DNA typing of birch: Development of a forensic STR system for Betula pendula and Betula pubescens.

Although botanical trace evidence is often encountered in case investigations, the utilization of such traces in forensic investigations is still limited. Development of a forensic STR system for the two species of Betula (birch) indigenous to and abundant in North West Europe is a step in enhancing the applicability of traces from these species. We describe six microsatellite markers developed for birch species in detail, including repeat structure, and we propose a nomenclature for the encountered alleles. To assess the population characteristics, the genetic composition of wild, planted and intermediate populations of Betula pendula (a diploid species) and Betula pubescens (a tetraploid species) were investigated. The genetic differences between these two species were larger than the differences between populations of one species, even when both species co-occurred at one location. Therefore allele frequencies were estimated for both species separately. General, conservative random match probabilities were estimated for wild trees based on these allele frequencies (5∙10-6 for the diploid B. pendula and 1∙10-13 for the tetraploid B. pubescens), illustrating the potential relevance if trace evidence secured from a suspect is found to match a birch tree growing on or near a crime scene. Apart from wild trees, planted Betula trees also occur that may not originate from seeds, but may have been propagated through cloning. Based on the studied Betula trees, the random match probability of a potentially planted profile might be as high as 1.4∙10-2.

Local populations and inaccuracies: Determining the relevant mitochondrial haplotype distributions for North West European cats.

Typing of different portions of the feline mitochondrial control region has illustrated pronounced differences in haplotype distributions between cats from the Netherlands and other parts of the world. To gain a better understanding of the haplotype distribution of North West Continental Europe, 605bp of mitochondrial DNA was typed from randomly selected cats from the Netherlands (N=146), Belgium (N=64) and South West Germany (N=128). The genetic differences between these randomly sampled European populations correlate to the geographical distances, with the Dutch and the South West German populations furthest apart and the Belgian population as an intermediate (Fst values 0.01-0.03). Comparison of North West European mainland distributions to published feline mitochondrial haplotype distributions illustrated moderate to large genetic differentiation (Fst values 0.01-0.32). In this comparison, the correlation between geographical and genetic distance was absent, leading to founder effects and human impact on cat population structure and dispersion being considered as important parameters. When an accurate estimation of feline haplotype distribution is required in forensics, care should be taken when deciding whether extrapolating the frequency data from a certain source to a larger area (country/continent) is justified or whether additional typing of local populations is necessary. This may differ from case to case as local frequencies can be relevant, but can also be deceitful. To improve the applicability of forensic feline mitochondrial DNA studies, documentation and publishing of sampling strategies is advised, as is the implementation of measures to help eliminate potentially erroneous haplotypes.

Forensic utility of the feline mitochondrial control region - A Dutch perspective.

Different portions of the feline mitochondrial DNA control region (CR) were evaluated for their informative value in forensic investigations. The 402bp region located between RS2 and RS3 described most extensively in the past is not efficient for distinguishing between the majority of Dutch cats, illustrated by a random match probability (RMP) of 41%. Typing of the whole region between RS2 and RS3, and additional typing of the 5'portion of the feline CR decreases the RMP to 29%, increasing the applicability of such analyses for forensic investigations. The haplotype distribution in Dutch random bred cats (N=113) differs greatly from the distributions reported for other countries, with a single haplotype NL-A1 present in 54% of the population. The three investigated breeds showed haplotype distributions differing from each other and the random bred cats with haplotype NL-A1 accounting for 4%, 29% and 32% of Maine Coon, Norwegian forest cats and Siamese & Oriental cats. These results indicate the necessity of validating haplotype frequencies within continents and regions prior to reporting the value a mtDNA match. In cases where known purebred cats are involved, further investigation of the breed may be valuable.

Influence of environmental conditions on the production of phenazine-1-carboxamide by Pseudomonas chlororaphis PCL1391.

Pseudomonas chlororaphis PCL1391 produces the secondary metabolite phenazine-1-carboxamide (PCN), which is an antifungal metabolite required for biocontrol activity of the strain. Identification of conditions involved in PCN production showed that some carbon sources and all amino acids tested promote PCN levels. Decreasing the pH from 7 to 6 or decreasing the growth temperature from 21 to 16 degrees C decreased PCN production dramatically. In contrast, growth at 1% oxygen as well as low magnesium concentrations increased PCN levels. Salt stress, low concentrations of ferric iron, phosphate, sulfate, and ammonium ions reduced PCN levels. Fusaric acid, a secondary metabolite produced by the soilborne Fusarium spp. fungi, also reduced PCN levels. Different nitrogen sources greatly influenced PCN levels. Analysis of autoinducer levels at conditions of high and low PCN production demonstrated that, under all tested conditions, PCN levels correlate with autoinducer levels, indicating that the regulation of PCN production by environmental factors takes place at or before autoinducer production. Moreover, the results show that autoinducer production not only is induced by a high optical density but also can be induced by certain environmental conditions. We discuss our findings in relation to the success of biocontrol in the field.