Validation of microsatellite markers for routine horse parentage testing

A parallel testing of 4803 routine Quarter Horse parentage cases, using 15 loci of blood group and protein polymorphisms (blood typing) and 11 loci of dinucleotide repeat microsatellites (DNA typing), validated DNA markers for horse pedigree verification. For the 26 loci, taken together, the theoretical effectiveness of detecting incorrect parentage was 99·999%, making it extremely unlikely that false parentage would fail to be recognized. The tests identified incorrect parentage assignment for 95 offspring (2% of cases). Despite fewer loci, DNA typing was as effective as blood typing and, in parentage exclusion cases, provided more systems to substantiate the genetic incompatibility. Five offspring presented potential genetic incompatibilities with their parents in only a single microsatellite system, but the parentage exclusions could not be confirmed with discordant results at additional loci. Two of these five incompatibilities could be explained as consequences of a null allele and three as fragment size increases or decreases (putative mutations). Provided that an exclusion assignment was based on at least two systems of genetic incompatibility, such rare genetic events did not lead to false exclusions. Notwithstanding the near 100% effectiveness estimations for either typing panel alone to identify incorrect parentage, this validation test showed an actual effectiveness of 97·3% for blood typing and 98·2% for DNA typing. The DNA-based test, however, may feasibly achieve higher efficacy than reported here by adding selected systems to the parentage test panel.     

Probability of random sire exclusion using microsatellite markers for parentage verification

Many microsatellite sequences have been described in the bovine genome. Being highly polymorphic these have been suggested as markers for parentage verification and individual identification in cattle. We have evaluated the use of five highly polymorphic microsatellite markers for parentage verification in 14 breeds of cattle in the UK. Three of the microsatellite loci occur within introns in genes: BoLA DRB3 , steroid 21-hydroxylase, and the beta subunit of the follicle-stimulating hormone. The other two are anonymous sites ETH131 and HEL6. Results were analysed by a statistical approach that takes in to account deviations from Hardy-Wienberg equilibrium and linkage disequilibrium for multiple loci. The method of determining the probability of random sire exclusion uses observed genotype frequencies instead of allele frequencies. Independently, the markers used have a probability of between 0.72 and 0.62 of identifying a parentage error, while used together the five markers give, on average across breeds, a probability of 0.99 of excluding an incorrect sire.     

Comparison of Thoroughbred and Arabian horses using RAPD markers

We compared pools of DNA from 10 Thoroughbred horses and 10 Arabian horses for the presence of randomly amplified polymorphic DNA (RAPD) markers which might be useful in distinguishing between the breeds. Using 212 decamer oligonucleotides and our polymerase chain reaction (PCR) conditions, 173 of the primers produced scoreable bands. The number of bands ranged from 0 to 9 with an average of 3·6. In family studies using 11 arbitrarily selected primers, five of the 11 primers produced polymorphic bands which exhibited Mendelian inheritance as dominant markers. When comparing the pooled DNA from Thoroughbred and Arabian horses we found 10 primers which identified markers present in the pooled DNA from one breed but absent in the pool from the other breed. Testing individual horses revealed that only two markers were wholly absent for one group while being present among members of the other. Primer UBC-85 (5′-GTGCTCGTGC-3′) detected a pair of markers absent in Thoroughbred horses but present among 11 of 31 Arabian horses. These markers were 1500 and 1700 base pairs (bp) long and designated UBC-85^C and UBC-85^D, respectively. Primer UBC-126 (5′-CTTTCGTGCT-3′) detected a 1000 bp marker (designated UBC-126^C) absent in 20 of 20 Thoroughbred horses but present in 31 of 31 Arabian horses. UBC-126^C would be particularly effective for breed comparisons, especially if the DNA band were cloned, sequenced and an allelic marker present in Thoroughbred horses but rare or absent among Arabian horses was identified. The distribution of such markers among other horse breeds might be useful to infer relationships among breeds. These kinds of markers may also be useful in detecting unwanted crossbreeding between two horse breeds.     

Exclusion probabilities of 22 bovine microsatellite markers in fluorescent multiplexes for semiautomated parentage testing

Six multiplexes developed for semiautomated fluorescence genotyping were evaluated for parentage testing. These multiplexes contained primer pairs for the amplification of 22 microsatellites on 17 bovine autosomes. Exclusion probabilities were determined from genotypes of 1022 Holstein cattle and 311 beef cattle belonging to five breeds. Two cases were considered: case 1, genotypes are known for an alleged parent and an offspring but genotypes of a confirmed parent are unknown; and case 2, genotypes are known for an alleged parent, a confirmed parent and an offspring. If the alleged parent is not the true parent, then the 22 markers will exclude the alleged parent with a probability of >0·9986 for case 1 and with a probability of >0·99999 for case 2. On the basis of these exclusion probabilities, the probability that an alleged parent will be falsely included as a parent is in the range of 1/716 to 1/2845 for case 1 and 1/1·2 million to 1/159753 for case 2. In addition to these results, a rapid and efficient non-organic method for extraction of DNA from semen is described.     

Resolving genetic relationships with microsatellite markers: a parentage testing system for the swallow Hirundo rustica

Eight polymorphic microsatellite markers from the swallow were isolated and characterized. Extraordinary variability was revealed at the HrU6 locus with 45 different alleles scored among 46 unrelated individuals. The probability that the same genotype combination would occur in two random and unrelated individuals at six selected loci was as low as 1.3 × 10^-8 and the combined exclusion probability was 0.9996. Stable Mendelian inheritance was observed in about 1000 meioses. No significant linkage was revealed and for almost all combinations of marker-pairs, linkage closer than 5 cM could be excluded. At two loci, null (nonamplifying) alleles were encountered. Thirteen (30%) extra-pair offspring were identified in 5 (56%) broods when applying the marker set on a nearly complete swallow colony. We were able to identify a single male from the other families in the colony as the most likely father for nine of the 13 extra-pair offspring.     

Power of 22 microsatellite markers in fluorescent multiplexes for parentage testing in goats (Capra hircus)

Two multiplex systems, each containing 11 microsatellite loci, were developed for semiautomated parentage testing in goats. Eight of the loci originate from goats, nine from cattle and five from sheep. Eighteen of the loci have been mapped to 16 different autosomes (in goats and cattle). Parentage exclusion probabilities were computed from allele frequencies in approximately 30 unrelated individuals from each of four economically important breeds: Mongolian Native Cashmere, Turkish Angora, Swiss Saanen, and Spanish Murciana-Grenadina. In cases where genotypes are known for one parent and an offspring, the 22 markers will exclude an (erroneously) alleged parent with a probability of > 0.999999 in the cashmere breed, > 0.99999 in Angora and Murciana-Grenadina, and > 0.9999 in Saanen. The multiplexes provide very high power for individual identification as the probability of finding two identical genotypes for the 22 loci is < 1 in 1.10(15) in each of the four breeds. The multiplexes will also be useful for studies of population structure, history, and diversity in goats and also in wild Capra species that represent important resources for genetic improvement of domestic breeds.     

Power of exclusion of 19 microsatellite markers for parentage testing in river buffalo (Bubalus bubalis)

In the present study, 19 microsatellite markers were assessed for their power of exclusion to test parentage in river buffalo. Microsatellite genotypes of 216 unrelated buffaloes belonging to five different breeds were utilized for the study. The probabilities of exclusion were calculated for three hypothetical situations viz. paternity testing (PE1), one parental genotype unavailable (PE2) and exclusion of both parents i.e. substituted offspring (PE3). The mean probability of exclusion across 19 investigated markers in buffalo was 0.578 (PE1), 0.405 (PE2) and 0.764 (PE3) respectively. The probability of exclusion for paternity (PE1) ranged between 0.297 and 0.814 across different markers. The exclusion probability for the cases one parent unavailable (PE2) and substituted offspring (PE3) varied from 0.143 to 0.688 and 0.465 to 0.946 respectively. Polymorphism information content and expected heterozygosity were found to have significantly high correlation with probability of exclusion of microsatellite markers. The cumulative PE1 of nine marker loci was estimated to be 0.9999 while in case of absence of one of the parental genotypes, a minimum of 11 markers were required to achieve a cumulative PE2 of 0.999. In conclusion, the present study proposes two multiplex sets with four and five markers respectively for routine parentage testing in buffalo and an additional set of four markers for doubtful cases of paternity.     

A parentage study of closely related Ukrainian wine grape varieties using microsatellite markers

Four bred grapevine varieties released for commercial cultivation in Ukraine, namely ‘Antey Magarachskii’, ‘Rubinovyi Magaracha’, ‘Granatovyi Magaracha’ and ‘Rubin Golodrigi’, and their putative parental forms were genotyped using six microsatellite loci. Genotypes were compared with breeding records to verify genetic relationships among varieties. Results of the analysis confirmed four of six parent-offspring relationships. Results of the analysis allow to assume that genotype ‘Seyve Villard 20347’ is the direct parent of ‘Antey Magarachskii’ instead of its grandparent. The first-studied accession believed to be that of ‘Granatovyi Magaracha’ was identified as impurity. In order to verify the parentage of ‘Granatovyi Magaracha’, rest accessions of that variety and its putative parent ‘Antey Magarachskii’ were additionally genotyped at 13 nuclear loci and at three chloroplast loci. The parent-offspring relationship was confirmed, as all ‘Granatovyi Magaracha’ accessions had a common allele with the parent variety ‘Antey Magarachskii’ at each locus and the same chlorotype A. Different ‘Granatovyi Magaracha’ accessions could have been obtained via vegetative propagation of two seedlings which arose from one crossing.     

Gametic phase disequilibrium between the syntenic multiallelic HTG4 and HMS3 markers widely used for parentage testing in Thoroughbred horses

Validation of parentage and horse breed registries through DNA typing relies on estimates of random match probabilities with DNA profiles generated from multiple polymorphic loci. Of the twenty-seven microsatellite loci recommended by the International Society for Animal Genetics for parentage testing in Thoroughbred horses, eleven are located on five chromosomes. An important aspect in determining combined exclusion probabilities is the ascertainment of the genetic linkage status of syntenic markers, which may affect reliable use of the product rule in estimating random match probabilities. In principle, linked markers can be in gametic phase disequilibrium (GD). We aimed at determining the extent, by frequency and strength, of GD between the HTG4 and HMS3 multiallelic loci, syntenic on chromosome 9. We typed the qualified offspring (n ₁ = 27; n ₂ = 14) of two Quarter Bred stallions (registered by the Brazilian Association of Quarter Horse Breeders) and 121 unrelated horses from the same breed. In the 41 informative meioses analyzed, the frequency of recombination between the HTG4 and HMS3 loci was 0.27. Consistent with genetic map distances, this recombination rate does not fit to the theoretical distribution for independently segregated markers. We estimated sign-based D′ coefficients as a measure of GD, and showed that the HTG4 and HMS3 loci are in significant, yet partial and weak, disequilibrium, with two allele pairs involved (HTG4*M/HMS3*P, D′(+) = 0.6274; and HTG4*K/HMS3*P, D′(−) = −0.6096). These results warn against the inadequate inclusion of genetically linked markers in the calculation of combined power of discrimination for Thoroughbred parentage validation.     

Power of microsatellite markers for fingerprinting and parentage analysis in Eucalyptus grandis breeding populations

We report the genetic analysis of 192 unrelated individuals of an elite breeding population of Eucalyptus grandis (Hill ex Maiden) with a selected set of six highly polymorphic microsatellite markers developed for species of the genus Eucalyptus. A full characterization of this set of six loci was carried out generating allele frequency distributions that were used to estimate parameters of genetic information content of these loci, including expected heterozygosity, polymorphism information content (PIC), power of exclusion, and probability of identity. The number of detected alleles per locus ranged from 6 to 33, with an average of 19.8 +/- 9.2. The average expected heterozygosity was 0.86 +/- 0.11 and the average PIC was 0.83 +/- 0.16. Using only three loci, it was possible to discriminate all 192 individuals. The overall probability of identity considering all six EMBRA microsatellite markers combined was lower than 1 in 2 billion. An analysis of the sample size necessary to estimate expected heterozygosity with minimum variance indicated that at least 64 individuals have to be genotyped to characterize this parameter with adequate accuracy for most microsatellites in Eucalyptus. The high degree of multiallelism and the clear and simple codominant Mendelian inheritance of the set of microsatellites used provide an extremely powerful system for the unique identification of Eucalyptus individuals for fingerprinting purposes and parentage testing.     

Nonamplifying alleles at microsatellite loci: a caution for parentage and population studies

While genotyping wild red deer (Cervus elaphus) at microsatellite loci for paternity assignment, we found three loci (MAP65, BOVIRBP and CelJP23) with segregating nonamplifying alleles. Nonamplifying alleles were detected through mismatches between known mother-offspring pairs and by significant deviations from Hardy-Weinberg equilibria. In a wide range of molecular ecology applications, and especially in parentage assignment, the possible existence of undetectable alleles must be taken into account; this may be particularly important for microsatellite data.     

Establishment of paternity testing system using microsatellite markers in Chinese Holstein

To estimate the efficiency of microsatellite markers in paternity testing among Chinese Holstein, 30 microsatellite loci were used to differentiate 330 Chinese Holstein genotypes, according to the calculation of the allele frequency, number of alleles, effective number of alleles, genetic heterozygosity, polymorphic information content (PIC), and the exclusion probability in this cattle population. The results demonstrated that the exclusion probability ranged from 0.620 in locus BM1818 to 0.265 in locus INRA005 with the average of 0.472 and 11 microsatellite markers exceeding 0.5. The combined exclusion probability of nine microsatellite markers was over 0.99. The result showed that paternity testing of Chinese Holstein was basically resolved using the nine microsatellite markers selected.     

Power of exclusion for parentage verification and probability of match for identity in American Kennel Club breeds using 17 canine microsatellite markers

DNA analysis of microsatellite markers has become a common tool for verifying parentage in breed registries and identifying individual animals that are linked to a database or owner. Panels of markers have been developed in canines, but their utility across and within a wide range of breeds has not been reported. The American Kennel Club (AKC) authorized a study to determine the power to exclude non-parents and identify individuals using DNA genotypes of 17 microsatellite markers in two panels. Cheek swab samples were voluntarily collected at Parent Breed Club National Specialty dog shows and 9561 samples representing 108 breeds were collected, averaging 88.5 dogs per breed. The primary panel of 10 markers exceeded 99% power of exclusion for canine parentage verification of 61% of the breeds. In combination with the secondary panel of seven markers, 100% of the tested breeds exceeded 99% power of exclusion. The minimum probability match rate of the first panel was 3.6 x 10(-5) averaged across breeds, and with the addition of the second panel, the probability match rate was 3.2 x 10(-8); thus the probability of another random, unrelated dog with the same genotype is very low. The results of this analysis indicated that, on average, the primary panel meets the AKC's needs for routine parentage testing, but that a combination of 10-15 genetic markers from the two panels could yield a universal canine panel with enhanced processing efficiency, reliability and informativeness.     

Genetic blood markers in Arabian, Barb and Arab-Barb horses in Morocco

Gene frequencies at 16 blood group and protein polymorphism loci (A, C, D, K, P, Q, U, Al, Gc, Es, A1B, Tf, PGD, PGM, GPI and Pi) are given for three horse breeds in Morocco (Arabian, Arab-Barb and Barb). These data are used to calculate average heterozygosity (h), Nei's standard genetic distance (D_N) and probability of exclusion (PE). Variability expressed as the average heterozygosity was lower in the Arabian (0.330 ± 0.066), while it was higher and almost the same in the Arab-Barb (0.413 ± 0.071) and the Barb (0.414 A ± 0–070). The shortest genetic distance was found between Barb and Arab-Barb. The 16 loci used are at least 95% effective for recognizing incorrect paternity in these breeds. The Barb and Arab-Barb genetic profiles obtained showed the rare variants interesting perhaps in the context of European and American breeds: notably D^cfgkm, D^dekl, Es-N, Tf-A and Pi-W.     

Population genetic analysis among five Indian population groups using six microsatellite markers

Genetic variation at six tetranucleotide microsatellites (HUMTHO1, HUMVWA, F13A01, D3S1359, D12S66, and D12S67) has heen determined in five endogamous ethnic population groups of India belonging to two major linguistic families. The populations analyzed were Konkanastha Brahmins and Marathas (Maharashtra state) from the Indo-Aryan linguistic family and Nairs, Ezhavas, and Muslims (Kerala state) from the Dravidian family. All six loci show high gene diversity, ranging from 0.63 +/- 0.04 to 0.84 +/- 0.02. The average GST value observed was 1.7%, indicating that the differences between the populations account for less than 2% of the diversity, while the genetic variation is high within the five population groups studied (>98%). The phylogenetic tree fails to show any clear cluster. The absence of any cluster along with low average GST is suggestive of substantial genetic similarity among the studied populations, in spite of clear geographical, linguistic, and cultural barriers. This similarity indicates either a greater gene flow between these groups or, alternatively, may reflect a recent evolution for them, considering that the Indian caste system evolved only about 3000 years ago.     

Development of microsatellite markers for parentage analysis in the great tinamou (Tinamus major)

Eighteen microsatellite loci were isolated from great tinamous (Tinamus major), which are large terrestrial birds found in the Neotropics. These are the first primers developed for the Order Tinamiformes. Paternity analyses are possible because the levels of heterozygosity are sufficiently high (0.29-0.90).