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.     

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.     

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.     

Microsatellite-based parentage control in cattle

As a new approach to parentage control we developed two multiplex coamplification polymerase chain reaction (PCR) systems containing a total of six different short tandem repeat (STR) loci; the microsatellite polymorphisms were visualized by automated fluorescence detection on the Applied Biosystems 373 DNA Sequencer with 672 Genescan Analysis software. Allele frequency data were determined from 238 animals. Thirty-five bovine parentage control cases not solvable by conventional blood typing could be solved.     

Comparison of microsatellites and amplified fragment length polymorphism markers for parentage analysis

This study compares the properties of dominant markers, such as amplified fragment length polymorphisms (AFLPs), with those of codominant multiallelic markers, such as microsatellites, in reconstructing parentage. These two types of markers were used to search for both parents of an individual without prior knowledge of their relationships, by calculating likelihood ratios based on genotypic data, including mistyping. Experimental data on 89 oak trees genotyped for six microsatellite markers and 159 polymorphic AFLP loci were used as a starting point for simulations and tests. Both sets of markers produced high exclusion probabilities, and among dominant markers those with dominant allele frequencies in the range 0.1-0.4 were more informative. Such codominant and dominant markers can be used to construct powerful statistical tests to decide whether a genotyped individual (or two individuals) can be considered as the true parent (or parent pair). Gene flow from outside the study stand (GFO), inferred from parentage analysis with microsatellites, overestimated the true GFO, whereas with AFLPs it was underestimated. As expected, dominant markers are less efficient than codominant markers for achieving this, but can still be used with good confidence, especially when loci are deliberately selected according to their allele frequencies.     

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.     

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).     

Efficient resolution of parentage in dogs by amplification of microsatellites

Parentage control has been performed for 15 litters from 12 different dog breeds by amplification of microsatellites. As it was possible to include all putative parents in all cases, they were solved by exclusion. Discrimination between parents/non-parents was made after genotyping of 6–9 microsatellite loci. In 12 of the cases all but one of the alleged fathers were excluded while in three cases it was unambiguously shown that superfecundation had taken place. Furthermore, one inclusion case concerning disputed maternity has been investigated. Maternity indices were calculated for 12 loci and probability of maternity was estimated to be 99-99%. These results testify that microsatellites can be applied very efficiently for resolution of parentage in dogs.     

Estimation of the number of SNP genetic markers required for parentage verification

Formulae were developed to compute exclusion probabilities for parentage confirmation for any number of diallelic markers under the assumption that the minor allele frequency (MAF) varied among markers, but has a uniform distribution. Three scenarios were analysed: a progeny with (1) a single putative parent; (2) two putative parents; and (3) one actual parent and one putative parent. Exclusion probabilities were computed for minimum values for the MAFs of 0.1, 0.2 and 0.3, and required either one or at least two conflicts for exclusion. The numbers of markers required to obtain 99% exclusion probabilities based on a single conflict for the three minimum MAFs were 54, 45 and 39 for scenario 1; 17, 16 and 15 for scenario 2; and 28, 25 and 24 for scenario 3. The requirement of at least two conflicts for exclusion increased the number of markers required by approximately 45% for all three scenarios and all three minimum MAFs. The results obtained by the analytical formulae were very close to results obtained by simulation and to values in the literature for specific marker sets.     

Validation of a microsatellite panel for parentage testing of locally adapted and commercial goats in Brazil

Brazilian goats are generally kept in small herds and extensive rearing systems, mainly in the northeastern region of the country. Despite production improvement in recent years, the lack of pedigree control has affected genetic progress. This study aimed to validate a panel of 16 microsatellites for parentage testing in locally adapted and commercial goats breeds raised in Brazil, as well as to compare its efficiency with the panel recommended by the Brazilian Ministry of Agriculture, Livestock and Supplies (MAPA) in 2004. The number of alleles and expected heterozygosity (He) per marker ranged from four to 18, and from 0.051 to 0.831, respectively. Using all markers, 100% of parentage cases of the validation dataset were resolved with a strict confidence level of 95%. The 16 microsatellites panel showed adequate exclusion power (99.99%) and identity accuracy (99.99%). Suggestions for improvement of the marker panel endorsed by MAPA are provided.     

Novel polymorphic microsatellite markers in section Caulorrhizae (Arachis,Fabaceae)

This work reports the characterization of 11 polymorphic microsatellite loci in section Caulorrhizae. The primer pairs were designed from Arachis pintoi and showed full transferability to Arachis repens species. These new markers were used to evaluate the genetic diversity in germplasm (accessions and cultivars) of section Caulorrhizae. This new set of markers detected greater gene diversity than morphological and molecular markers such as AFLP (amplified fragment length polymorphism) and RAPD (rapid analysis of polymorphic DNA) previously used in this germplasm.     

Highly polymorphic microsatellite markers for Radix balthica (Linnaeus 1758)

We present data for eight polymorphic microsatellite markers isolated from a microsatellite-enriched DNA library for the freshwater snail Radix balthica. Three of them were specific for R. balthica while five also amplified polymorphic products in two congeneric species. Test application on populations from all over the species range has shown that these loci are highly informative for analysing population structure and estimating migration rates. Observed deviations from Hardy-Weinberg equilibrium are attributed to a mixed mating system.     

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.     

Developing 23 new polymorphic microsatellite markers and simulating parentage assignment in the Pacific bluefin tuna, Thunnus orientalis

Twenty-three new polymorphic microsatellite markers were isolated in the Pacific bluefin tuna, Thunnus orientalis. Each locus comprised three to 34 alleles. The expected and observed heterozygosities ranged between 0.46 and 0.96 and between 0.44 and 0.97, respectively. The Kto9, Kto11, and Kto42 markers demonstrated significant deviation from Hardy-Weinberg equilibrium; high null allele frequencies (0.08-0.14) were observed in the deviating group. From the results of simulation of parentage assignment, a combination of four loci (i.e. Kto15, Kto23, Kto38, and Kto39) was considered the best for parentage assignment.     

Characterization of 10 microsatellite markers for the understorey Amazonian herb Heliconia acuminata

We characterized 10 microsatellite loci for the plant Heliconia acuminata from the Biological Dynamics of Forest Fragments Project (Manaus, Brazil). Markers were screened in 61 individuals from one population and were found to be polymorphic with an average of eight alleles per locus. We found moderate to high levels of polymorphic information content, and observed and expected heterozygosities. All 10 markers are suitable for spatial genetic structure and parentage analyses and will be used for understanding H. acuminata dynamics across a fragmented landscape.     

Isolation and characterization of microsatellite DNA markers in the Pacific abalone,Haliotis discus hannai

We developed 17 new microsatellite markers in Haliotis discus hannai. All loci were found to be polymorphic with an average of 13.1 alleles per locus (range 3–28). The mean observed and expected heterozygosities were 0.77 (range 0.17–1.00) and 0.79 (range 0.42–0.96), respectively. Six loci deviated significantly from Hardy–Weinberg proportions, and thus should be used with caution. The high variabilities revealed in this study suggest that these microsatellite loci should provide useful markers for studies of trait mapping, kinship and population genetics.     

New microsatellite markers for the snow crab Chionoecetes opilio (Brachyura: Majidae)

Nine novel microsatellite loci were isolated from Chionoecetes opilio by screening an enriched genomic library using nonradioactive polymerase chain reaction techniques, and the polymorphisms were examined to estimate genetic variability. The genetic variabilities varied depending on the locus. All loci were found to be polymorphic with an average of 9.7 alleles per locus (range 3–25). The observed and expected heterozygosities ranged from 0.80 to 0.98 and from 0.56 to 0.95, respectively. Five loci showed significant Hardy–Weinberg disequilibrium at the P < 0.05 level. The high variabilities revealed in this study suggest that these microsatellite loci should provide useful markers for genetic variation monitoring of C. opilio.     

Comparison of protein markers and microsatellites in differentiation of cattle populations

Five cattle populations, representing four breeds, were analysed for 14 protein markers and five microsatellite loci. The breeds studied were Brown Swiss and three autochthonous Spanish cattle: Avileña-Negra Ibérica (A-NI), two populations (A-NI 1 and A-NI 2) from different, reproductively isolated, locations; Sayaguesa; and Morucha. A total of 752 animals were examined for biochemical polymorphisms, of which 488 were also DNA typed. Genetic parameters and phylogenetic trees were obtained separately for each group of markers and results were compared. Estimates of heterozygosity and genetic distances from microsatellites were greater than those obtained using protein markers. The overall topology of the two dendrograms was similar. A-NI 1 and A-NI 2 populations were grouped together, related to Morucha, and the three of them related to Sayaguesa. Brown Swiss appeared in a separate branch from Spanish cattle. These results support the usefulness of microsatellites in the study of genetic relationships among closely related populations and breeds.     

Isolation of 10 microsatellite markers for mongoose lemurs (Eulemur mongoz)

We present here 10 new microsatellite markers for the mongoose lemur (Eulemur mongoz), nine of which were isolated from E. mongoz and one from Lemur catta. At least 60 individuals were genotyped for each of the 10 loci. Mendelian inheritance at each locus was tested by genotyping five captive families with known pedigrees. All loci were polymorphic and demonstrated simple Mendelian inheritance. The microsatellite markers presented here will be useful for genetic surveys of wild E. mongoz, to gain insights into the genetic background of the captive population, and to aid in the conservation of the species’ genetic diversity.     

Parentage testing in alpacas (Vicugna pacos) using semi-automated fluorescent multiplex PCRs with 10 microsatellite markers

The aim of this study was to assess and apply a microsatellite multiplex system for parentage determination in alpacas. An approach for parentage testing based on 10 microsatellites was evaluated in a population of 329 unrelated alpacas from different geographical zones in Perú. All microsatellite markers, which amplified in two multiplex reactions, were highly polymorphic with a mean of 14.5 alleles per locus (six to 28 alleles per locus) and an average expected heterozygosity ( H _E) of 0.8185 (range of 0.698–0.946). The total parentage exclusion probability was 0.999456 for excluding a candidate parent from parentage of an arbitrary offspring, given only the genotype of the offspring, and 0.999991 for excluding a candidate parent from parentage of an arbitrary offspring, given the genotype of the offspring and the other parent. In a case test of parentage assignment, the microsatellite panel assigned 38 (from 45 cases) offspring parentage to 10 sires with LOD scores ranging from 2.19 × 10⁺¹³ to 1.34 × 10⁺¹⁵ and Δ values ranging from 2.80 × 10⁺¹² to 1.34 × 10⁺¹⁵ with an estimated pedigree error rate of 15.5%. The performance of this multiplex panel of markers suggests that it will be useful in parentage testing of alpacas.