Genetic polymorphism of local Abkhazian grape cultivars

Local grape cultivars from different countries of the world are an important part of the gene pool of this culture. Of particular interest are the genotypes of the most ancient regions of viticulture. The territories of the subtropical zone of Georgia and the central part of Abkhazia belong to one of the centers of origin of the cultural grapevine. The purpose of the work was to genotype native Abkhazian grape cultivars, to study their genetic diversity based on DNA profiling data and to compare them with the genotypes of local varieties of other viticultural regions. Samples of plants were taken on the territory of the Republic of Abkhazia in private farmsteads and in the collection of the agricultural firm “Vina i Vody Abkhazii“ (“Wines and Waters of Abkhazia”). The genotyping of the Abkhazian cultivars Avasirhva, Agbizh, Azhapsh, Azhizhkvakva, Azhikvaca, Atvizh, Atyrkuazh, Achkykazh, Kachich was carried out using 14 DNA markers, 9 of which are standard microsatellite markers recommended for the identification of grape varieties. To improve our knowledge about the sizes of the identified alleles, we used the DNA of grape cultivars with a known allelic composition at the analyzed loci. Statistical analysis of the data showed that the observed heterozygosity for the analyzed loci exceeded expected values, which indicates a genetic polymorphism of the studied sample of varieties. Evaluation of genetic similarity within the analyzed group based on the results of genotyping at 14 loci showed that the cultivars Kachich and Azhapsh differed from the other Abkhazian varieties. The obtained DNA profiles of the Abkhazian cultivars were checked for compliance with DNA-fingerprints of grape varieties in the Vitis International Variety Catalogue. The Georgian varieties Azhizhkvakva and Tsitska turned out to be synonyms according to DNA profiles, two varieties from the Database (Italian Albana bianca and Georgian Ojaleshi) have differences in DNA-fingerprints from the varieties Atyrkuazh and Azhikvatsa only in one allele, respectively. When comparing the identified Abkhazian grape genotypes, their difference from the sample of Dagestan, Don, Greek, Turkish, Italian, Spanish, and French varieties and genetic similarity with the genotypes of Georgian grapes were shown.

Technical note: Development of DNA quantitation and STR typing systems for Panthera tigris species determination and individual identification in forensic casework

The aim of this technical note is to provide an overview of methodical approaches used to develop molecular systems for species determination/DNA quantification called Ptig Qplex and individual identification called Ptig STRplex of Panthera tigris samples. Both systems will help to combat the illegal trade of endangered species and create a worldwide shared database of DNA profiles.

Optimisation of an Automated DNA Extraction Method for Bone and Teeth Samples and Applicability to Two Forensic Cases

Bones and teeth are highly challenging sources of DNA in forensic science and human remains identification, requiring multiple laborious processing steps. In this study, we compared an organic phenol–chloroform method to the QIAamp® DNA Investigator and PrepFiler Express BTA™ methods in order to identify the most efficient automated DNA extraction method for bones and teeth. Results from individual tooth powder replicates showed that the PrepFiler Express BTA™ method extracted the highest yields of DNA per mg of tooth powder, returning a minimum of 20/21 PowerPlex® 21 loci. Samples extracted using the organic extraction or QIAamp® DNA Investigator methods produced PowerPlex® 21 profiles displaying a ski-slope morphology. The improved DNA quality and yield from the PrepFiler Express BTA™ method was verified using aged samples, where higher DNA yields per mg of powder and more informative profiles were obtained. Furthermore, the PrepFiler Express BTA™ method subsequently provided useful DNA profiles for two forensic cases involving degraded bone samples. Overall, this study showed that the PrepFiler Express BTA™ chemistry is a reliable and robust method for DNA extraction from bone and teeth samples, and will allow larger numbers of samples to be efficiently extracted in the event of a Disaster Victim Identification event.

Probabilistic Genotyping of Single Cell Replicates from Complex DNA Mixtures Recovers Higher Contributor LRs than Standard Analysis

DNA mixtures are a common source of crime scene evidence and are often one of the more difficult sources of biological evidence to interpret. With the implementation of probabilistic genotyping (PG), mixture analysis has been revolutionized allowing previously unresolvable mixed profiles to be analyzed and probative genotype information from contributors to be recovered. However, due to allele overlap, artifacts, or low-level minor contributors, genotype information loss inevitably occurs. In order to reduce the potential loss of significant DNA information from donors in complex mixtures, an alternative approach is to physically separate individual cells from mixtures prior to performing DNA typing thus obtaining single source profiles from contributors. In the present work, a simplified micro-manipulation technique combined with enhanced single-cell DNA typing was used to collect one or few cells, referred to as direct single-cell subsampling (DSCS). Using this approach, single and 2-cell subsamples were collected from 2-6 person mixtures. Single-cell subsamples resulted in single source DNA profiles while the 2-cell subsamples returned either single source DNA profiles or new mini-mixtures that are less complex than the original mixture due to the presence of fewer contributors. PG (STRmixTM) was implemented, after appropriate validation, to analyze the original bulk mixtures, single source cell subsamples, and the 2-cell mini mixture subsamples from the original 2-6-person mixtures. PG further allowed replicate analysis to be employed which, in many instances, resulted in a significant gain of genotype information such that the returned donor likelihood ratios (LRs) were comparable to that seen in their single source reference profiles (i.e., the reciprocal of their random match probabilities). In every mixture, the DSCS approach gave improved results for each donor compared to standard bulk mixture analysis. With the 5- and 6- person complex mixtures, DSCS recovered highly probative LRs (> 1020) from donors that had returned non-probative LRs (<103) by standard methods.

Application of SCoT markers for accessing of genetic diversity of gramineous forage grass species

Using the SCoT marker system, 8 varieties of cereal grasses belonging to 5 species were analyzed: Festuca pratensis, Lolium perenne, Lolium multiflorum, Festuca rubra, Festulolium. Of the 10 tested SCoT markers, 7 informative markers were selected that reveal interspecies genetic polymorphism. According to the results of the analysis, DNA profiles characteristic of each studied species were obtained, and primers allowing to detect intervarietal differences for subsequent identification and molecular genetic passportization were selected.

Evaluation of a custom QIAseq targeted DNA panel with 164 ancestry informative markers sequenced with the Illumina MiSeq

Introduction of new methods requires meticulous evaluation before they can be applied to forensic genetic case work. Here, a custom QIAseq Targeted DNA panel with 164 ancestry informative markers was assessed using the MiSeq sequencing platform. Concordance, sensitivity, and the capability for analysis of mixtures were tested. The assay gave reproducible and nearly concordant results with an input of 10 and 2 ng DNA. Lower DNA input led to an increase in both locus and allele drop-outs, and a higher variation in heterozygote balance. Locus or allele drop-outs in the samples with less than 2 ng DNA input were not necessarily associated with the overall performance of a locus. Thus, the QIAseq assay will be difficult to implement in a forensic genetic setting where the sample material is often scarce and of poor quality. With equal or near equal mixture ratios, the mixture DNA profiles were easily identified by an increased number of imbalanced heterozygotes. For more skewed mixture ratios, the mixture DNA profiles were identified by an increased noise level. Lastly, individuals from Great Britain and the Middle East were investigated. The Middle Eastern individuals showed a greater affinity with South European populations compared to North European populations.

Source Level Attribution: DNA Profiling from the ABAcard® HemaTrace® Kit

ABAcard® HemaTrace® kits have been used for crime scene stains for confirmation of human blood for many years. However, when the stain is too small to allow for separate testing, confirmatory testing may be forgone to preference DNA analysis. This can lead to court challenges as to the biological source and therefore probative value of the DNA profile. This research aimed to develop a protocol for DNA analysis of a minute blood stain subsequent to HemaTrace® testing. Stains were collected and subjected to HemaTrace® testing. Swabs were then removed from the HemaTrace® buffer solution and processed. DNA yields and STR DNA profiles were analysed for both quantity and quality. Full profiles were reliably obtained from stains with diameters of 0.6 mm–0.7 mm, reflecting DNA concentrations between 0.0036 ng/μL and 0.007 ng/μL, varying according to substrate characteristics. However, stains below a diameter of 0.6 mm should proceed directly for DNA profiling. This protocol was also successfully performed on blood stains which had undergone UV irradiation, although use of the reporting peak height threshold (lower than the routine analytical threshold) was required to obtain useable profiles. We have been able to demonstrate a protocol which, with minor adjustments to crime scene procedures, allows for both the confirmation of the presence of human blood, together with the generation of useful DNA profiles.

INTERNATIONAL EXPERIENCE IN ANTI-THEFT OF CATALYTIC CONVERTERS INSTALLED IN CARS.

Recently, there has been increase in the number of property crimes in the world, including theft of external car parts. The number of thefts of catalytic converters installed in cars of various brands has sharply increased due to a significant increase in the value of precious metals such as platinum, palladium and rhodium. The reason for a sharp increase in the number of crimes of this particular car part is small amounts of the mentioned precious metals in the catalytic convertors. The purpose of this article is to consider this problem faced by law enforcement and legislative bodies in many countries and to propose certain ways for its solution. The article provides an overview of the situation with thefts of catalytic converters in the countries of the European Union, the United States and Israel. Detailed reasons for the occurrence of this problem are provided, as well as measures to protect a car from the theft are suggested. Possible legislative and investigative-forensic actions to prevent this type of crime are considered: - law enforcement agencies investigating this type of crime should clearly understand that we are not talking about isolated, unrelated cases, but about well-planned actions of criminal groups. - increase in control over purchase and sale of metal carried out without accounting and corresponding documentation and amendments to the administrative procedural code also require strengthening. - applying a special forensic marking on parts, including on a catalytic converter, will allow to track its location and provide full information to law enforcement agencies from which vehicle it was stolen in the event of its theft. - inspection and examination by a forensic expert of vehicles and other physical evidence left by criminals at crime scenes for criminals’ fingerprints and DNA profiles will help to significantly increase the detection rate of this type of crime.

COVID-19 in Patients with Active Tuberculosis

Data on the coincidence of tuberculosis (TB) and COVID-19 are limited, and previous observations are based on the results of just a few studies, which has led to polarized views on the course of infection with SARS-CoV-2 in patients with active TB. We present the first two cases of TB and COVID-19 coinfection in the population of patients in Poland, diagnosed shortly after the outbreak of the global pandemic. In the first patient, TB was very advanced at the time of infection with SARS-CoV-2. From the third day of hospitalisation, respiratory failure was increasing, with no improvement after the use of high-flow oxygen therapy and mechanical ventilation. On the seventh day of hospitalization, the patient died. In the second presented case, therapeutic success was achieved despite the coincidence of COVID-19, infection with HIV, and extrapulmonary and pulmonary TB. The patient had symptoms of renal failure and the SARS-CoV-2 infection was mild and asymptomatic. Because both patients were in the care of a homeless shelter, a molecular epidemiological investigation was carried out. Different DNA profiles of Mycobacterium tuberculosis complex isolates detected in clinical materials from patients ruled out the transmission of tuberculosis. Based on our analysis, it is impossible to clearly define the influence of active TB on the course of SARS-CoV-2 infection. We can only suggest that coinfection is particularly dangerous for socially disadvantaged people, the elderly, and people with other comorbidities. In the coming years, a negative impact of the current pandemic on control programmes will be observed for many infectious diseases, including TB.