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孙典巧, 孙悦娜, 王日昕, 徐田军. 鮸鱼EST 序列中微卫星标记的初步筛选及特征分析[J]. 水生生物学报, 2011, 35(5): 753-760. DOI: 10.3724/SP.J.1035.2011.00753
引用本文: 孙典巧, 孙悦娜, 王日昕, 徐田军. 鮸鱼EST 序列中微卫星标记的初步筛选及特征分析[J]. 水生生物学报, 2011, 35(5): 753-760. DOI: 10.3724/SP.J.1035.2011.00753
SUN Dian-Qiao, SUN Yue-Na, WANG Ri-Xin, XU Tian-Jun. THE CHARACTERISTIC ANALYSIS OF MICROSATELLITES FROM ESTS IN MIICHTHYS MIIUY[J]. ACTA HYDROBIOLOGICA SINICA, 2011, 35(5): 753-760. DOI: 10.3724/SP.J.1035.2011.00753
Citation: SUN Dian-Qiao, SUN Yue-Na, WANG Ri-Xin, XU Tian-Jun. THE CHARACTERISTIC ANALYSIS OF MICROSATELLITES FROM ESTS IN MIICHTHYS MIIUY[J]. ACTA HYDROBIOLOGICA SINICA, 2011, 35(5): 753-760. DOI: 10.3724/SP.J.1035.2011.00753

鮸鱼EST 序列中微卫星标记的初步筛选及特征分析

THE CHARACTERISTIC ANALYSIS OF MICROSATELLITES FROM ESTS IN MIICHTHYS MIIUY

  • 摘要: 为大规模发掘和利用Ⅰ型微卫星标记, 通过建立全长均一化cDNA 文库和随机序列测定, 对鱼鮸转录基因组进行较大规模的微卫星序列特征分析和初步筛选。研究从4609 条高质量ESTs 中筛选到382 条微卫星序列, 筛出率为8.29%, 平均每318 bp 的ESTs 中就有一段不小于14 bp 的微卫星序列。筛选到的微卫星全部在低拷贝区间, 且重复类型丰富, 其中, 二核苷酸和三核苷酸重复类型出现频率较高, 分别占微卫星总序列的37.43%和32.98%。这两种类型的微卫星序列占到所有微卫星序列数目的70.41%, 而每种重复类型中的优势拷贝类型又存在差异, 两核苷酸微卫星中AC 型含量最高, 达到75.4%; 三核苷酸重复的优势重复类型是A、G 组合的基元重复类型(AAG 和AGG), 占三核苷酸类型的44.75%。根据设计的45 对引物的多态检测, 在可以扩增的33 对引物中筛选到9 个多态位点, 多态位点的比例达到20%。这表明黑鮸EST-SSR 中的多态位点比较丰富, 适于进行大规模EST-SSR 多态标记的筛选。以上结果为近缘物种间的微卫星分布频率和丰度的比较、鮸鱼及近缘物种可用Ⅰ型微卫星标记开发等工作提供基础和分析平台。

     

    Abstract: To explore large-scale and use the type I of microsatellite markers, microsatellite sequences in Miichthys miiuy genome transcription were analyzed and selected of polymorphism by establishing full homogenization cDNA library and random sequencing. Program of Tandem Repeat Finder (TRF) was used to select the sequences contained simple sequence repeats (SSR) from the expressed sequence tags (ESTs) of M. miiuy. Then some characterizations, such as the repeat type, time and number, the percentage of SSR and ESTs and total length, were added up based on the motif. Totally, 382 sequences contained not less than 14 bp simple repeats were obtained from 4609 ESTs of M. miiuy in our study, about 8.29% in the total database and on average about 318 bp length of EST contained a repeat sequence not less than 14 bp. All of the SSR selected were low-copies and the repeat types were abundant, including dinucleotide, trinucleotide, four-nucleotide, five-nucleotide and six-nucleotide repeat. Among the kinds of microsatellite repeats, these types, dinucleotide and trinucleotide repeat were the most abundant and it was 37.43% and 32.98% of the total microsatellite repeats, respectively. These types of microsatellites were 70.41% of the total, but the advantage motif type of each were different. The AC repeat was the most common type (75.4%) in dinucleotide repeats, while in trinucleotide repeats, the A and G combination motifs (AGG and AAG) was the most common, which was 44.75% of all motifs. At the same time, the initiative polymorphism was tested to examine the value of EST-SSR loci selected in our study. Finally, 33 pairs of primers were successfully amplified of 45 pairs of designed primers, and nine loci were polymorphism. The polymorphism loci were about 20% of the total primers. So the type of microsatellite we selected was great abundant and useful for the further study. The results provided the basis for comparing the frequency and abundance of the microsatellites distribution between species and the development of type I microsatellite markers in M. miiuy and some allied species. The nine polymorphism loci could be used in genetic diversity analysis, genome mapping and molecular assisted breeding in M. miiuy.

     

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