›› 2018, Vol. 61 ›› Issue (7): 815-824.doi: 10.16380/j.kcxb.2018.07.008

• RESEARCH PAPERS • Previous Articles     Next Articles

Development of SSR primers for Simulium (Eusimulium) angustipes (Diptera: Simuliidae) based on RNA-seq dataset

GUO Huan, WANG Gang, ZHANG Shu-Tian, HUANG Min    

  1. (Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education, Entomological Museum, Northwest A&F University, Yangling, Shaanxi 712100, China)
  • Online:2018-07-20 Published:2018-07-20

Abstract: 【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 BlastX 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)n was the most abundant mononucleotide motif (86.93%). SSR primers were synthesized, and 15 primers were amplified successfully, of which 12 loci were polymorphic. BlastX 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.

Key words: Simulium (Eusimulium) angustipes, RNA-seq dataset, microsatellite DNA, SSR molecular marker, polymorphism