A case of multiple assignments (paternity/maternity) in an equine-out breeding system

Recently, the use of DNA markers has provided a more accurate method of identifying individuals and verifying parentage. In this report, we describe foal assignment in a farm bred jumping horses (Silla argentino). Ten mares were freely served by two stallions, resulting in nine foals. Weaning occurred without registration of the mare of each offspring, resulting in a failure to identify either the mare or the sire of each foal. Animals were typed using 12 microsatellite systems and four biochemical polymorphisms in order to determine the paternity/maternity of each foal. We used the CERVUS program to evaluate the parentage of each offspring. It was possible to determine maternity in eight cases, and paternity in all of them. We concluded that this set of codominant markers analyzed following a likelihood-based approach included in the CERVUS package, are useful tools to solve parentage assignments in domestic horses.     

Performance evaluation and optimization of multiplex PCRs for the highly discriminating OSU 10-locus set Y-STRs

In a previous study, a new set of Y-chromosome short tandem repeats, the OSU 10-locus set (MPM1 and MPM2), was shown to have a higher discrimination power when evaluated against the 10 SWGDAM loci on a common population panel. Here, we describe the optimization of the multiplex reactions using dye-labeled primers followed by performance evaluations. The loci exhibited high precision, human male specificity, reliability in different body fluids, high sensitivity, stability, and the ability to amplify nonprobative casework and mixture samples. Stutter for the all of the loci, with the exception of the highly polymorphic locus DYS688, was similar to that observed for autosomal loci. The results of the performance evaluations reinforced the utility of these loci.     

Phylogenetic evidence for multiple independent duplication events at the DYS19 locus

Duplication events at Y chromosome STR loci have been repeatedly described in human populations. DYS19 is probably the best known example and it exhibits duplicate state in individuals from all continents. Despite the large amount of available data, evolutionary relationship between DYS19 duplication-bearing chromosomes has not been so far investigated. We address the genealogical correlation among such chromosomes by analysing newly identified DYS19 duplicated Y chromosomes by SNP genotyping and microsatellite-based network analysis. SNP and network analysis show that DYS19 duplicated Y chromosomes associate with different Y chromosome lineages. These results indicate that DYS19 duplication occurred more than once during human evolution.     

Canine Population Data Generated from a Multiplex STR Kit for Use in Forensic Casework*

Abstract: Canine biological specimens are often part of the physical evidence from crime scenes. Until now, there have been no validated canine‐specific forensic reagent kits available. A multiplex genotyping system, comprising 18 short tandem repeats (STRs) and a sex‐linked zinc finger locus for gender determination, was developed for generating population genetic data assessing the weight of canine forensic DNA profiles. Allele frequencies were estimated for 236 pedigreed and 431 mixed breed dogs residing in the U.S. Average random match probability is 1 in 2 × 10³³ using the regional database and 1 in 4 × 10³⁹ using the breed dataset. Each pedigreed population was genetically distinct and could be differentiated from the mixed breed dog population but genetic variation was not significantly correlated with geographic transition. Results herein support the use of the allele frequency data with the canine STR multiplex for conveying the significance of identity testing for forensic casework, parentage testing, and breed assignments.     

Development of a Nomenclature System for a Canine STR Multiplex Reagent Kit*†

Abstract: Despite the popularity of dogs in US households, canine DNA evidence remains largely untapped in forensic investigations partially because of the absence of well‐defined forensic short tandem repeats (STRs), lack of standardized and validated PCR protocols, STR reagent kits, and poorly developed nomenclature. A nomenclature system was established based on internationally recognized recommendations for human forensic STRs for a recently developed canine STR reagent kit. Representative alleles were sequenced from each of the 18 STRs and the sex‐typing marker included in the kit. This study also reflects on the impact of point mutations, insertions, and deletions within and outside the STR core repeat structures. An understanding of the STRs’ sequence and repeat structures will enable development of a robust and reliable allele nomenclature and improve the accuracy and precision of allele fragment sizing in canine forensic profiling. The expected allele sizes have been calculated, and their repeat stuctures defined based on sequence information.     

Substitution of human for horse urine disproves an accusation of doping*

In order to detect switching and/or manipulation of samples, the owner of a stallion asked our lab to perform a DNA test on a positive doping urine sample. The objective was to compare the urine DNA profile versus blood and hair DNA profiles from the same stallion. At first, 10 microsatellite markers were investigated to determine the horse identity. No results were obtained when horse specific markers were typed in the urine sample. In order to confirm the species origin of this sample we analyzed the mitochondrial cytochrome b gene. This analysis from blood and hair samples produced reproducible and clear PCR-RFLP patterns and DNA sequence match with those expected for horse, while the urine sample results were coincident with human. These results allowed us to exclude the urine sample from the questioned stallion and determine its human species origin, confirming the manipulation of urine sample.     

Exploiting expressed sequence tag databases for the development and characterization of gene-derived simple sequence repeat markers in the opium poppy (Papaver somniferum L.) for forensic applications

Simple sequence repeat (SSR) markers in the opium poppy (Papaver somniferum L.) were identified from an expressed sequence tag (EST) database comprised of 20,340 sequences. In total, 2780 SSR-containing sequences were identified. The most frequent microsatellite had an AT/TA motif (37%). Twenty-two opium poppy EST-SSR markers were presently developed and polymorphisms of six markers (psom 2, 4, 12, 13, 17, and 22) were utilized in 135 individuals under narcotic control investigation. An average of three alleles per locus (range: 2-5 alleles) with a mean heterozygosity of 0.167 was detected. Six loci identified 29 unique profiles in 135 individuals. The EST-SSR markers exhibited small degrees of genetic differentiation (fixation index = 0.727, p < 0.001). Other variable markers will be needed to facilitate the forensic identification of the opium poppy for future cases. To determine the potential for cross-species amplification, six markers were tested in five Papaver genera species and two Eschscholzia genera. The psom 4 and psom 17 primer pair was transferable. This is the first study to report SSR markers of the opium poppy.