基于转录组数据的意大利蝗微卫星位点分析与分子标记开发

Analysis of microsatellite loci from Calliptamus italicus(Orthopera:Acrididae)based on a transcriptome dataset

【目的】意大利蝗Calliptamus italicus(L.)是新疆荒漠半荒漠草原重要害虫。本研究利用已获得的意大利蝗转录组数据,鉴定其微卫星位点。【方法】使用MISA筛选SSR位点,利用Primer Premier 5设计引物,通过PCR扩增对引物进行验证。【结果】在意大利蝗转录组数据库中,共检测出156500个SSR位点,分布在126369条unigene中。其中,单核苷酸重复为60.88%,二核苷酸重复为23.58%,三核苷酸重复和四核苷酸重复分别为12.99%和2.04%。单核苷酸重复主要为A/T(44.38%),二核苷酸重复主要为AC/GT(12.99%)和AG/CT(8.05%)。基于筛选的SSR位点设计引物,随机挑选24对引物,对10个不同地理种群意大利蝗成虫DNA样品进行PCR扩增,共有6对引物扩增成功。【结论】本研究利用转录组数据发掘意大利蝗SSR位点,为意大利蝗分子标记、种群遗传及功能基因等研究奠定基础。

[Objectives]To investigate genetic variation in Calliptamus italicus(L.),an important pest of desert and semi-desert grasslands in Xinjiang.[Methods]Microsatellite loci were analyzed from the transcriptome dataset of C.italicus,and primers designed for PCR amplification.SSR loci were identified using MISA,and primers were designed using Primer 5 for experimental validation.[Results]In total,156500 SSR loci distributed in 126369 unigenes were detected.The majority of these were mono-nucleotides(60.88%),followed by di-nucleotides(23.58%),tri-nucleotides(12.99%),and tetra-nucleotides(2.04%).The most abundant mono-nucleotide repeat motif was A/T(44.38%).The dominant motif among the di-nucleotide repeat units was AC/GT(12.99%),followed by AG/CT(8.05%).24 primer pairs were randomly selected to validate SSR loci detected with genomic DNA from multiple individuals from 10 different geographic populations.6 primer pairs amplified the expected products.[Conclusion]Based on transcriptome data,156500 SSR loci were identified which can provide a foundation for research on molecular markers,population genetics and functional genes of C.italicus.