp Carassius auratus[J].Journal of Fishery Sciences of China,1995,2(4):8-15(in Chinese).
[15]沈俊宝,王国瑞,范兆廷.黑龙江主要水域鲫鱼倍性及其地理分布[J].水产学报,1983,7(2):87-94.Shen J B,Wang G R,Fan Z T.The ploidy of crucian carp and its geographical distribution in Heilongjiang main river[J].Journal of Fisheries of China,1983,7(2):87-94(in Chinese).
[16]庄怡,刘必谦,唐杰.利用PCR-RFLP技术对鲫鱼6个不同品系的鉴定[J].生物技术通报,2012(11):144-149.Zhuang Y,Liu B Q,Tang J.Identifying six strains of Carassius auratus by PCR-RFLP on mtDNA ND3/ND4L/ND4 gene[J].Biotechnology Bulletin,2012(11):144-149(in Chinese).
[17]闫路路,秦艳杰,闫喜武,等.基于转录组平台的蛤仔微卫星标记筛选[J].生态学报,2015,35(5):1573-1580.Yan L L,Qin Y J,Yan X W,et al.Development of microsatellite markers in Ruditapes philippinarum using next generation sequencing[J].Acta Ecologica Sinica,2015,35(5):1573-1580(in Chinese).
[18]Slabbert R,Prins N,Brink D.A microsatellite panel for triploid verification in the abalone Haliotis midae[J].African Journal of Marine Science,2010,32(2):259-264.
[19]张小敏,马徐发,吴文化,等.利用微卫星遗传标记探讨达氏鳇的多倍体倍性[J].动物学杂志,2013,48(4):507-512.Zhang X M,Ma X F,Wu W H,et al.Polyploidy of Huso dauricus as revealed by microsatellite genetic markers[J].Chinese Journal of Zoology,2013,48(4):507-512(in Chinese).
[20]Lampert K P,Lamatsch D K,Schories S,et al.Microsatellites for the gynogenetic Amazon molly,Poecilia formosa:useful tools for detection of mutation rate,ploidy determination and overall genetic diversity[J].Journal of Genetics,2006,85(1):67-71.
[21]Zheng X H,Lu C Y,Zhao Y Y,et al.A set of polymorphic trinucleotide and tetranucleotide microsatellite markers for silver crucian carp(Carassius auratus gibelio)and cross-amplification in crucian carp[J].Biochemical Genetics,2010,48(7-8):624-635.
[22]林义浩.快速获得大量鱼类肾细胞中期分裂相的PHA体内注射法[J].水产学报,1982,6(3):201-208.Lin Y H.A PHA injection method in vivo for the rapid obtainment of large numbers of metaphase figures from kideny cells of teleosts[J].Journal of Fisheries of China,1982,6(3):201-208(in Chinese).
[23]Levan A,Fredga K,Sandberg A A.Nomenclature for centromeric position on chromosomes[J].Hereditas,1964,52(2):201-220.
[24]冼健安,王安利,孙敬锋.流式细胞术在水产动物研究中的应用[J].水产科学,2009,28(5):290-294.Xian J A,Wang A L,Sun J F.Applications of flow cytometry in aquatic animal research[J].Fisheries Science,2009,28(5):290-294(in Chinese).
[25]Castillo A,Budak H,Varshney R K,et al.Transferability and polymorphism of barley EST-SSR markers used for phylogenetic analysis in Hordeum chilense[J].BMC Plant Biology,2008,8:97.
[26]Feng B,Yi S V,Zhang M M,et al.Development of novel EST-SSR markers for ploidy identification based on de novo transcriptome assembly for Misgurnus anguillicaudatus[J].PLoS One,2018,13(4):e0195829.
[27]文亚峰,Uchiyama K,韩文军,等.微卫星标记中的无效等位基因[J].生物多样性,2013,21(1):117-126.Wen Y F,Uchiyama K,Han W J,et al.Null alleles in microsatellite markers[J].Biodiversity Science,2013,21(1):117-126(in Chinese).
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