Species identification in forensic samples using the SPInDel approach: A GHEP-ISFG inter-laboratory collaborative exercise.

DNA is a powerful tool available for forensic investigations requiring identification of species. However, it is necessary to develop and validate methods able to produce results in degraded and or low quality DNA samples with the high standards obligatory in forensic research. Here, we describe a voluntary collaborative exercise to test the recently developed Species Identification by Insertions/Deletions (SPInDel) method. The SPInDel kit allows the identification of species by the generation of numeric profiles combining the lengths of six mitochondrial ribosomal RNA (rRNA) gene regions amplified in a single reaction followed by capillary electrophoresis. The exercise was organized during 2014 by a Working Commission of the Spanish and Portuguese-Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG), created in 2013. The 24 participating laboratories from 10 countries were asked to identify the species in 11 DNA samples from previous GHEP-ISFG proficiency tests using a SPInDel primer mix and control samples of the 10 target species. A computer software was also provided to the participants to assist the analyses of the results. All samples were correctly identified by 22 of the 24 laboratories, including samples with low amounts of DNA (hair shafts) and mixtures of saliva and blood. Correct species identifications were obtained in 238 of the 241 (98.8%) reported SPInDel profiles. Two laboratories were responsible for the three cases of misclassifications. The SPInDel was efficient in the identification of species in mixtures considering that only a single laboratory failed to detect a mixture in one sample. This result suggests that SPInDel is a valid method for mixture analyses without the need for DNA sequencing, with the advantage of identifying more than one species in a single reaction. The low frequency of wrong (5.0%) and missing (2.1%) alleles did not interfere with the correct species identification, which demonstrated the advantage of using a method based on the analysis of multiple loci. Overall, the SPInDel method was easily implemented by laboratories using different genotyping platforms, the interpretation of results was straightforward and the SPInDel software was used without any problems. The results of this collaborative exercise indicate that the SPInDel method can be applied successfully in forensic casework investigations.

Evidence of three maternal lineages in Near Eastern sheep supporting multiple domestication events.

The variability of mtDNA was analysed in local sheep breeds reared throughout Turkey, for which a fragment of the D-loop region and the complete cytochrome b were sequenced. Phylogenetic analyses performed independently for the D-loop and the Cyt b gene revealed three clearly separated clusters indicating three major maternal lineages, two of which had been previously described as types B and A. The new type, C, was present in all the breeds analysed and showed considerable mtDNA variability. Divergence time was obtained on the basis of Cyt b gene and was estimated to be around 160,000-170,000 years ago for lineages B and A, whereas the divergence of lineage C proved to have occurred earlier (between 450,000 and 750,000 years ago). These times greatly predate domestication and suggest that the origin of modern sheep breeds was more complex than previously thought and that at least three independent sheep domestication events occurred. Our results, together with archaeological information and the current wild sheep populations in the Near East region support the high importance of this area in the sheep domestication process. Finally, the evidence of a third maternal lineage has important implications regarding the history of modern sheep.

Numerical calculation of the electrophoretic mobility of colloidal particles in weak electrolyte solutions.

A numerical calculation of the electrophoretic mobility of colloidal particles in weak electrolyte solution is presented. It is based on a previous work (C. Grosse, V.N. Shilov, J. Colloid Interface Sci. 211 (1999) 160-170), where the analytical theory of the thin double layer concentration polarization is generalized to the case of weak electrolytes, i.e., when the dissociation-recombination equilibrium and rate constants both have finite values. The analytical results are first completed by including terms corresponding to co-ions that were neglected in the original presentation. It is shown that these terms that have little bearing in the case of strong electrolytes, become quite important in the case when the electrolyte is weak. The problem is then solved using the network method, leading to numerical results for the electric potential and the concentrations of counterions, co-ions, and neutral ion pairs. Finally, the electrophoretic mobility is calculated both analytically and numerically. It is shown that the hypothesis of a weak electrolyte leads to changes of mobility with respect to the classical results that are even stronger than predicted analytically.

Corrected results for the influence of size, zeta potential, and state of motion of dispersed particles on the conductivity of a colloidal suspension.

A correction of a recent work on the dependence of the DC conductivity of diluted colloidal suspensions on the size, zeta potential, and state of motion of dispersed particles (C. Grosse, S. Pedrosa, V.N. Shilov, J. Colloid Interface Sci. 251 (2002) 304) is presented. It is shown that the procedure used in that work to calculate the contribution of the particles to the conductivity of the suspension leads to a result that includes the variation of the conductivity of the dispersion medium. Revised analytical and numerical calculations are presented, which strongly reinforce the conclusions reached in the original work: The expression for the conductivity increment based on the value of the dipolar coefficient of the suspended particles (calculated taking into account their electrophoretic motion) appears to be valid over the whole range of particle sizes.

On the influence of size, zeta potential, and state of motion of dispersed particles on the conductivity of a colloidal suspension.

The dependence of the DC conductivity of diluted colloidal suspensions on the size, zeta potential, and state of motion of the dispersed particles is analyzed both theoretically and numerically. It is shown that the simple formula that represents the conductivity as a sum of products: charge times mobility, taken over all the carriers present in the suspension, is only valid for exceedingly low values of the product kappaa. In contrast, the formulation based on the value of the dipolar coefficient of the suspended particles seems to be valid for all the range of particle sizes. This assertion is only true if the dipolar coefficient is calculated taking into account the electrophoretic motion of the particles. For very low values of the product kappaa, the dipolar coefficient of particles free to move can be several orders of magnitude larger than that of immobile particles.

Mitochondrial diversity and the origin of Iberian sheep.

Mitochondrial DNA diversity was analysed in 19 Iberian and six foreign sheep breeds. Three mtDNA lineages (B, A and C) were found in the Iberian sheep, with type B clearly predominating over the others. The results were analysed for each of the morphologically determined breed groups in Iberian sheep: Merino, Entrefino, Churro and Iberian trunks. MtDNA lineage C was found only in the Iberian trunk composed of Montesina and Ojalada. These two populations had high mtDNA variability, and in the Iberian sheep only Merino Branco had more variation. The other three Merino types studied showed moderate variability, including the most authentic Merino, the Spanish Merino. These three Merinos clustered closely in a multidimensional scaling representation of distances, while the fourth breed (Merino Branco) showed a clear separation. As for the other two trunks, breeds from the Churro group showed greater maternal uniformity while results for populations included in the so-called Entrefino trunk seemed to have a more heterogeneous maternal origin. The results obtained are discussed with available data from nuclear markers and with morphological classifications, and all this information is analysed in relation to the origin of the different Iberian sheep breeds.