基于RNA-seq数据的窄足真蚋SSR分子标记开发
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摘要
【目的】窄足真蚋Simulium(Eusimulium)angustipes属嗜血蚋种,并传播禽类疾病。本研究旨在通过RNA-seq技术获得窄足真蚋的转录组数据,进而开发其微卫星标记,为窄足真蚋的种群遗传学研究提供可靠的分子标记。【方法】以采自陕西宝鸡和新疆阿勒泰地区的窄足真蚋幼虫为材料,构建转录组数据库。使用软件MISA(microsatellite identification tool)搜索窄足真蚋unigenes库中的所有SSR位点,并随机挑选SSR位点,利用软件Primer Premier 5.0和Oligo 6.7设计微卫星引物。经PCR扩增和电泳检测,用Cervus 3.0.7等生物信息学软件进行统计分析,筛选多态性微卫星引物。利用Blast X比对Nr和Swiss-Prot蛋白质数据库,对含多态性SSR位点的unigenes进行功能注释。【结果】共鉴定出29 471个SSRs。最丰富的重复基序是单核苷酸重复类型,占总SSR数的87.05%;其次是三核苷酸重复类型,占7.95%。共发现34种碱基重复基序,主要是A/T基序重复,占86.93%。15对微卫星引物均成功扩增出特异性产物,含12对具多态性的微卫星分子标记。比对结果显示,7个SSR位点来自注释基因序列。【结论】基于RNA-seq二代测序技术可实现窄足真蚋SSR分子标记的高效率开发,对窄足真蚋的种群遗传学研究具有重要意义。
【Aim】Simulium( Eusimulium) angustipes is a bloodthirsty species that transmits poultry diseases. In this study,S.( E.) angustipes transcriptomes were sequenced using RNA-seq technology,and the SSRs were developed using transcriptome datasets,which may serve as reliable molecular markers for population genetics research of simuliids. 【Methods】Transcriptome database of S.( E.) angustipes larvae collected from Baoji of Shaanxi and Altay of Xinjiang was constructed. The software MISA was used to explore all the microsatellite loci in the unigene database of S.( E.) angustipes. Primers were designed using software Primer Premier 5. 0 and Oligo 6. 7 from the SSR loci chosen randomly. After PCR procedure and electrophoresis detection,the primers with polymorphism for SSR were screened and analyzed using Cervus 3. 0. 7 and other bioinformatics softwares. Unigenes with polymorphic SSR loci were annotated using Blast X against Nr and Swiss-Prot databases. 【Results】A total of 29 471 SSRs were identified in S.( E.) angustipes transcriptome. The most frequent repeat motifs were mononucleotide motifs( 87. 05%),followed by trinucleotide motifs( 7. 95%). Among all the 34 SSR motifs,( A/T) nwas the most abundant mononucleotide motif( 86. 93%). SSR primers were synthesized,and 15 primers were amplified successfully,of which 12 loci were polymorphic. Blast X results indicated that seven SSR loci were identified from the annotated genes. 【Conclusion】Based on RNA-seq sequencing technology,we can better realize the development of SSRs for S.( E.) angustipes,which is of great significance to the study of population genetics of this group.
【Aim】Simulium( Eusimulium) angustipes is a bloodthirsty species that transmits poultry diseases. In this study,S.( E.) angustipes transcriptomes were sequenced using RNA-seq technology,and the SSRs were developed using transcriptome datasets,which may serve as reliable molecular markers for population genetics research of simuliids. 【Methods】Transcriptome database of S.( E.) angustipes larvae collected from Baoji of Shaanxi and Altay of Xinjiang was constructed. The software MISA was used to explore all the microsatellite loci in the unigene database of S.( E.) angustipes. Primers were designed using software Primer Premier 5. 0 and Oligo 6. 7 from the SSR loci chosen randomly. After PCR procedure and electrophoresis detection,the primers with polymorphism for SSR were screened and analyzed using Cervus 3. 0. 7 and other bioinformatics softwares. Unigenes with polymorphic SSR loci were annotated using Blast X against Nr and Swiss-Prot databases. 【Results】A total of 29 471 SSRs were identified in S.( E.) angustipes transcriptome. The most frequent repeat motifs were mononucleotide motifs( 87. 05%),followed by trinucleotide motifs( 7. 95%). Among all the 34 SSR motifs,( A/T) nwas the most abundant mononucleotide motif( 86. 93%). SSR primers were synthesized,and 15 primers were amplified successfully,of which 12 loci were polymorphic. Blast X results indicated that seven SSR loci were identified from the annotated genes. 【Conclusion】Based on RNA-seq sequencing technology,we can better realize the development of SSRs for S.( E.) angustipes,which is of great significance to the study of population genetics of this group.
引文
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