尼罗罗非鱼六个性别相关标记的FISH分析

FISH analysis of six sex-related markers in Nile tilapia,Oreochromis niloticus

从尼罗罗非鱼(Oreochromis niloticus)BAC基因文库5个克隆中提取和纯化含有6个性别连锁或相关标记(CLC5,GM204,GM271,GM354,UNH995和UNH104)的重组质粒DNA作为模板,以简并核苷酸为引物,通过PCR制备原位杂交探针。探针用荧光素进行标记,并与尼罗罗非鱼中期相染色体进行荧光原位杂交以确定这些标记在尼罗罗非鱼染色体上的位置和分布。结果显示,这些性别连锁或相关标记都位于尼罗罗非鱼第一对染色体长臂近末端,从分子细胞学角度验证了第一对染色体是尼罗罗非鱼的性染色体。另外由于这些标记的荧光信号在XY个体的2条性染色体上都有,一方面说明这些标记在罗非鱼上还不是性别特异的;另一方面也验证了尼罗罗非鱼的性染色体还处于分化的早期阶段。[中国水产科学,2006,13(4):525-529]

Tilapia has one pair of relative undifferential sex chromosome, which can not be distinguished from morphological appearance on the metaphase spread. The first pair of chromosome was confirmed as the sex chromosome of Nile tilapia by synaptonemal complex analysis. Genetic linkage maps were also constructed in tilapia and some of markers were revealled to be related with sex determination. By bulked segregant analysis, it was demonstrated that primary sex-controlling loci were located near marker UNH995 and UNH104 in the Nile tilapia. W loci in the blue tilapia were located near marker GM271 and GM354. Marker CLC5 and GM204 were also involved in X and W loci. All of these evidences showed that loci near these markers were probably the primary sex-controlling loci in tilapia. In our experiment, degenerated oligo-nucleotide primed PCR and fluorescence in situ hybridization were adopted to construct FISH maps of these six sex-linked or sex-related markers in order to unveil the relationship between these markers and chromosomes in Nile tilapia. Further to discuss the sex determination and differentiation mechanism in Nile tilapia form the molecular cytological level. In the paper, the recombinant plasmid DNAs containing six sex-related markers （CLCS, GM204, GM271, GM354, UNH995＆UNH104） were extracted and purified from BAC library of Nile tilapia, Oreochrornis niloticus. The probes for the in situ hybridization were prepared from the plasmid DNAs and labelled with fluoreseeins by degenerate oligo-nucleotide primed PCR （DOP-PCR）. The DOP-PCR was performed in 1x buffer, 2.5 mmol/L MgCl2, 0.4 mmol/L dNTPs, 4 mmol/L primer, 1 U Taq enzyme and 100 ng template DNA. The reaction parameters were predenaturing for 3 ： 00 at 94℃, 9 cycles of denaturing for 1 ： 00 at 94℃, annealling for 1 ： 30 at 30℃, prolonging for 3 ： 00 at 72 ℃, other 30 cycles of denaturing for 1 ： 00 at 95 ℃, annealling for 1 ： 00 at 62 ℃, prolonging for 1 ： 30 at 72 ℃. The labelled probes were then applied in the fluorescence in situ hybridization （FISH） to the mitotic metaphase chromosomes of Nile tilapia in order to localize these markers on the Nile tilapia chromosome by the following protocol. Metaphase spreads were dehydrated by passage through an ethanol series, denatured in 70 % formamide, 2 × SSC at 70 ℃ for 2 min, incubated in ice-cold 70 % ethanol for 3 min, dehydrated by passage through a second ethanol series and air dried. For each hybridization, 20ng of labelled probe, dissolved in 15μL of hybridisation solution （50% deionised formamide, 10% dextran sulphate in 2 × SSC, pH 7.0）, was denatured at 72 ℃ for 5 min and added to the prepared metaphase spreads. Coverslips were applied to the slides, which were incubated overnight at 37 ℃ in a moist chamber. After hybridisation, slides were washed three times in 50 % formamide, 2 × SSC at 42 ℃, twice in 2 × SSC at 42 ℃ and once in 4 × SSC, 0.05 % Tween 20 at room temperature. The hybridization of labelled probe was detected by incubation of the slides in 4 × SSC, 0.05% Tween 20 containing cyanine 3.29 （Cy3）-conjugated streptavidin. The slides were counterstained with 4,6-diamidino-2-phenylindole （DAPI） and antifade. Cy3 and DAPI signals were captured and observed by using a Cytovision Image Analysis System. The FISH results showed that all of these sex-linked or sex-related markers were located on the sub-terminal of the long arm of the first pair of chromosome and indicated the first pair of chromosome was the sex chromosome of Nile tilapia from this molecular cytological evidence. The signals of these markers observed on both chromosomes of XY individual sex chromosome implied they were not sex specific markers. On the other hand, it was verified that sex chromosome of Nile tilapia was on the early stage of differentiation. [Journal of Fishery Sciences of China, 2006,13 （4） ： 525 - 529 ]