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In mass fatality events, the need to identify large numbers of deceased persons using DNA can be a significant drain on already overburdened forensic practitioners, both in the field setting and the laboratory. The laboratory may be required to extract DNA from a variety of postmortem sample types, family or direct reference samples related to the missing, and perform matching of these results in a short period of time. While most forensic institutions are well equipped to handle both family and direct reference samples, postmortem samples such as bone or heterogeneous tissue samples can be difficult for labs to analyze. We have devised an easily deployable, efficient, and inexpensive method for collecting postmortem DNA samples on commercially available DNA preservation cards (“FTA ® ” cards). FTA ® cards are already widely used in forensic labs and are convenient for shipping due to their small volume and stability at room temperature. We evaluated the suitability of a protocol involving swabbing of incisions made on cadavers and sample deposition onto FTA ® cards over various postmortem intervals and under different environmental conditions. Each trial took place during a different point in the calendar year to evaluate the effects of seasonal weather patterns and temperature on decomposition, DNA yield, and rates of DNA degradation. To further account for the effects of seasonality (temperature and humidity), the progression of body decomposition was recorded following the Total Body Score (TBS) method [1]. DNA degradation was assessed either through STR amplification of 1.2 mm FTA punches or DNA extraction from 3.0 mm punches followed by real-time PCR quantification and STR amplification and genotyping. No consistent relationship was observed between postmortem interval and DNA degradation. Instead, the TBS score, which captures the stage of body decomposition, was shown to correlate well with DNA quantity. A TBS of 15 and below consistently yielded strong partial or full profiles (20 STR loci and Amelogenin using the PowerPlex 21 System) from all individuals from either 1.2 mm or 3.0 mm punches. Transfer of sample swabs to FTA cards is shown to be a simple and effective method for both field and laboratory operations over a range of conditions that can be evaluated by field forensic practitioners based on a body decomposition score. The approach could be beneficially integrated into mass fatality response plans.

STR-based DNA analysis is still the main tool for human DNA identification in most forensic DNA laboratories. DNA typing of aged human skeletal elements faces well-known interpretation challenges characteristic of degraded and low copy number DNA samples. Analyzing tens of thousands of human bone and teeth samples, we found that the occasional presence of artefactual peaks of presumed microbial origin adds another layer of complexity to the interpretation of STR profiles. In this paper, we present our approach and suggest guidelines for identifying and distinguishing non-human peaks, developed over the last 18 years. Additionally, we report a compendium of artefact peaks of presumed microbial origin recorded in human STR profiles obtained from bone and teeth samples, originating from Iraq, Chile, Maldives, Brazil and Western Balkans. Our experience has shown that these artefacts are not uncommon in bone STR testing, suggesting the possibility of occurrence in other forensic contexts, particularly trace DNA samples. Raising awareness among the forensic DNA community and accounting for this phenomenon is important for accurate STR interpretation.

The International Commission on Missing Persons (ICMP) conducts high throughput STR profiling on degraded skeletal remains, primarily recovered from mass graves relating to conflicts from 1992 to 1999 in the former Yugoslavia. To date, over 11,000 individuals have been identified through comparison of bone profiles to a large database of profiles from family members of the missing. To increase success rates in STR recovery, three short amplicon STR multiplexes (a 7-plex, a 6-plex, and a 5-plex) have been devised and implemented. These target loci from large commercial multiplexes, with an average decrease in amplicon size of 144 bp. The ICMP “miniplexes” have proven to provide substantially greater recovery of DNA data from a certain subset of difficult samples. However, the circumstances under which miniplexes provide additional data are restricted, and their advantages do not outweigh those of large commercial multiplexes for a majority of cases. The miniplexes, however, also have a very powerful use in DNA testing to support large scale reassociation of commingled, partial skeletons recovered from secondary mass graves.

In winter wheat, low temperature hardening induces a non-specific resistance to snow moulds and other fungi. In a suppressive subtractive hybridization cDNA library between winter wheat unhardened and hardened at 2°C, 17% of 186 sequenced cDNA clones were homologous to the small plant defence related proteins, γ-thionin, γ-purothionin or non-specific lipid transfer proteins (LTPs), based on their nucleotide sequences or deduced from amino acid sequences. Several partial- and full-length cDNA clones homologous to two defensin proteins (γ-thionin, subfamily B-2, γ-purothionin, subfamily I), and non-specific LTPs were isolated. Regulation of defensin transcripts was clearly different from that of the LTPs under controlled environments in the field. The γ-thionin and γ-purothionin transcripts were not expressed in unhardened plants grown at 20°C, strongly upregulated after 1–3 days hardening at 2°C, remained upregulated for 28 days hardening, and disappeared following 1 day of exposure to dehardening conditions at 20°C. The LTPs transcripts were constitutively expressed in plants growing at 20°C, gradually increased to maximum levels following 14–28 days at hardening, and remained upregulated following 1 and 7 days dehardening. In the field, the γ-thionin and γ-purothionin transcripts rapidly increased to maximum levels by mid-November and decreased gradually during the winter until mid-March LTP transcripts were highly expressed in the early autumn, further increased during December–January, then decreased during late winter and early spring. Differences in the pattern and level of expression of these transcripts were evident among cultivars; γ-thionin expression was associated with freezing resistance among the genotypes tested under both controlled environment and field conditions.

The first seedling or all-stage resistance (R) R gene against stripe rust isolated from Moro wheat (Triticum aestivum L.) using a map-based cloning approach was identified as Yr10. Clone 4B of this gene encodes a highly evolutionary-conserved and unique CC-NBS-LRR sequence. Clone 4E, a homolog of Yr10, but lacking transcription start site (TSS) and putative TATA-box and CAAT-box, is likely a non-expressed pseudogene. Clones 4B and 4E are 84% identical and divergent in the intron and the LRR domain. Gene silencing and transgenesis were used in conjunction with inoculation with differentially avirulent and virulent stripe rust strains to demonstrate Yr10 functionality. The Yr10 CC-NBS-LRR sequence is unique among known CC-NBS-LRR R genes in wheat but highly conserved homologs (E = 0.0) were identified in Aegilops tauschii and other monocots including Hordeum vulgare and Brachypodium distachyon. Related sequences were also identified in genomic databases of maize, rice, and in sorghum. This is the first report of a CC-NBS-LRR resistance gene in plants with limited homologies in its native host, but with numerous homologous R genes in related monocots that are either host or non-hosts for stripe rust. These results represent a unique example of gene evolution and dispersion across species.

This article presents the multidisciplinary effort in trying to identify the skeletal remains of 100 Norwegian soldiers serving in the German army, killed in Karelia Russia in 1944, from the recovery of the remains through the final identification using DNA. Of the 150 bone samples sent for DNA testing, 93 DNA profiles were obtained relating to 57 unique individuals. The relatives could not be directly contacted as the soldiers were considered as traitors to Norway; therefore, only 45 reference samples, relating to 42 cases of the missing, were donated. DNA matches for 14 soldiers and 12 additional body part re-associations for these individuals were found. Another 24 bone samples were re-associated with 16 individuals, but no familial match was found. More than six decades after the end of WWII, DNA analysis can significantly contribute to the identification of the remains.

Due to the large number of fragmented and commingled bodies recovered from mass graves related to the war in the former Yugoslavia, DNA typing has become an essential tool for the identification of missing persons from these conflicts. The International Commission on Missing Persons (ICMP) has identified more than 18,500 individuals using DNA analysis of tooth and bone. This wide body of experience permitted an empirical evaluation of the relative preservation of DNA in different skeletal elements. In this chapter, we present the ICMP’s experiences in sampling skeletal remains for DNA analysis, and a resulting detailed protocol for cost-effective DNA-based reassociation in commingled cases.

STR analysis of DNA extracted from skeletal samples can play an important role in the identification of missing persons. Here we present a method for the extraction of DNA from skeletal samples involving complete demineralization and digestion of the sample, followed by purification by silica binding. This method, together with the multiplex STR typing approach also presented, has proven highly successful in the recovery of DNA profiles from degraded, aged skeletal remains from a wide range of environmental contexts. The methodological steps presented include bone decontamination and grinding, DNA extraction, repurification in the case of highly inhibited samples, quantification, STR multiplex amplification, and profile reporting guidelines. However, the conditions applied for amplification and the criteria for allele calling and profile submission must be based on the results of each laboratory's internal validation experiments involving the type of samples relevant to the project at hand. The methods presented here have permitted large-scale DNA-based identification of persons missing from mass disasters and armed conflict.

We report the results of an inter-laboratory exercise on typing of autosomal single nucleotide polymorphisms (SNP) for forensic genetic investigations in crime cases. The European DNA Profiling Group (EDNAP), a working group under the International Society for Forensic Genetics (ISFG), organised the exercise. A total of 11 European and one US forensic genetic laboratories tested a subset of a 52 SNP-multiplex PCR kit developed by the SNPforID consortium. The 52 SNP-multiplex kit amplifies 52 DNA fragments with 52 autosomal SNP loci in one multiplex PCR. The 52 SNPs are detected in two separate single base extension (SBE) multiplex reactions with 29 and 23 SNPs, respectively, using SNaPshot kit, capillary electrophoresis and multicolour fluorescence detection. For practical reasons, only the 29 SBE multiplex reaction was carried out by the participating laboratories. A total of 11 bloodstains on FTA cards including a sample of poor quality and a negative control were sent to the laboratories together with the essential reagents for the initial multiplex PCR and the multiplex SBE reaction. The total SNP locus dropout rate was 2.8% and more than 50% of the dropouts were observed with the poor quality sample. The overall rate of discrepant SNP allele assignments was 2.0%. Two laboratories reported 60% of all the discrepancies. Two laboratories reported all 29 SNP alleles in all 10 positive samples correctly. The results of the collaborative exercise were surprisingly good and demonstrate that SNP typing with SBE, capillary electrophoresis and multicolour detection methods can be developed for forensic genetics.