中国明对虾基因组微卫星重复单元类型与其多态性关系
RELATI ONSHIPS BETW EEN POLYMORPHISM AND TYPES OF SSRMOTIF IN THE GENOM E OF CHINESE SHRI M P FENNEROPENAEUS CH INENSIS
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摘要: 利用超声波粉碎中国明对虾Fenneropenaeus chinensis基因组后建立随机基因组文库,对其测序后获得了1996个克隆序列,经SeqmanⅡ(DNAstar)拼装后获得独立克隆数目为1900个,每个序列长度从400-700bp不等.利用重复序列分析软件对这些序列中含有微卫星重复序列的序列进行分析,共找到136个包含完整侧翼序列的重复序列.利用引物设计软件从以上重复序列中设计出34对引物,合成引物后,通过PCR扩增和聚丙烯酰胺凝胶电泳的方法获得了各个微卫星位点的等位基因数目.34对引物中,除4个没有扩增出产物外,其他都有较好的扩增结果,可以分辨出多态性信息情况,并据此分析了不同微卫星重复序列类型与其对应的位点多态性之间的关系.结果表明,两碱基重复类型具有较高的遗传多态性,而三碱基和四碱基以及复合型重复类型的平均多态性不高;两碱基重复序列类型各拷贝类别间的多态性信息没有明显的差异.进一步对两碱基的重复拷贝数目与多态性信息(等位基因数目)的相关关系进行分析,以考察拷贝数多少与等位基因数目之间的关系.利用SPSS软件进行相关分析,结果表明重复拷贝数目和等位基因数目呈一定相关(相关系数0.121),但相关性不显著(P=0.621).Abstract: The microsatellite (SSR) is a kind of co-dominant, and specialmolecularmarker, and used widely in many ge-netics analysis, such as genetic distance between different populations of organisms. Though papers about the isolation of microsatellite can be often found in journals, no detailed reports about which motif type of microsatellite might be more polymorphism were found so far. Here, we wanted to isolate the microsatllite sequences from random genomic library of Chinese shrimp (F. Chinensis), and obtained polymorphism microsatellite loci, then seek after the correlation between the polymorphism and the repeat motifs of microsatellites. Using the ultrasonic to break the genome DNA, the length of about 800-1500 bp DNA were selected to establish the random clone DNA library, and 1996 sequenceswere sequenced using MegaBace 1000 sequencer (Amersham Biosciences). After the biosoft SeqmanⅡ(DNAstar) assembling, 1990 in-dependent sequenceswere got, and the length of each clone sequence was about 400-700bp, from which 136 microsatel-lite-containing sequenceswere found. W ith the help of primer designed software (Premier Primer 5. 0), thirty-four primer pairswere designed and used to amplify the genome of F. Chinensis by PCR. The criterion for primer design was that the repeat numberofmotifwasmore than fifteen and the length of primer sequenceswas between 20 and 24. The PCR amplifi-cation results were further detected by AgNO3 staining methods and subsequently scanning by scanner. In these primer pairs, the detected results were as following: four primer pairs had no any amplification production, and thirty primers worked effectively, inwhich the polymorphism (allele) was observed. So, the success rate of primer design is88.-%. In the 34 primerpairs, though 30 microsatellite primerpairs generated amplification products, 1-primerpairs got vivid prod-uct bands and abundant polymorphism. So, the method trying to get microsatellite primers from randomly sequenced ge-nome sequences is inefficient. In the research, the lowest repeat numberofmotif is sixteen, and the corresponding number of allele is four. Using SPSS (Version 13. 0), we analyzed further the relationships between polymorphism and types of microsatellite motif, and the resultswere as follows: the polymorphism wasmore for dinucleotide motif than for trinucleoti-de, tetranucleotide, and compound repeat motifs. For the dinucleotide type, there was no significance difference among different classes (i.e. AT, AG, etc.) of dinucleotide motif. Furthermore, we analyzed the correlativity between polymor-phism and the repeat number of dinucleotide motif, and the results showed that there existed correlation between them (r=0.121), but no significance (P=0.621). So, more studies are needed for further clarifying their relationship be-tween polymorphism and types of SSR motif.
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Keywords:
- Fenneropenaeus chinensis /
- Microsatellites /
- MicrosatelliteMotif /
- Polymorphism
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