qPCR-based mitochondrial DNA quantification: Influence of template DNA fragmentation on accuracy

Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA (λnDNA) and mtDNA (λmtDNA) we present an approach to possibly correct measurements in degraded samples in the future. To our knowledge this is the first time different degradation impact of the two genomes is demonstrated and which evaluates systematically the impact of DNA degradation on quantification of mtDNA copy number.

A landscape mosaics approach for characterizing swidden systems from a REDD+ perspective

Swidden agriculture is often deemed responsible for deforestation and forest degradation in tropical regions, yet swidden landscapes are commonly not visible on land cover/use maps, making it difficult to prove this assertion. For a future REDD+ scheme, the correct identification of deforestation and forest degradation and linking these processes to land use is crucial. However, it is a key challenge to distinguish degradation and deforestation from temporal vegetation dynamics inherent to swiddening. In this article we present an approach for spatial delineation of swidden systems based on landscape mosaics. Furthermore we introduce a classification for change processes based on the change matrix of these landscape mosaics. Our approach is illustrated by a case study in Viengkham district in northern Laos. Over a 30-year time period the swidden landscapes have increased in extent and they have degraded, shifting from long crop–fallow cycles to short cycles. From 2007 to 2009 degradation within the swidden system accounted for half of all the landscape mosaics change processes. Pioneering shifting cultivation did not prevail. The landscape mosaics approach could be used in a swidden compatible monitoring, reporting and verification (MRV) system of a future REDD+ framework.