Abstract
We developed and optimized a method using Chelex DNA extraction followed by whole genome amplification (WGA) to overcome problems conducting molecular genetic studies due to the limited amount of DNA obtainable from individual small organisms such as predatory mites. The DNA from a single mite, Phytoseiulus persimilis Athias-Henrot (Acari: Phytoseiidae), isolated in Chelex suspension was subjected to WGA. More than 1000-fold amplification of the DNA was achieved using as little as 0.03 ng genomic DNA template. The DNA obtained by the WGA was used for polymerase chain reaction followed by direct sequencing. From WGA DNA, nuclear DNA intergenic spacers ITS1 and ITS2 and a mitochondrial DNA 12S marker were tested in three different geographical populations of the predatory mite: California, the Netherlands, and Sicily. We found a total of four different alleles of the 12S in the Sicilian population, but no polymorphism was identified in the ITS marker. The combination of Chelex DNA extraction and WGA is thus shown to be a simple and robust technique for examining molecular markers for multiple loci by using individual mites. We conclude that the methods, Chelex extraction of DNA followed by WGA, provide a large quantity of DNA template that can be used for multiple PCR reactions useful for genetic studies requiring the genotypes of individual mites.
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Acknowledgements
We thank Prof. Salvatore Ragusa, Universita degli Studi di Palermo, Palermo, Italy for supplying P. persimilis from Sicily; Xiaoli Wu for help with colony maintenance; Ju Lin Weng for helping assess yield of DNA; and Srini Kambhampati, Mark Ungerer and Kun Yan Zhu for their valuable comments on an earlier draft of this manuscript. This study was funded under the Kansas NSF EPSCoR, Ecological Genomics Project. This is Contribution No. 07-71-J from the Kansas Agricultural Experiment Station.
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Konakandla, B., Park, Y. & Margolies, D. Whole genome amplification of Chelex-extracted DNA from a single mite: a method for studying genetics of the predatory mite Phytoseiulus persimilis . Exp Appl Acarol 40, 241–247 (2006). https://doi.org/10.1007/s10493-006-9042-1
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DOI: https://doi.org/10.1007/s10493-006-9042-1