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.