A microsatellite within the bovine K-casein gene reveals a polymorphism correlating strongly with polymorphisms previously described at the protein as well as the DNA level

The polymorphism of a (TA)n(CA)n repeat microsatellite present in the third intaron of the bovine K-casein gene (CASK) has been investigated. The existence of six alleles differing only in the number of dinucleotide repeats has been established. A total of 330 animals belonging to nine different pure bred Bos Taurus French breeds or to a cross-bred Bos taurus*Bos indicus population (Créole) were genotyped. The distribution of the microsatellite alleles was examined and clear breed differences were noted. Genotyping of animals by isoelectric focusing (IEF) or restriction fragment length polymorphism (RFLP) (TaqI) was performed, in order to examine the relationship of the microsatellite polymorphism to other previously described CASK polymorphisms, at the protein and DNA levels. Strong correlation was seen, indicating that evolution of the various polymorphisms was not independent, and nine CASK haplotypes were observed.     

Hypervariability of intronic simple (gt)n(ga)m repeats in HLA-DRB genes

We have investigated the extent of DNA variability in intronic simple (gt)_n(ga)_m repeat sequences and correlated this to sequence polymorphisms in the flanking exon 2 of HLA-DRB genes. The polymerase chain reaction (PCR) was used to amplify a DNA fragment containing exon 2 and the repeat region of intron 2. The PCR products were separated on sequencing gels in order to demonstrate length hypervariability of the (gt)_n(ga)_m repeats. In a parallel experiment, the PCR products were cloned and sequenced (each exon 2 plus adjacent simple repeats) to characterize the simple repeats in relation to the HLA-DRB sequences. In a panel of 25 DRB1, DRB4, and DRB5 alleles new sequences were not detected. Restriction fragment length polymorphism (RFLP) subtyping of serologically defined haplotypes corresponds to translated DNA sequences in 85% of the cases, the exceptions involving unusual DR/DQ combinations. Many identical DRB1 alleles can be distinguished on the basis of their adjacent simple repeats. We found group-specific organization of the repeats: the DRw52 supergroup repeats differ from those of DRB1*0101, DRB4*0101, and DRB5*0101 alleles and from those of pseudogenes. Finally, we amplified baboon DNA and found a DRB allele with extensive similarity to DRB1 sequences of the DRw52 supergroup. The simple repeat of the baboon gene, however, resembles that of human pseudogenes. In addition to further subtyping, the parallel study of polymorphic protein and hypervariable DNA alleles may allow conclusions to be drawn on the relationships between the DRB genes and perhaps also on the theory of trans-species evolution.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M 34258.     

Creation of a SINE enriched library for the isolation of polymorphic (AGC)n microsatellite markers in the bovine genome

Trinucleotide (AGC)_n microsatellites are found as 3′ tails of the artiodactyl short interspersed nuclear element (SINE) A-dimer. We describe a polymerase chain reaction (PCR)-based method for the construction of a plasmid library enriched for SINE (AGC)_n microsatellites. By amplifying Sau3AI inserts with a conserved SINE primer and a flanking vector primer, a 35-fold enrichment of (AGC)_n microsatellites over a conventional genomic library was obtained. The SINE primer was used for both sequencing of AGC-containing inserts and analysis of polymorphism. Twenty-three unique reverse primers were synthesized and used on bovine genomic DNA, 21 producing PCR products of expected size. Five polymorphic (AGC)_n microsatellites with 2–4 alleles each were characterized. Allele sizes differed by a 3 bp motif and lacked the stutter bands associated with dinucleotide repeats. A tendency of increased polymorphism for longer AGC repeat arrays was observed. High stringency selection for positive clones containing eight or more AGC repeats can thus facilitate the isolation of polymorphic (AGC)_n microsatellites, Enrichment for (AGC), microsatellites by SINE-vector PCR can be applied to other bovidae species, such as sheep or goat, containing the artiodactyl SINE elements.     

Comparison of ISSR polymorphism among cattle breeds

Polymorphism analysis of DNA fragments flanked by (AG)₉C and (GA)₉C inverted dinucleotide microsatellite repeats in 766 animals of 19 cattle breeds and one breeding type revealed 66 fragments, of which 64 were polymorphic. The breeds proved to differ in the frequency and presence or absence of amplified DNA fragments at the genomic level, indicating that ISSR fingerprinting is informative for differentiating the PCR product spectra and cattle breeds. Multilocus ISSR polymorphism analysis identified the group of fragments that can be used as Bos taurus and B. indicus species markers to describe the standards of breeds, their genetic profiles, and breed-specific patterns. Based on ISSR polymorphism, a prototypal gene pool of cattle was constructed and the breeds closest to it were identified. Genetic diversity analysis made it possible to assume that an optimal mean heterozygosity is characteristic of cattle breeds and that deviations from this optimum are indicative of various processes occurring in the population (breed).     

Novel DNA polymorphism in the mouse tumor necrosis factor receptors type 1 and type 2

The introduction of the polymerase chain reaction (PCR) provides an entirely new means of analyzing DNA polymorphism and makes practical the analysis of length variation in simple-sequence tandem repeats of dinucleotides. In the process of cloning and sequencing the mouse genomic DNA for tumor necrosis factor (TNF) receptors type 1 and type 2, we identified two simple dinucleotide repeats within the noncoding regions of TNF receptor type 1 and three such sequences within TNF receptor type 2. PCR analysis of these sequences, using genomic DNA from 21 different inbred and wild mouse strains, as demonstrated by running the amplified products on sequencing gels, showed that the repeats are highly polymorphic. We identified seven alleles of TNF receptor type 2 and five alleles of TNF receptor type 1. Using these polymorphic markers in two sets of recombinant inbred strains of mice, the chromosomal localization of Tnfr-1 was mapped to mouse chromosome 6 and Tnfr-2 was located to the distal portion of mouse chromosome 4.     

Microsatellite DNA loci for Western Hemlock [Tsuga heterophylla (Raf.) Sarg]

Polymorphic microsatellite loci were developed for Western Hemlock [Tsuga heterophylla (Raf.) Sarg], a prominent forest tree species in Western North America. Microsatellite‐enriched libraries were screened for (CA)_n dinucleotide repeats from which 33 positive clones were sequenced. Polymerase chain reaction (PCR) primers for 16 microsatellite loci were prepared and tested against DNA from unrelated Western Hemlock trees. The 12 most informative microsatellite loci are reported here. From four to 22 alleles per locus were observed, with an average expected heterozygousity of 0.799.     

Polymorphism and phylogeny of dinucleotide repeats in human T-cell receptor Vb6 genes

The Vb6 subfamily is the largest reported subfamily of human T-cell receptor (Tcr) genes with as many as 14 possible members based on variation in reported DNA sequences. A study of the genomic organization of four distinct Vb6 genes indicated that they contained within their introns theuniterrupted dinucleotide repeat (GT)_n, with n>8. DNA amplification primers and conditions were determined which amplified the intron of these four different Vb6 gene segments. All four Vb6 genes tested showed length polymorphism when examined in a group of unrelated individuals. Careful sizing and DNA sequencing showed that the alleles of each gene differed in size by multiples of two base pairs (bp), due to different repeat numbers of the dinucleotide (GT)_n. These four microsatellite polymorphisms had from three to ten alleles, and individual heterozygosities of 26% to 83%. The large number of alleles and the high heterozygosity make these polymerase chain reaction (PCR)-based polymorphisms very attractive genetic markers for segregation studies which postulate the presence of autoimmune susceptibility genes within the Tcrb region. Vb6 hybridization to genomic DNA confirmed the relatively large size of the Vb6 subfamily in several hominoid species. Nucleotide sequencing of an intron of the Vb6 genes from other primates revealed the presence of dinucleotide repeats similar to those found in human Vb6 genes. Thus, the (GT)_n microsatellite was not only present in the Vb6 intron before Vb6 gene duplication, but was present before speciation of the hominoids.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers L07638, L07640, and X07641.     

Coding versus intron variability: extremely polymorphic HLA-DRB1 exons are flanked by specific composite microsatellites, even in distant populations

Although microsatellite typing is the dominant method in genome research and indirect gene diagnosis, precise relationships of exonic and adjacent simple repeat polymorphisms are not known. We investigated exon 2 sequences of HLA-DRB1 genes and their neighbouring (GT)_n(GA)_m repeats including the intervening single copy spacer. DRB1 is the most polymorphic protein-coding locus in man and all vertebrates investigated. The entire DRB1 variability exists in exon 2. DRB1 genes in different haplotype groups (DR1, DR51, DR52, DR8 and DR53) are accompanied by characteristic modifications of the (GT)_n(GA)_m block (3′ to group-specific single copy spacers). Among more than 520 alleles analysed, > 100 different types of microsatellites were observed. The perfect (GT)_n and (GA)_m blocks vary in length and may be partly ‘degenerated’, mostly in a subgroup-specific manner. Interestingly, the extent of microsatellite diversity varies in given DRB1 alleles. While the microsatellites of the DR7, DR9 alleles and in the DR1 group are virtually invariant, in DR4 and DR13, in particular, simple repeats appear hypervariable with at least 15 or 17 different length alleles, respectively. Comparing Caucasians, Bushmen and South American Indians, the microsatellite variation in identical DRB1 alleles (e.g. DRB1*0102, 03 011, 1302) is smaller than within any of the DR groups in Caucasians. Taken together, extremely polymorphic DRB1 exons evolve in concert with certain variants of an exceptionally well-preserved microsatellite. Received: 8 October 1996     

A set of 99 cattle microsatellites: characterization,synteny mapping,and polymorphism

Cattle microsatellite clones (136) were isolated from cosmid (10) and plasmid (126) libraries and sequenced. The dinucleotide repeats were studied in each of these sequences and compared with dinucleotide repeats found in other vertebrate species where information was available. The distribution in cattle was similar to that described for other mammals, such as rat, mouse, pig, or human. A major difference resides in the number of sequences present in the bovine genome, which seemed at best one-third as large as in other species. Oligonucleotide primers (117 pairs) were synthesized, and a PCR product of expected size was obtained for 88 microsatellite sequences (75%). Synteny or chromosome assignment was searched for each locus with PCR amplification on a panel of 36 hamster/bovine somatic cell hybrids. Of our bovine microsatellites, eighty-six could be assigned to synteny groups of chromosomes. In addition, 10 other microsatellites—HEL 5, 6, 9, 11, 12, 13 (Kaukinen and Varvio 1993), HEL 4, 7, 14, 15—as well as the microsatellite found in the -casein gene (Fries et al. 1990) were mapped on the hybrids. Microsatellite polymorphism was checked on at leat 30 unrelated animals of different breeds. Almost all the autosomal and X Chr microsatellites displayed polymorphism, with the number of alleles varying between two and 44. We assume that these microsatellites could be very helpful in the construction of a primary public linkage map of the bovine genome, with an aim of finding markers for Economic Trait Loci (ETL) in cattle.     

Sequence and PCR-RFLP analysis of 14 novel BoLA-DRB3 alleles

The genetic diversity of the bovine class IIDRB3 locus was investigated by polymerase chain reaction (PCR) amplification and DNA sequencing of the first domain exon. Studying 34 animals of various cattle breeds, 14 previously unrecognized DRB3 alleles were identified. In three alleles, amino acid substitutions were observed that had not been previously found in bovine DRB3, but occurred at the same position in bovine DQB and in the DRB alleles of other mammals. For all newly identified alleles, the restriction fragment length polymorphism (RFLP) patterns of PCR products obtained with the enzymes Rsa I, Bst YI, and Hae III were compared with patterns of 38 previously described alleles. Altogether, eleven novel PCR-RFLP types were defined. Twelve out of the 42 PCR-RFLP types identified so far were not found to be fully informative because they corresponded to more than one allelic sequence. PCR-RFLP may therefore be a rapid and useful method for DRB3 typing in cattle families, but for studies on outbred populations, sequencing and hybridization techniques are required.     

Extensive polymorphism of the BOLA-DRB3 gene distinguished by PCR-RFLP

A polymerase chain reaction (PCR)-based method is described for typing of alleles of the bovine lymphocyte antigen (BoLA)-DRB3 gene. A total of 30 DRB3 alleles were distinguished by digestion of PCR amplification products of BoLA-DRB3 exon 2 with RsaI, BstYI and HaeIII (PCR-RFLP). All restriction fragment patterns, with the exception of one HaeIII pattern, were consistent with restriction sites that were found among 14 previously sequenced DRB3 alleles. The PCR-RFLP typing method was evaluated on 168 genomic DNA samples collected from animals of 10 cattle breeds, 48 of which were typed in the Fourth International BoLA Workshop for BoLA-DRB and -DQ by conventional restriction fragment length polymorphism (RFLP) analysis using heterologous and homologous DNA probes. Thirty-one DRB/DQ haplotypes containing 23 DRB3 alleles were identified among the 48 workshop animals analysed. Using PCR-RFLP, 11 DRB3 alleles were identified in 18 workshop animals for which DRB RFLPs were not informative. PCR-RFLP typing of additional animals revealed five new DRB3 alleles, of which three contained a putatively located three basepair deletion in the identical position as found for the sequenced allele DRB*2A. PCR-RFLP was shown to be a rapid and sensitive method for the detection of polymorphism in a functionally relevant domain of the BoLA-DRB3 gene and should be useful for studying the evolution of DRB polymorphism in cattle and other Bovidae.     

Characterization of highly variable (GA/CT) n microsatellites in the bur oak,Quercus macrocarpa

The objective of this study was to ascertain the usefulness of polymerase chain reaction (PCR)-based microsatellite analysis for studying pollination and parentage in a wind-pollinated temperate tree. A small insert genomic library of the bur oak (Quercus macrocarpa) was constructed and screened for the presence of (CA/GT) _n and (GA/CT) _n repeats. The proportion of positive clones yielded estimates of 3×10⁵ such dinucleotide repeats per genome, roughly comparable to abundances reported in other eukaryotic genomes. Thirteen positive clones were sequenced. In contrast to mammalian genomes, the (GA/CT) _n motif was more abundant than the (CA/GT) _n motif in these clones. The (GA/CT) _n repeats also showed longer average repeat length (mean n=16.2 versus 7.3), suggesting that they are better candidates for yielding polymorphic genetic markers in oak genomes. Indeed, a survey of adult bur oaks and offspring in a small stand in northern Illinois at 3 of these (GA/CT) _n microsatellite loci revealed Mendelian inheritance and extremely high levels of polymorphism, with the number of alleles at each locus ranging from 11–20 and heterozygosity ranging from 0.66 to 0.75. These results, indicating that (GA/CT) _n microsatellites are both abundant and highly polymorphic in the bur oak genome, suggest that such genetic markers have tremendous potential for applications for studies of parentage, pollination and dispersal in temperate trees.     

Abundant (A)n·(T)n mononucleotide repeats in the pig genome: linkage mapping of the porcine APOB, FSA, ALOX12, PEPN and RLN loci

A computer analysis revealed that the mononucleotide repeat (A)_n-(T)_n is about five times as common as (CA)_n-(GT)_n repeats in the porcine genome, with frequency estimates of one every 7kb and 30kb, respectively. Seven mononucleotide repeats with n= 12–25 located close to coding sequences were analysed for polymorphism using polymerase chain reaction (PCR) amplification and polyacrylamide gel electrophoresis. All loci were variable with 3–6 alleles and heterozygosities of 0.26–0.69 based on investigation of 10 unrelated pigs (two wild boars and eight domestic sows). Repeat length correlated with degree of polymorphism. A comparison with (CA)_n-(GT)_n polymorphisms suggested that the number of repeat units rather than the total length of the repeat region is the common denominator that governs polymorphism at both mono- and dinucleotide repeat loci. (A)_n-(T)_n polymorphisms allowed linkage mapping of relaxin to chromosome 1, apolipoprotein B to chromosome 3, aminopepti-dase N to chromosome 7, arachidonate 12-lipoxygenase to chromosome 12, and follistatin to chromosome 16. The rich abundance of potentially informative (A)_n-(T)_n repeats will increase the chances of finding a PCR-based marker near any DNA sequence of interest.     

Identification of a conserved microsatellite site in the porcine and bovine insulin-like growth factor-I gene 5'' flank

Examination of published rat and human sequences for the insulin-like growth factor-I (IGF-I) gene indicated the presence of CA dinucleotide repeats in corresponding segments of each. Presence of similar microsatellite sequences in the porcine and bovine IGF-I genes was hypothesized. A 1200-bp segment upstream of the porcine and bovine IGF-I genes was amplified using the polymerase chain reaction (PCR) with primers developed from a consensus of human, rat and bovine sequences. Both porcine and bovine PCR products contained similar microsatellite sequences. Amplified pIGF-I DNA was cloned and sequenced, and an additional primer was developed specifically for microsatellite marker detection. Six allelic variants of 124, 130, 132, 134, 136 or 138 bp were observed in pigs with differing frequencies between breeds (P < 0.01). The same primers were used to amplify the corresponding bovine microsatellite. Three alleles of 126, 128 and 130 bp were observed in a genetically diverse cattle population with estimated frequencies of 0.06, 0.68 and 0.26, respectively. Results of this study indicate sequence information from the human and laboratory species can be used to facilitate genetic marker development in livestock species.     

Abundance, variability and chromosomal location of microsatellites in wheat

The potential of microsatellite sequences as genetic markers in hexaploid wheat (Triticum aestivum) was investigated with respect to their abundance, variability, chromosomal location and usefulness in related species. By screening a lambda phage library, the total number of (GA)_n blocks was estimated to be 3.6 x 10⁴ and the number of (GT)_n blocks to be 2.3 x 10⁴ per haploid wheat genome. This results in an average distance of approximately 270 kb between these two microsatellite types combined. Based on sequence analysis data from 70 isolated microsatellites, it was found that wheat microsatellites are relatively long containing up to 40 dinucleotide repeats. Of the tested primer pairs, 36% resulted in fragments with a size corresponding to the expected length of the sequenced microsatellite clone. The variability of 15 microsatellite markers was investigated on 18 wheat accessions. Significantly, more variation was detected with the microsatellite markers than with RFLP markers with, on average, 4.6 different alleles per microsatellite. The 15 PCR-amplified microsatellites were further localized on chromosome arms using cytogenetic stocks of Chinese Spring. Finally, the primers for the 15 wheat microsatellites were used for PCR amplification with rye (Secale cereale) and barley accessions (Hordeum vulgare, H. spontaneum). Amplified fragments were observed for ten primer pairs with barley DNA and for nine primer pairs with rye DNA as template. A microsatellite was found by dot blot analysis in the PCR products of barley and rye DNA for only one primer pair.     

Isolation and characterization of microsatellite loci in the flour mite Acarus siro

Thirteen microsatellites with dinucleotide repeats were isolated from genomic DNA in solution by hybridization capture. Eleven were evaluated against Acarus siro using a multiple‐tube approach to assess polymorphism and reliability of the results. The multiple‐tube method demonstrated the presence of null alleles due to low levels of DNA. Seven of the 11 microsatellite loci were polymorphic and six produced reliable amplification products, if results only from individuals that scored at all six loci were used in the analysis. These were evaluated in eight strains of A. siro. Two of the samples showed some genetic divergence using F_ST values.     

Development,characterization and utilization of GenBank microsatellite markers in <Emphasis Type="Italic">Phyllostachys pubescens</Emphasis> and related species

Public sequence databases provide a rapid, simple and cost-effective source of microsatellite markers. We analyzed 1,532 bamboo (Phyllostachys pubescens) sequences available in public domain DNA databases, and found 3,241 simple sequence repeat (SSR) loci comprising repeats of two or more nucleotides in 920 genomic survey sequences (GSSs) and 68 cDNA sequences. This corresponded to one SSR per 336 bp of GSS DNA and one SSR per 363 bp of cDNA. The SSRs consisted of 76.6 and 74.5% dinucleotide repeats, 20.0 and 22.3% trinucleotide repeats, and 3.4 and 3.2% higher-number repeats in the GSS DNA and cDNA sequences, respectively. The repeat motif AG/CT (or GA/TC) was the most abundant. Nineteen microsatellite markers were developed from Class I and Class II SSRs, showing that the limited polymorphism in Ph. pubescens cultivars and provenances could be attributed to clonal propagation of the bamboo plant. The transferability of the microsatellites reached 75.3%, and the polymorphism of loci successfully transferred was 66.7% for six additional Phyllostachys species. Microsatellite PBM014 transferred successfully to all six species, showed rich polymorphism, and could serve as species-specific alleles for the identification of Phyllostachys interspecies hybrids.     

Stability of triplet repeats of myotonic dystrophy and fragile X loci in human mutator mismatch repair cell lines

At least nine human genetic diseases, including myotonic dystrophy (DM) and fragile X syndrome have been associated with the expansion of CTG or CGG trinucleotide repeats within the disease loci. Little is known about the molecular mechanisms or the genetic control of the expansion of triplet repeats. Mutations in human mismatch repair genes are associated with the increased polymorphism of many microsatellites, including dinucleotide repeats. The effect of mutations in two mismatch repair genes on the size of trinucleotide repeats in the DM and FRAXA loci has been analyzed. PCR and Southern analysis of the triplet repeat regions of the DM and fragile X mental retardation (FRAXA) loci in cell lines HTC116 and LoVo, which contain mutations in both alleles of the hMLH1 and hMSH2 genes, respectively, indicated that the size of the endogenous (CTG)_n and (CGG)_n tracts fall within the range observed in the normal population. This suggests that mutations in hMLH1 or hMSH2 do not result in the instability of CTG or CGG tracts to the levels observed in individuals with myotonic dystrophy or fragile X syndrome. Received: 4 December 1995 / Revised: 29 January 1996, 7 March 1996     

Analysis and Frequency of Bovine Lymphocyte Antigen (BoLA-DRB3) Alleles in Iranian Holstein Cattle

The bovine lymphocyte antigen (BoLA-DRB3) gene encodes cell surface glycoproteins that initiate immune response by presenting processed antigenic peptides to CD4 T helper cells. DRB3 is the most polymorphic bovine MHC class II gene which encodes the peptide-binding groove. DRB3 gene has been extensively evaluated as a candidate marker for association with various bovine diseases and immunological traits. This study describes genetic variability in the BoLA-DRB3 in Iranian Holstein cattle. This is the first study of the DNA polymorphism of the BoLA-DRB3 gene in Iranian Holstein cattle. Hemi-nested PCR-RFLP method is used for identification the frequency of BoLA-DRB3 alleles. The BoLA-DRB3 locus is highly polymorphic in the studied herd (26 alleles). Almost 67% of the alleles were accounted for four alleles (BoLA-DRB3.2*8, *24, *11, and *16) in Iranian Holstein cattle. The DRB3.2*8 allele frequency (26.6%) was higher than the others. The frequencies of the DRB3.2*54, *37, *36, *28, *25, *14, *13, *10, *1 alleles were lower than 1%. Significant distinctions have been found between Iranian Holstein cattle and other cattle breeds studied. In Iranian Holstein cattle the alleles (BoLA-DRB3.2*22, *2, and *16) associated with a lower risk of cystic ovarian disease in Holstein cattle are found. The alleles associated with the resistance to mastitis and to bovine leukemia virus infection BoLA-DRB3.2*11 and *23 are detected with the frequencies 10.4 and 4.4%, respectively. Thus, in the Iranian Holstein cows studied alleles associated with resistance to various diseases are found. The method of DNA-typing of animals can be used in agricultural practice for BoLA-DRB3 allele genotyping of cattle in order to reduce spreading of alleles providing susceptibility to mastitis or leukemia in cattle herds.__________From Genetika, Vol. 41, No. 6, 2005, pp. 817–822.Original English Text Copyright © 2005 by Nassiry, Eftekhar Shahroodi, Mosafer, Mohammadi, Manshad, Ghazanfari, Mohammad Abadi, Sulimova.The article was submitted by the authors in English.     

Parentage testing and linkage analysis in the horse using a set of highly polymorphic microsatellites

Ten (TG)_n positive clones, isolated from an equine genomic library and sequenced, contained 12–19 uninterrupted TG repeats. Primers for polymerase chain reaction (PCR) were synthesized and nine of these (TG)_n loci (HTG7-15) were successfully amplified and utilized in this study together with five previously reported equine microsatellite loci (HTG2-6). The PCR products were analysed by polyacrylamide gel electrophoresis followed by automated laser fluorescence detection or autoradiography. All microsatellites showed polymorphism and stable Mendelian inheritance. Differences in microsatellite variability between horse breeds were detected. A linkage analysis comprising HTG2-15, one coat colour gene and 16 genetic blood markers enabled addition of HTG2 to linkage group U2 and a new linkage group (U6) was established comprising the loci HTG7 and HTG12. Close linkage was excluded within a set of eight microsatellites. The estimated probability of exclusion in four breeds for a parentage test based on these eight loci varied between 0.96 and 0.99.