两种书虱微卫星富集文库的构建及比较

Construction and comparative analysis of enriched microsatellite library from Liposcelis bostrychophila and L. entomophila genome

利用链霉亲和素与生物素之间的强亲和性原理,将链霉亲和素偶联的磁珠与微卫星探针(AC)12、(TC)12、(ATC)8、(ATG)8、(AAC)8、(ATAC)6及(GATA)6退火结合后,再亲和捕捉含接头和微卫星序列的单链书虱基因组DNA限制性酶切目的片段,经PCR扩增形成双链后进行克隆、建库。成功构建了嗜卷书虱和嗜虫书虱共13个微卫星富集文库,包括6个嗜卷书虱文库,7个嗜虫书虱文库,其平均阳性克隆率为71.17%。经检测发现共得到两种书虱260个微卫星位点。这两种书虱微卫星富集文库的建立和高多态性微卫星位点的筛选将为嗜卷书虱和嗜虫书虱的种群遗传与进化、基因连锁图谱构建、分子系统发育研究等提供大量分子遗传标记,对其在实仓中的持续控制提供遗传学信息。

The psocids Liposcelis bostrychophila and Liposcelis entomophila are major pests of stored grain and commonly occur on a wide range of stored products.Their world-wide prevalence and increasing importance as pests contaminating food and agricultural commodities have been documented in recent years.Intensive use of chemical insecticides for pest control has facilitated resistance development in psocids,especially,resistance to PH3.Control of these pests has proven difficult because they do not respond to management tactics that have been developed for other stored-product pests.Previous research has focused on psocids physiology,biochemistry,and basic biology in grain storage systems.However,the population genetic structure has not been well categorized,which may be useful to understand the formation of resistance mechanisms of psocids to insecticides.Therefore,a detailed understanding of L.bostrychophila and L.entomophila gene flow patterns,determining the geographical origins and dispersal of source populations and the resultant genetic structure among populations within China is needed.Microsatellites or simple sequence repeats（SSRs） are tandemly repeated motifs of 1-6 genetic base pairs.Because of their ubiquity,neutrality,hyper-variability,co-dominance,sensitivity and high polymorphism,microsatellite markers have become a powerful tool for revealing genetic variation and subsequently the population structure in recent decades.Up to now,only 11 microsatellite loci have been isolated from Liposcelis decolor（Pearman）. In the present paper,microsatellite-enriched libraries of L.bostrychophila and L.entomophila were constructed utilizing methodologies that exploit the strong affinity between biotin（vitamin B7） and the protein streptavidin.We propose a fast and easy protocol which is combination of two different published methods.Briefly,high-quality genomic DNA was digested by the restriction enzyme MseI and then ligated to designed adaptors.250-1000 bp microsatellite-containing DNA fragments were captured by streptavidin-coated magnetic beads.The beads affinity capture of microsatellite repeats using biotinylated oligonucleotide probes（AC）12,（TC）12,（ATC）8,（ATG）8,（AAC）8,（ATAC）6,and（GATA）6.Subsequently,PCR was used to amplify the captured molecules for transferring single strand DNA to double strand DNA.The PCR products（enriched microsatellite DNA fragments） were ligated to pGEM-T Easy vector and transformed into Trans5α competent cells.In total,13 microsatellite enriched libraries were constructed： 6 for L.bostrychophila,and 7 for L.entomophila. A total of 5218 clones were PCR screened for microsatellite content.The clones of L.bostrychophila and L.entomophila were 2542,2676,respectively.The percentage of the microsatellite positive clones ranged 9.38%-100%.The number of microsatellites detected for these two species was 260.In L.bostrychophila′s microsatellite-enriched libraries,the ratio of microsatellite positive clones ranked from the highest to lowest were tri-nucleotide libraries di-nucleotide tetra-nucleotide.Compared to L.bostrychophila′s microsatellite-enriched libraries,the ratio of microsatellite positive clones of L.entomophila′s enriched libraries ranked from the highest to lowest were di-nucleotide libraries tri-nucleotide libraries tetra-nucleotide libraries.Comparative analysis of microsatellite sequences for these two psocids revealed that the microsatellite sequences exist in multiple copies in the psocid genome.In addition,they were found to have similar or almost identical flanking regions.From our present study,the tri-nucleotide libraries yielded greater results for determining gene flow. Accumulation of multiple polymorphic microsatellite loci will greatly facilitate studies of individual identification and they are crucial to investigations of population genetics and evolution,gene linkage mapping,and elucidating the molecular phylogeny of psocids in China and internationally.