Chen Jianjun, Wang Youli, Yue Yuanyuan, Xia Xiaohua, Du Qiyan, Chang Zhongjie
College of Life Science, Henan Normal University, Henan, P. R. China.
Mol Cell Probes. 2009 Oct;23(5):235-9. doi: 10.1016/j.mcp.2009.04.004. Epub 2009 May 4.
In this study, we identified a sex-specific DNA marker using randomly amplified polymorphic DNA (RAPD) fingerprinting in Yellow River carps (Cyprinus carpio from the Yellow River). Two hundred and twenty random primers were used in pooled DNA samples and individual DNA samples from male and female fish for RAPD fingerprinting. When using the primer S2107, a novel sex-specific PCR product was identified in all male individuals. DNA sequencing revealed that this 909 bp long DNA fragment has a low similarity to a repetitive sequence in zebrafish Danio rerio. In order to confirm the amplification results, two primers were designed within the male-specific sequence in order to amplify the sex-specific fragment from genomic DNA of male and female carps for sexing by PCR. The results indicated bands specific for males but not females. Respective results were obtained in dot blot and Southern blot hybridization experiments when using this sex-specific fragment as the probe. The sex-specific pattern was observed in 30 individuals from three separate common carp stocks, suggesting that the sequence is conserved in common carp species. However, no hybridization signals were found in grass carps Ctenopharyngodon idella, which are related to common carps. We submit to use this sex-specific fragment as a marker to rapidly and accurately identify the gender of Yellow River C. carpio. Furthermore, the sex-specific chromosome region may be characterized and used to study mechanisms of chromosome evolution in this fish species.
在本研究中,我们利用随机扩增多态性DNA(RAPD)指纹图谱技术,在黄河鲤鱼(Cyprinus carpio from the Yellow River)中鉴定出一种性别特异性DNA标记。使用了220种随机引物对雄性和雌性鱼类的混合DNA样本及个体DNA样本进行RAPD指纹图谱分析。当使用引物S2107时,在所有雄性个体中鉴定出一种新的性别特异性PCR产物。DNA测序显示,这个909 bp长的DNA片段与斑马鱼Danio rerio中的一个重复序列相似度较低。为了确认扩增结果,在雄性特异性序列内设计了两个引物,以便从雄性和雌性鲤鱼的基因组DNA中扩增性别特异性片段,通过PCR进行性别鉴定。结果显示仅雄性有特异性条带,雌性没有。当使用这个性别特异性片段作为探针时,在斑点杂交和Southern杂交实验中分别得到了相应结果。在来自三个不同鲤鱼种群的30个个体中观察到了性别特异性模式,这表明该序列在鲤鱼物种中是保守的。然而,在与鲤鱼亲缘关系较近的草鱼Ctenopharyngodon idella中未发现杂交信号。我们建议使用这个性别特异性片段作为标记,以快速准确地鉴定黄河鲤鱼的性别。此外,这个性别特异性染色体区域可以被表征,并用于研究该鱼类物种的染色体进化机制。