基于454 GS FLX高通量测序的南疆沙蜥微卫星特征分析及其候选引物设计

Characterization of Microsatellite DNA Loci and Design of Candidate Primers to Amplify These Regions for Phrynocephalus forsythii by Using 454 GS FLX

南疆沙蜥Phrynocephalus forsythii是我国特有的一种小型爬行动物,分布于塔里木盆地。利用Roche 454 GS FLX高通量测序对该物种基因组测序,获得了55 909条高质量序列。利用Krait搜索并初步统计和分析基因组微卫星序列,共得到1~6个碱基重复类型的完美型微卫星12 109个。不同类型微卫星中,四碱基重复类型数目最多,有4 037个,约占总数的33.34%,其次是二碱基,约占总数的28.09%,再是三碱基、单碱基、五碱基和六碱基,分别约占总数的18.72%、13.91%、4.48%和1.46%。单碱基微卫星中C最多,二碱基微卫星中AC最多,三碱基、四碱基、五碱基和六碱基中最多的分别是AAC、AAAT、AAAAT和AACCCT。AC、AAAT、C、AG、A、AAC、AAT、AAAC、ACC和ACG是数量最多的10种重复拷贝类别。挑选部分三、四碱基重复类型的微卫星序列设计了100对可用于后续对南疆沙蜥微卫星标记开发的候选引物。本研究开启了对南疆沙蜥基因组微卫星特征的了解,为利用微卫星标记研究南疆沙蜥种群遗传结构奠定了基础。

The Forsyth s toad-headed agama,Phrynocephalus forsythii,is an endemic small lizard in the Tarim Basin in northwest China.The genome data of P.forsythii was generated using Roche 454 sequencing platform,and 55 909 high quality sequences were obtained.A total of 12 109 simple sequence repeats(SSRs)with 1-6 bp nucleotide motifs were identified by using Krait.Among the different repeat types of screened microsatellite DNA,tetranucleotide,having 4 037(33.34%),was the most common repeat unit followed by the di-(28.09%),tri-(18.72%),mono-(13.91%),penta-(4.48%)and hexanucleotides(1.46%).Additionally,C,AC,AAC,AAAT,AAAAT,and AACCCT were the most common repeat units among the mono-hexanucleotides,respectively.In the genome of P.forsythii,there were 10 predominant repeat types,including AC,AAAT,C,AG,A,AAC,AAT,AAAC,ACC and ACG.One hundred pairs of candidate primers containing a part of tri-and tetranucleotide repeat types were designed to amplify the microsatellite DNA loci in P.forsythii.This study sheds new light on understanding the feature of genomic microsatellite DNA in P.forsythii,and lays a foundation for further studying the population genetic structure of P.forsythii by using microsatellite markers.