Population studies of 16 bovine STR loci for forensic purposes.

As a consequence of the close integration of cattle into the food chain of humans, forensically relevant cases involving cattle (Bos taurus) DNA analysis are common. However, scientific publications reporting the information content of the commonly used bovine short tandem repeat (STR) loci remains scarce. Population studies were performed for 16 polymorphic STR loci (BM1818, BM1824, BM2113, CSRM60, CSSM66, ETH3, ETH10, ETH225, HAUT27, ILSTS006, INRA023, SPS115, TGLA53, TGLA122, TGLA126, and TGLA227) including 4,162 randomly selected cattle representing 20 distinct breeds. The power of parental exclusion, expected and observed heterozygosity, probability of identity, and non-amplifying ("null") allele frequencies were calculated. Major differences existed in the information content between different cattle breeds. The selection of 16 STR loci, partially recommended by International Society for Animal Genetics as the minimum standard needed for bovine STR typing, was sufficient for forensic analysis. Furthermore, the efficacy of the loci was assessed in assigning unknown individuals to the correct breed based on genotype data. The individual assignment tests provided excellent success in several breeds.

Population studies of 17 equine STR for forensic and phylogenetic analysis.

As a consequence of the close integration of horses into human society, equine DNA analysis has become relevant for forensic purposes. However, the information content of the equine Short Tandem Repeat (STR) loci commonly used for the identification or paternity testing has so far not been fully characterized. Population studies were performed for 17 polymorphic STR loci (AHT4, AHT5, ASB2, ASB17, ASB23, CA425, HMS1, HMS2, HMS3, HMS6, HMS7, HTG4, HTG6, HTG7, HTG10, LEX3 and VHL20) including 8641 horses representing 35 populations. The power of parental exclusion, polymorphic information content, expected and observed heterozygosity and probability of identity were calculated, showing that the set of 17 STRs has sufficient discriminating power for forensic analysis in almost all breeds. We also explored the reliability of individual assignment tests in identifying the correct breeds of origin for unknown samples. The overall proportion of individuals correctly assigned to a population was 97.2%. Finally, we demonstrate the phylogenetic signal of the 17 STR. We found three clusters of related breeds: (i) the cold-blooded draught breeds Haflinger, Dutch draft and Friesian; (ii) the pony breeds Shetland and Miniature horse with the Falabella, Appaloosa and Icelandic; and (iii) The Warmblood riding breeds, together with the hot-blooded Standard-bred, Thoroughbred and Arabian.

A proposal for standardization in forensic equine DNA typing: allele nomenclature for 17 equine-specific STR loci.

In this study, a proposal is presented for the allele nomenclature of 17 polymorphic STR loci (AHT4, AHT5, ASB2, ASB17, ASB23, CA425, HMS1, HMS2, HMS3, HMS6, HMS7, HTG4, HTG6, HTG7, HTG10, LEX3 and VHL20) for equine genotyping (Equus caballus). The nomenclature is based on sequence data of the polymorphic region of the STR loci as recommended by the DNA commission of the International Society for Forensic Genetics for human DNA typing. For each STR locus, several alleles were selected and animals homozygous for those alleles were subjected to sequence analysis. The alleles of the 17 STR loci consisted either of simple (10), compound (6) or complex repeat patterns (1). Only a limited number of alleles with the same fragment size showed different repeat structures. The allele designation described here was based on the number of repeats, including all variable regions within the amplified fragment.

A proposal for standardization in forensic bovine DNA typing: allele nomenclature of 16 cattle-specific short tandem repeat loci.

In this study, a proposal is presented for the allele nomenclature of 16 polymorphic short tandem repeat (STR) loci (BM1824, BM2113, ETH10, ETH225, INRA023, SPS115, TGLA122, TGLA126, TGLA227, ETH3, TGLA53, BM1818, CSRM60, CSSM66, HAUT27 and ILSTS006) for bovine genotyping (Bos taurus). The nomenclature is based on sequence data of the polymorphic region(s) of the STR loci as recommended by the DNA commission of the International Society of Forensic Genetics for human DNA typing. To cover commonly and rarely occurring alleles, a selection of animals homozygous for the alleles at these STR loci were analysed and subjected to sequence studies. The alleles of the STR loci consisted either of simple or compound dinucleotide repeat patterns. Only a limited number of alleles with the same fragment size showed different repeat structures. The allele designation described here was based on the number of repeats including all variable regions within the amplified fragment. The set of 16 STR markers should be propagated for the use in all bovine applications including forensic analysis.

Detection of universal variable fragments as markers for genetic studies. A novel technology for DNA fingerprinting.

A novel DNA technology enables the detection of universal variable fragments (UVF), thus revealing genetic variation without a priori sequence information. The detection of UVF markers is based on two amplifications of genomic DNA with the polymerase chain reaction. In the first amplification, two short oligonucleotide primers produce a large number of fragments. One primer is based on a microsatellite sequence, whereas the second primer can have any sequence. In the second amplification, the length of the primers is increased in order to decrease the number of amplicons. This enables the selection of polymorphic fragments. Restriction digestion can be used to further increase the number of polymorphisms. Until now, we have demonstrated UVF in several different species. In addition, with the present study we have contributed to the linkage map of the rabbit by localizing 11 UVF markers on different linkage groups. Mendelian inheritance was shown in this linkage study through a backcross of two inbred rabbit strains. The power of the UVF technique is based on the selection for microsatellite variation in combination with the detection of single-nucleotide polymorphisms. UVF thus offers the possibility of increasing the clustering of markers and localizing genes in species for which sequence information is either not present or only scarcely present.

A single nucleotide polymorphism set for paternal identification to reduce the costs of trait recording in commercial pig breeding.

In animal breeding, recording of correct pedigrees is essential to achieve genetic progress. Markers on DNA are useful to verify the on-farm pedigree records (parental verification) but can also be used to assign parents retrospectively (parental identification). This approach could reduce the costs of recording for traits with low incidence, such as those related to diseases or mortality. In this study, SNP were used to assign the true sires of 368 purebred animals from a Duroc-based sire line and 140 crossbred offspring from a commercial pig population. Some of the sires were closely related. There were 3 full sibs and 17 half sibs among the true fathers and 4 full sibs and 35 half sibs among all putative fathers. To define the number of SNP necessary, 5 SNP panels (40, 60, 80, 100, and 120 SNP) were assembled from the Illumina PorcineSNP60 Beadchip (Illumina, San Diego, CA) based on minor allele frequency (>0.3), high genotyping call rate (≥90%), and equal spacing across the genome. For paternal identification considering only the 66 true sires in the data set, 60 SNP resulted in 100% correct assignment of the sire. By including additional putative sires (n = 304), 80 SNP were sufficient for 100% correct assignment of the sire. The following criteria were derived to identify the correct sire for the current data set: the logarithm of odds (LOD) score for assigning the correct sire was ≥5, the number of mismatches was ≤1, and the difference in the LOD score between the first and the second most likely sire was >5. If the correct sire was not present among all putative sires, the mean LOD for the most likely sire was close to zero or negative when using 100 SNP. More SNP would be needed for paternal identification if the number of putative sires increased and the degree of relatedness was greater than in the data set used here. The threshold for the number of mismatches can be adjusted according to the practical situation to account for the trade-off between false negatives and false positives. The latter can be avoided efficiently, ensuring that the correct father is being sampled. Nevertheless, a restriction on the number of putative sires is advisable to reduce the risk of assigning close relatives.