Evaluation of Five Methods for Total DNA Extraction from Western Corn Rootworm Beetles

Background: DNA extraction is a routine step in many insect molecular studies. A variety of methods have been used to isolate DNA molecules from insects, and many commercial kits are available. Extraction methods need to be evaluated for their efficiency, cost, and side effects such as DNA degradation during extraction. Methodology/Principal Findings: From individual western corn rootworm beetles, Diabrotica virgifera virgifera, DNA extractions by the SDS method, CTAB method, DNAzol reagent, Puregene solutions and DNeasy column were compared in terms of DNA quantity and quality, cost of materials, and time consumed. Although all five methods resulted in acceptable DNA concentrations and absorbance ratios, the SDS and CTAB methods resulted in higher DNA yield (ng DNA vs. mg tissue) at much lower cost and less degradation as revealed on agarose gels. The DNeasy kit was most time-efficient but was the costliest among the methods tested. The effects of ethanol volume, temperature and incubation time on precipitation of DNA were also investigated. The DNA samples obtained by the five methods were tested in PCR for six microsatellites located in various positions of the beetle’s genome, and all samples showed successful amplifications. Conclusion/Significance: These evaluations provide a guide for choosing methods of DNA extraction from western corn rootworm beetles based on expected DNA yield and quality, extraction time, cost, and waste control. The extraction conditions for this mid-size insect were optimized. The DNA extracted by the five methods was suitable for further molecular applications such as PCR and sequencing by synthesis

Establishment success in a forest biodiversity and ecosystem functioning experiment in subtropical China (BEF-China)

Experimental forest plantations to study biodiversity–ecosystem functioning (BEF) relationships have recently been established in different regions of the world, but subtropical biomes have not been covered so far. Here, we report about the initial survivorship of 26 tree species in the first such experiment in subtropical China. In the context of the joint Sino–German–Swiss Research Unit “BEF-China,” 271 experimental forest plots were established using 24 naturally occurring tree species and two native commercial conifers. Based on the survival inventories carried out in November 2009 and June 2010, the overall survival rate was 87 % after the first 14 months. Generalized mixed-effects models showed that survival rates of seedlings were significantly affected by species richness, the species’ leaf habit (deciduous or evergreen), species identity, planting date, and altitude. In the first survey, seedling establishment success decreased with increasing richness levels, a tendency that disappeared in the second survey after replanting. Though evergreen species performed less well than deciduous species with establishment rates of 84 versus 93 % in the second survey, their planting success exceeded the general expectation for subtropical broad-leaved evergreen species. These results have important implications for establishing mixed-species plantations for diversity conservation and improvement of ecosystem functioning in the Chinese subtropics and elsewhere. Additional costs associated with mixed-species plantations as compared to conventional plantations also demonstrate the potential of upscaling BEF experiments to large-scale afforestation projects