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ABSTRACT The human X-chromosome non-coding markers, such as short tandem repeats (STRs), single nucleotide polymorphisms (SNPs), insertion-deletions (INDELs) and Alu insertions, are useful for revealing relationships among populations and for the identification of individuals. In the last decades, a number of studies have been performed to determine the genetic structure of Latin American populations by using X-chromosome markers. These studies provided useful information regarding the genetic composition of these populations and their relationship with Native American, Asian and European populations. One of the most interesting findings achieved by X-chromosome studies is the bias in the sex ratio of individuals that gave rise to the current Latin American populations, as it was previously observed through the analysis of uniparental markers, and which is undoubtedly evidenced in the differential inheritance of X-chromosome in comparison to autosomes. Besides, the genetic drift process that affected Native American populations is more pronounced in X-chromosome markers than in autosomes. The present review summarizes our current knowledge concerning X-chromosome non-coding polymorphisms studied in Latin American populations.

RESUMEN Los marcadores no codificantes del cromosoma X humano, como las repeticiones cortas en tándem (STR), los polimorfismos de un solo nucleótido (SNP), las inserciones-deleciones (INDEL) y las inserciones Alu, son útiles para revelar la relación existente entre poblaciones, y también para la identificación de personas. En las últimas décadas, se han realizado una serie de estudios para determinar la estructura genética de las poblaciones latinoamericanas, utilizando marcadores de cromosoma X. Estos estudios proporcionaron información útil sobre la composición genética de estas poblaciones y su relación con las poblaciones nativas americanas, asiáticas y europeas. Uno de los hallazgos más interesantes logrados en estos estudios es el sesgo en la proporción de sexos de los individuos que originaron las poblaciones latinoamericanas actuales, tal como se observó previamente a través del análisis de marcadores uniparentales, y que queda evidenciado por la herencia diferencial del cromosoma X en comparación con los autosomas. Además, el proceso de deriva genética que afectó a las poblaciones nativas americanas actuó de manera más pronunciada en los marcadores del cromosoma X que en los autosomas. La presente revisión resume nuestro conocimiento actual sobre los polimorfismos no codificantes del cromosoma X estudiados en poblaciones latinoamericanas.

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This work presents the results of a DNA test aimed to determine a possible biological link of paternal half brotherhood of two males. The combined use of biparentally inherited markers (autosomal STRs) and a panel of 27 Y-STRs allowed us to determine the existence of a biological relationship of kinship, even after detecting three mutations at their Y-STR haplotypes along the analyses, constituting an infrequent multiple mutation situation. This case is an example illustrating the importance of having different analytical markers sets and strategies for clarifying complex kinship cases where mutations occur.

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BACKGROUND: Argentinean population is the result of admixture between South Amerindians, Europeans and to a lesser degree, Africans. Since the advent of forensic molecular genetics, the construction of local reference databases became mandatory. Aiming to further extend the technical quality reference database of Argentina, we present herein the allele frequencies for 24 autosomal STRs, including D22S1045, and SE33 (not previously reported for Argentina in STRidER). CONCLUSIONS: Genotypes of 6454 unrelated individuals (3761 males and 2694 females) from 13 out of 23 provinces were analysed. Forensic parameters were calculated for each marker. The observed heterozygosity ranged from 0.661 (TPOX) to 0.941 (SE33). The locus SE33 was revealed to be the most informative marker showing the highest values for PIC (0.955), GD (0.952), TPI (8.455) and PE (0.879). On the other hand, TPOX turned out to be the least informative marker: PIC (0.618), GD (0.669), and PE (0.371). The high number of analyzed individuals allowed detecting low frequency alleles and microvariants in CSF1PO; D16S539 and D21S11 D18S51; PENTA D; PENTA E and at locus D6S1043. METHODS AND RESULTS: This study is the most extensive for Argentina and complements the already reported information concerning the autosomal STRs commonly used in forensic identification. The results were submitted passing STRidER quality control standards (QC), receiving the reference number STR000327 v.2.

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When DNA profile comparisons between a crime scene trace and a reference sample generate correspondence, the match probability has to be estimated, so that evaluation of the strength of the forensic DNA evidence can be made. The random match probability estimations require information on allele frequencies and an adjustment factor, referred to as theta (θ) or Fst, a co-ancestry correction factor for subpopulation effects. The θ value has been standardized for urban and isolated populations, but inconsistencies have been reported when it is specifically calculated for smaller and isolated populations, including Amerindian populations. Notably, attempts to characterize forensic markers of these minor populations have been extensively limited and more conservative estimates of the correction factor may be generated for each of them. Therefore, we estimate allele frequencies of 21 autosomal STR markers used for forensic testing and calculated relevant forensic parameters for the set. In addition, we featured the possible structure of five Brazilian Amerindian populations that have been genetically isolated for centuries so we could obtain the appropriate θ value for them. The sample consisted of 319 individuals: (1) 121 Kaingang, from two communities: Ivaí (KIV=61) and Rio das Cobras (KRC=60); and (2) 198 Guaranis from three communities: Mbya from Rio das Cobras (GRC=51), Guarani Ñandeva (GND=71) and Guarani Kaiowá (GKW=76). Between Guarani populations low (Rst=0.0402, p < 10-4) to high (Rst=0.1557, p < 10-5) differentiation was found. Regarding Guarani and Kaingang populations, intermediate (Rst=0.0590, p < 10-5) to high (Rst=0.1604, p < 10-5) differentiation was found. The two Kaingang populations showed very low differentiation between them (Rst=0.0017, p = 0.27), which justifies the union of both genetic data for forensic databases and calculations. The combined power of discrimination (PD) and the combined power of exclusion (PE) were calculated for each population, demonstrating the usefulness of this set of markers in forensic and kinship analysis regarding these populations. Considering the demographic heterogeneity of Amerindian populations in general, the Fst mean value (0.03) was evaluated regarding 43 different indigenous populations from the Americas, including Guaranis and Kaingangs. This result confirms the adequacy of the standardized θ value for the forensic random match probability estimations involving Amerindian populations.

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With the advent of expanded STR (short tandem repeats) typing kits, it was necessary to determine allele frequencies and other appropriate population data parameters for El Salvador. Samples were collected from the central, east, and west regions of the country and typed for 21 forensically relevant STR loci. The data indicate that all loci are highly polymorphic, the three regions are genetically similar, and the population data are similar to those of US Hispanics. The results of this study support that the allele frequency data described herein can be used for statistical calculations for human identity testing in El Salvador.

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The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected millions of people worldwide. SARS-CoV-2 belongs to the Betacoronavirus genus, containing the mouse hepatitis virus (MHV), an extensively studied animal coronavirus. Since MHV and SARS-CoV-2 share the same genus, MHV could offer insights relative to SARS-CoV-2 studies. MHV-3 strain causes hepatitis and cellular injury, making MHV-3 infection one of the best models for this debilitating disease. Surrogate coronaviruses have been used for virus resistance and inactivation studies, and although real-life conditions using SARS-CoV-2 should be encouraged, their use needs to be balanced with safety and costs. MHV can be manipulated under BSL2 laboratory conditions, unlike SARS-CoV-2, making it a model for studying the virucidal effects on coronaviruses. In this study, we used the betacoronavirus MHV-3 as a model to investigate the virucidal activity of an air disinfection equipment named STR Solution®, an air sterilizer with patented technology. MHV-3 was dried on different surfaces and exposed at varying distances from the STR Solution® equipment and at different exposure times. The residual infectivity was evaluated using the endpoint method. There was not a significant reduction (mean p-value = 0.4) of the viral titer under STR Solution® exposition. STR Solution® caused a slight decrease of the infectious particles' titer (> 1 log10) only under the following conditions: polypropylene at 3 m, for 1 and 3 h (1.2 log10 reduction TCID50) and Sus domesticus skin at 0.05 m, for 1 h (1.3 log10 reduction TCID50), and at 3 m for 1 h (1.2 log10 reduction TCID50). These and other studies confirm the usefulness of this model to evaluate virucidal activity.

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Forensic DNA typing typically relies on the length-based (LB) separation of PCR products containing short tandem repeat loci (STRs). Massively parallel sequencing (MPS) elucidates an additional level of STR motif and flanking region variation. Also, MPS enables simultaneous analysis of different marker-types - autosomal STRs, SNPs for lineage and identification purposes, reducing both the amount of sample used and the turn-around-time of analysis. Therefore, MPS methodologies are being considered as an additional tool in forensic genetic casework. The PowerSeq™ Auto/Y System (Promega Corp), a multiplex forensic kit for MPS, enables analysis of the 22 autosomal STR markers (plus Amelogenin) from the PowerPlex® Fusion 6C kit and 23 Y-STR markers from the PowerPlex® Y23 kit. Population data were generated from 140 individuals from an admixed sample from Rio de Janeiro, Brazil. All samples were processed according to the manufacturers' recommended protocols. Raw data (FastQ) were generated for each indexed sample and analyzed using STRait Razor v2s and PowerSeqv2.config file. The subsequent population data showed the largest increase in expected heterozygosity (23%), from LB to sequence-based (SB) analyses at the D5S818 locus. Unreported allele was found at the D21S11 locus. The random match probability across all loci decreased from 5.9 × 10-28 to 7.6 × 10-33. Sensitivity studies using 1, 0.25, 0.062 and 0.016 ng of DNA input were analyzed in triplicate. Full Y-STR profiles were detected in all samples, and no autosomal allele drop-out was observed with 62 pg of input DNA. For mixture studies, 1 ng of genomic DNA from a male and female sample at 1:1, 1:4, 1:9, 1:19 and 1:49 proportions were analyzed in triplicate. Clearly resolvable alleles (i.e., no stacking or shared alleles) were obtained at a 1:19 male to female contributor ratio. The minus one stutter (-1) increased with the longest uninterrupted stretch (LUS) allele size reads and according to simple or compound/complex repeats. The haplotype-specific stutter rates add more information for mixed samples interpretation. These data support the use of the PowerSeqTM Auto/Y systems prototype kit (22 autosomal STR loci, 23 Y-STR loci and Amelogenin) for forensic genetics applications.

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The franciscana (Pontoporia blainvillei) is the most threatened small cetacean in the South Atlantic. In this study we report the development of 13 microsatellite markers for franciscanas through next-generation sequencing, and the characterization of those loci in 38 samples from the species' northernmost population (Espírito Santo, Brazil). Besides providing diversity indices for the new, specific loci, we also report on the transferability of heterologous loci which had not been screened in franciscanas before, and review all loci used in previous studies. Expected heterozygosity in the new loci ranged between 0.107 and 0.595, and all but one were in Hardy Weinberg Equilibrium. These are the first microsatellite loci isolated from franciscanas, and they are an important addition to heterologous markers that were available previously.

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BACKGROUND: Short Tandem repeats (STRs) existed as popular elements in both eukaryotic and prokaryotic genomes. RESULTS: In this study, we analyzed the characteristics, distributions, and motif features of STRs within whole-genomes of 140 plant species. The results showed that STR density was negatively correlated with the genome size. Hexanucleotide repeat was the most abundant type of STRs. The distribution of algae shows a preference different from that of other plants. By analyzing GC contents of STRs and genome, it was concluded that STR motif was influenced by GC contents. Analysis of the long STRs in genome (length 1000 bp) found that dicots have the more long STRs. For STR types, di- and tri-nucleotide accounted for the highest proportion. Analyzing and designing long STRs in CDS (length 500 bp) was to verify the role of long STRs in Gossypium hirsutum TM-1 and Solanum tuberosum. By comparing the long STRs found in Fragaria x ananassa with other species, some evolutionary characteristics of the long STRs were obtained. CONCLUSIONS: We got the characteristics, distribution, and motif features of STRs in the whole genome of 140 plants and obtained some evolutionary characteristics of long STRs. The study provides useful insights into STR preference, characteristics, and distribution in plants.

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In order to determine the population allele frequencies of autosomal STR markers of forensic interest in the Zimbabwean population, we analyzed a sample of 478 individuals from 19 different ethnic groups using the PowerPlex® Fusion 6C Kit (Promega Corp, Madison, Wisconsin). The data obtained were compared among the different Zimbabwean ethnic groups as well as with several African populations to establish whether significant differences exist among them. No significant differences were found among the ethnic groups in Zimbabwe. Statistically significant differences were observed between allele frequencies in Zimbabwe and some other African populations, although FST with neighboring Bantu populations from South and Southeast regions were low (below 0.005 in most single locus comparisons).

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To establish the diversity, structure, and phylogenetic relationships among Colombian Creole cattle, six native breeds and one introduced breed were genotyped for 20 microsatellite loci. The average number of alleles per breed ranged from 7050 (Romosinuano) to 10,100 (Casanareño), and the expected heterozygosity ranged from 0.691 (San martinero) to 0.785 (Casanareño). The deviation from the Hardy-Weinberg equilibrium (HWE) was statistically significant (p < 0.05) in 59 out of 120 tests carried out in the six breeds for the 20 microsatellite loci analyzed. Colombian Creole bovine breeds have maintained a high level of genetic differentiation within the same populations (93%), and the rest is explained by differences between breeds (7%). The differentiation pattern and the genetic relationships between the Colombian Creole bovine breeds showed high consistency with the evolutionary history of each. Both the Bayesian grouping analysis and the neighbor-joining tree exhibited a reliable grouping pattern, which revealed two main groups: one comprised by the breeds Blanco Orejinegro, Hartón del Valle, Costeño Con Cuernos, Romosinuano, and San Martinero, and the other one by the Creole breed Casanareño and Zebu. These were probably caused by different historical, reproductive, and geographic isolation precedents, as well as by different levels of inbreeding. This study will help understand the genetic characteristics of Colombian Creole cattle and will benefit future conservation programs.

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Population data of the Aymara in the province of Puno were established for 23 autosomal STR markers. DNA was obtained from unrelated individuals (n = 190) who reside in three areas of the Floating Islands of Lake Titicaca, residents on the border with Bolivia and residents who are not from the border with Bolivia. The PENTA E marker presented the highest PD (0.9738), PIC (0.8793), and PM (0.7847) values. The combined PD was greater than 0.99999999 and the combined PE was 0.99999994. The largest distance, based on Fst values, was between the Aymara population and the Ashaninca population (0.04022), and the smallest distance was with the populations of Bolivia (0.00136) and Peru (0.00525).

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BACKGROUND: When the mother's DNA profile is not available for paternity testing, there is a smaller probability that a locus will exclude an alleged father. This study aims to evaluate the risk of potential false paternity inclusions in motherless cases. STUDY DESIGN AND METHODS: More than 20 000 duos were generated by removing the maternal genotypes from exclusion trios. After recalculating paternity in these duos, any found inclusions would be false. RESULTS: The use of an appropriate number of loci, mutation model, and mutation rates to analyze motherless paternity cases was robust against false inclusions. A single potential false inclusion was observed in a case wherein kinship plays a role. This result highlights the importance of testing the mother when available and of obtaining information on family circumstances for the proper handling of cases involving related individuals. CONCLUSION: The guidelines we used here were sufficient to avoid false inclusions in a data set of more than 20 000 motherless cases.

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In this study, allele frequencies were determined in a Peruvian population for application to human identification. A population of 601 unrelated individuals was analyzed (400 individuals with the GlobalFiler Express kit and 201 individuals with the VeriFiler Express kit). The locus with the highest power of discrimination (PD) was SE33 (0.9851, 31 alleles), while the least polymorphic locus was D22S1045 (0.75810, 11 alleles). The PE in a similar fashion ranged from 0.2421 (D22S1045) to 0.7818 (SE33). Under the assumption of independence, the combined PD was > 0.9999999999 while the combined PE = 0.9999999933. When comparing the population studied with different populations of Latin America, the greatest Fst genetic distance was obtained with a Venezuelan population (0.052), and the shortest distance was with a Bolivian and Peruvian population (0.004).

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Brucella canis, a Gram-negative coccobacilli belonging to the genus Brucellae, is a pathogenic bacterium that can produce infections in dogs and humans. Multiple studies have been carried out to develop diagnostic techniques to detect all zoonotic Brucellae. Diagnosis of Brucella canis infection is challenging due to the lack of highly specific and sensitive diagnostic assays. This work was divided in two phases: in the first one, were identified antigenic proteins in B. canis that could potentially be used for serological diagnosis of brucellosis. Human sera positive for canine brucellosis infection was used to recognize immunoreactive proteins that were then identified by performing 2D-GEL and immunoblot assays. These spots were analyzed using MALDI TOF MS and predicted proteins were identified. Of the 35 protein spots analyzed, 14 proteins were identified and subsequently characterized using bioinformatics, two of this were selected for the next phase. In the second phase, we developed and validated an indirect enzyme-linked immunosorbent assays using those recombinant proteins: inosine 5' phosphate dehydrogenase, pyruvate dehydrogenase E1 subunit beta (PdhB) and elongation factor Tu (Tuf). These genes were PCR-amplified from genomic DNA of B. canis strain Oliveri, cloned, and expressed in Escherichia coli. Recombinant proteins were purified by metal affinity chromatography, and used as antigens in indirect ELISA. Serum samples from healthy and B. canis-infected humans and dogs were used to evaluate the performance of indirect ELISAs. Our results suggest that PdhB and Tuf proteins could be used as antigens for serologic detection of B. canis infection in humans, but not in dogs. The use of recombinant antigens in iELISA assays to detect B. canis-specific antibodies in human serum could be a valuable tool to improve diagnosis of human brucellosis caused by B. canis.

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Chimerism testing by short tandem repeats (STRs) is used to monitor engraftment after allogeneic hematopoietic stem cell transplantation (HSCT). Generally, STR alleles are stable and transferred from parent to child or from donor to recipient. However, 3 cases did not follow this norm. Additional work-up with help from forensic literature solved these mysteries. In case 1, the patient received HSCT from his son. The son shared STR alleles in 22/23 loci except Penta E, which was explained by repeat expansion in the son. In case 2, the patient had been in remission for 14 years after HSCT for lymphoma and developed repeat expansion in CSF1PO in granulocytes. In case 3, a pre-HSCT patient demonstrated 3 alleles, with 2 peaks taller than the third, in the FGA locus (chromosome 4). A combination of a triallelic variant and leukemia-associated trisomy 4 explained the finding. STR number variants are rare and clinically inconsequential but can overlap malignancy-associated, clinically significant changes.

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Latent fingerprints are commonly found in crime scenes, and currently used in forensic analysis to obtain STR profiles from DNA recovered from finger contact. Analysis of STR profiles obtained from touch DNA has been very useful to elucidate crimes and the extraction method may be determinant for the recovery of genetic material collected from different surfaces. This study aimed to verify and compare the efficiency of two different extraction kits for processing touch DNA samples obtained from fingerprints deposited on computer keyboards, knife handles and exterior door handles and steering wheels of cars. One hundred and four experiments were conducted to simulate crime scenes and evaluate the efficiency of two extraction kits for touch DNA samples: the DNA IQ™ System and the Casework Direct Kit (both Promega Corporation). Each experiment was conducted with two individuals in order to obtain a mixture profile. The genetic material deposited was collected by double swab method (Sweet et al. 1997) and DNA quantification was conducted using Quantifiler Trio™ (ThermoFisher Scientific). Samples were amplified by PowerPlex® Fusion System kit (Promega). It was possible to obtain STR profiles for 32 (61.5%) out of the 52 extracted using DNA IQ and 51 (98.1%) out of the 52 extracted using the Casework Direct Kit. Samples extracted by DNA IQ had higher average of quantification values for long targets (>200bp) across all tested surfaces. That seems to be due to an incompatibility between the Quantifiler Trio and the Casework Direct Kit. Samples with positive quantification but without STR profile, as well as samples without quantification but with STR profiles were also observed. Statistical analysis showed that the Casework Direct Kit produced significantly more useful profiles than DNA IQ (p-value = 0.001), since these profiles had more STR markers with allelic correspondence to second donators present in the mixture. This study provides insights about the effect of different surfaces and extraction methods on recovery and generation of STR profiles. Limitations for the quantification step for these samples with a low quantity of DNA were highlighted as well. We concluded that the Casework Direct Kit was much more efficient for processing touch DNA samples than DNA IQ.

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We report one complex paternity case presenting a presumable paternal four-step STR mutation between the alleged father (AF) and child; the complexity of the case required the AF-brother hypothesis to be discarded without including this DNA sample. A total of 23 autosomal STR loci included in the Powerplex Fusion® and Globalfiler™ kits confirmed one isolated mismatch for D22S1045 between the AF (17/17) and the male child (13/15) in the presence of the mother (15/15). In this case, the STR structure and father's age do not seem to have contributed to promote the observed multistep mutation. The Paternity Index (PI) based on 23 autosomal STRs did not favor the AF paternity over the AF-brother hypothesis based on a flat prior (PI = 0.1217; W = 10.85%). For that reason, we included 38 autosomal human identification (HID) insertions-deletions (indels) and 20 retrotransposon insertion polymorphisms (RIPs) contained in the InnoTyper® 21 kit. Although these biallelic markers favored the AF paternity rather than the AF-brother hypothesis (LR = 110.3; W = 99.1%), the global PI based on 81 autosomal markers supported moderately the AF paternity hypothesis (LR = 13.4; W = 93.1%). The application of different mutation models showed a consistent support to the AF paternity hypothesis (PI = 93.1-99.95%), which could be useful for interpretation in these multistep STR mutation cases. In brief, we showed the impact of a four-step mutation at D22S1045 to obtain definitive paternity conclusions, particularly under a complex scenario when the AF-brother hypothesis is assessed. Forensic genomics arises as the next option for similar complex paternity cases.

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The Thraupidae family is one of the most wanted by bird breeders in Brazil because it is represented by its diverse, colorful and melodious singers. The Great-billed Seed-finch, Sporophila maximiliani, is the only representative of the genus Sporophila considered critically endangered in Brazil. Due to the demands of environmental agencies and of conservation programs, there is a need to increase the number of molecular markers available for the genus and specially for S. maximiliani. Therefore, this work aimed to provide a new set of microsatellite markers for S. maximiliani in order to help bird breeders and environmental agencies on fulfilling its demands as well as contributing with extra genetics tools for conservation programs of the S. maximiliani. Of the 30 markers developed, 25 successfully amplified, and 22 were polymorphic. Annealing temperature varied from 52 to 64 °C, number of alleles from 2 to 13, and the medium allele richness was 7.25 and medium expected, observed heterozygosity and PIC were, respectively, 0.812, 0.661 and 0.752. The probability of identity estimate was 8.54 × 10-27 and all the other probabilities of non-exclusion (sib-identity, parent pair and first-parent) were < 0.001, indicating that this set of microsatellite markers have high genetic variability and high power of individual genetic differentiation for S. maximiliani. Therefore, this work increases the options of molecular markers to be used on inspection for environmental agencies and for conservation programs on analyzing genetic variability and population studies for S. maximiliani.

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During World War II, many nations took part in the war. Among the supporters of the Alliance there was also Brazil. In August 1944, under the leadership of President Getúlio Vargas, Brazil declared war on Nazi Germany and took part in the Italian campaign by sending many troops to support the Allies in the Central Italy. Once the conflict was over, the deceased Brazilian soldiers were buried in Pistoia, a few kilometers from Florence. But only in 1960 the Brazilian government authorized the transfer of the dead soldiers to their homeland. Five years later, during the building of the Brazilian Military Votive Monument, still in the Pistoia cemetery, a last body was found but could not be identified: so he was buried as an "unknown soldier". In December 2012, the Brazilian Embassy in Italy asked for performing forensic genetics analysis for identification purposes on the remains of this last unknown Brazilian soldier. After almost 70 years a complete short tandem repeats (STR) profile was obtained, useful for any relatives searching. Key points: Identification of the last Brazilian Unknown Soldier buried in Italy.DNA analysis on 70 years old skeletal remains.Brazilian soldier's history during World War II.