DNA-based parentage verification in two Australian goat herds

This study aimed to evaluate a set of DNA markers for their effectiveness in parentage inference, to quantify the level of pedigree errors in Australian Angora and Cashmere goat herds using different pedigree recording methods, and to investigate genotype mismatches between parent and offspring. The 14 microsatellite markers evaluated in this study provided a high level of power (probability of exclusion, PE >99.70%) for parentage testing. The extent of PE depended on polymorphic information content (PIC) and number of alleles for each marker. The minimum number of MS markers essential for accurate determination of parentage was 12, when neither parent is known (PE1) and 10, when one parent is known (PE2). In both populations, the error rates of recorded sire and dam pedigree were significant, averaging around 12%. The error rates of sire and dam pedigree varied considerably between the two populations, reflecting management differences on the two properties. Of 14 MS markers, one locus, SRCRSP07, had null alleles present in the heterozygous state. This null allele was revealed by mismatches of genotypes of parent-offspring pairs. Highly significant deviation from Hardy–Weinberg Equilibrium and significant heterozygote deficiency was also observed at this locus.     

Characterization of new microsatellite loci in Pieris amamioshimensis (Ericaceae), a species nearly extinct in the wild

A set of 11 polymorphic microsatellite markers has been developed and characterized for the critically endangered species Pieris amamioshimensis. Fifty‐nine individuals of an ex‐situ population were used to identify these markers. The total number of alleles for each locus ranged from 3 to 9, with an average of 5.4. The expected heterozygosities (H_S) and observed heterozygosities (H_O) ranged from 0.47 to 0.77 and 0.22 to 0.88, respectively. In total, four loci exhibited significant deviations from Hardy–Weinberg equilibrium: two loci showed significant heterozygosity excess and the other two loci showed significant heterozygosity deficit. The polymorphism information content (0.43 ≤ PIC ≤ 0.73), the probability of exclusions (PE₁ = 0.9565, PE₂ = 0.9969 and PE₃ = 0.9999) and probabilities for identity (PI = 3.78 × 10^?9 and PI‐Sib = 2.35 × 10^?4) suggest that these markers are useful for estimating not only genetic diversity but also parentage, for the ex‐situ conservation management of populations.     

Effects of genotyping errors on parentage exclusion analysis

Genetic markers are widely used to determine the parentage of individuals in studies of mating systems, reproductive success, dispersals, quantitative genetic parameters and in the management of conservation populations. These markers are, however, imperfect for parentage analyses because of the presence of genotyping errors and undetectable alleles, which may cause incompatible genotypes (mismatches) between parents and offspring and thus result in false exclusions of true parentage. Highly polymorphic markers widely used in parentage analyses, such as microsatellites, are especially prone to genotyping errors. In this investigation, I derived the probabilities of excluding a random (related) individual from parentage and the probabilities of Mendelian-inconsistent errors (mismatches) and Mendelian-consistent errors (which do not cause mismatches) in parent-offspring dyads, when a marker having null alleles, allelic dropouts and false alleles is used in a parentage analysis. These probabilities are useful in evaluating the impact of various types of genotyping errors on the information content of a set of markers in and thus the power of a parentage analysis, in determining the threshold number of genetic mismatches that is appropriate for a parentage exclusion analysis and in estimating the rates of genotyping errors and frequencies of null alleles from observed mismatches between known parent-offspring dyads. These applications are demonstrated by numerical examples using both hypothetical and empirical data sets and discussed in the context of practical parentage exclusion analyses.     

Population estimators or progeny tests: what is the best method to assess null allele frequencies at SSR loci?

Nuclear SSRs are notorious for having relatively high frequencies of null alleles, i.e. alleles that fail to amplify and are thus recessive and undetected in heterozygotes. In this paper, we compare two kinds of approaches for estimating null allele frequencies at seven nuclear microsatellite markers in three French Fagus sylvatica populations: (1) maximum likelihood methods that compare observed and expected homozygote frequencies in the population under the assumption of Hardy-Weinberg equilibrium and (2) direct null allele frequency estimates from progeny where parent genotypes are known. We show that null allele frequencies are high in F. sylvatica (7.0% on average with the population method, 5.1% with the progeny method), and that estimates are consistent between the two approaches, especially when the number of sampled maternal half-sib progeny arrays is large. With null allele frequencies ranging between 5% and 8% on average across loci, population genetic parameters such as genetic differentiation (F _ST) may be mostly unbiased. However, using markers with such average prevalence of null alleles (up to 15% for some loci) can be seriously misleading in fine scale population studies and parentage analysis.     

Development of polymorphic microsatellite markers for the zebra finch (Taeniopygia guttata)

To study the population genetics as well as the mating system of captive zebra finch (Taeniopygia guttata) populations, we developed primers for 12 microsatellite loci and screened them in 529 individuals from two successive generations of a single captive population. All markers were polymorphic with five to 14 alleles per locus. We checked all markers for Mendelian inheritance in 307 offspring whose parents were known for sure. Four markers showed evidence for the presence of null alleles. Once allowing for null alleles, we found no mismatches between offspring and parents, suggesting a very low rate of mutation. Average observed and expected heterozygosities across the eight loci showing no evidence for null‐alleles was 0.819 and 0.812, respectively.     

Isolation and characterization of 149 novel microsatellite DNA markers for striped bass,Morone saxatilis,and cross‐species amplification in white bass,Morone chrysops,and their hybrid

To support detailed genetic analysis of striped bass (Morone saxatilis) and white bass (Morone chrysops), we isolated 153 microsatellite loci from repeat‐enriched striped bass DNA libraries. Of these, 147 markers amplified in striped bass (average 4.7 alleles per locus) and 133 in white bass (average 2.2 alleles per locus). One hundred twenty‐two markers amplified in their hybrid. Development of new microsatellite markers will facilitate evaluations of genetic structure in wild populations and will support pedigree analysis and linkage mapping for selective breeding.     

Development and diversity of microsatellite markers for endangered species Clinostigma savoryanum

A set of microsatellite markers was developed for Clinostigma savoryanum, an endemic palm species distributed in the Bonin Islands. We obtained 233 sequences that were unique, containing microsatellites from an enriched library. Twelve loci were screened for their feasibility to be used as high resolution genetic markers using each 30 individuals from two insular populations, Haha-jima and Mukou-jima. They showed polymorphisms of two to eight alleles per locus and expected heterozygosities of 0.124–0.789. There is no evidence for significant scoring error due to stuttering, large allele dropout and null alleles at 95% confidence interval except for the presence of null alleles in CLS00-77 of Mukou-jima population.     

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.     

Development of 81 new polymorphic EST-derived microsatellite markers for the olive flounder,Paralichthys olivaceus

Expressed sequence tags (ESTs) can be used to identify microsatellite markers. We developed 81 polymorphic microsatellite markers from 4,940 ESTs of the olive flounder, Paralichthys olivaceus. Out of 100 EST-derived microsatellites for which PCR primers were designed, 81 loci were polymorphic in 30 individuals from a single natural population with 2–28 (mean 10.6) alleles per locus. The observed and expected heterozygosities of these loci were 0.033–1.000 and 0.033–0.965, respectively. Segregation analysis within a mapping family revealed non-amplifying null alleles at five loci. These new EST-derived microsatellite markers should be useful for population genetic analyses, pedigree tracing and constructing a linkage map for olive flounder.     

Microsatellite markers for the whelk Buccinum undatum

Five microsatellite loci are described for the commercially exploited marine gastropod, Buccinum undatum. Levels of polymorphism were variable with three to 19 alleles per locus and expected heterozygosities of 0.26–0.94 in 60 individuals of the population from which the loci were isolated. Homozygote excess at two of the loci might be attributable to null alleles, and these loci should not be used in, for example, parentage analysis. Nevertheless, because null allele frequencies can be estimated and their effects partitioned, all are useful markers for studies of population differentiation.     

Parentage testing of racing camels (Camelus dromedarius) using microsatellite DNA typing

The camel racing industry would have added value in being able to assign parentage with high certainty. This study was aimed at assessing and applying microsatellite multiplexes to construct a parentage testing system for camels. An efficient system of 17 loci from 700 camel samples was used to construct a database of unrelated adults. Based on this, we estimated measures of polymorphism among the markers. In three multiplex reactions, we detected a total of 224 alleles, with 5–23 alleles/locus (mean = 13.18 ± 6.95 SD) and an average heterozygosity (H_E) of 0.54 (range 0.032–0.905). The total parentage exclusion probability was 0.99999 for excluding a candidate parent from parentage of an arbitrary offspring, given only the genotype of the offspring, and 0.9999 for excluding a candidate parent from parentage of an arbitrary offspring, given the genotype of the offspring and the other parent. We used 15 juveniles for parentage testing, as well as 17 sires (bull camels) and 21 dams (cows). In the case of parentage assignment, the microsatellite panel assigned all 15 offspring parentage with high confidence. Overall, these findings offer a set of microsatellite markers that are easy, simple and highly informative for parentage testing in camels.     

Inheritance of Microsatellite DNA Markers in the Pacific Abalone <Emphasis Type="Italic">Haliotis discus hannai</Emphasis>

Microsatellite markers have been developed for a variety of abalones, and locus-specific homozygote excesses at population level have been recorded for microsatellite loci. To ascertain whether null alleles exist at microsatellite loci in the Pacific abalone, we studied the mode of inheritance of 7 microsatellite loci in 4 families with a reciprocal cross of 2 females × 2 males. All loci segregated codominantly, but only 3 loci (Hdh1321, Hdh78, and Hdd108C) conformed to Mendelian segregation and can be used for parental analysis and population genetic studies. When null alleles were considered, 2 loci (Hdh1761 and Hdh1457) confirmed Mendelian expectations in all families, while the remaining 2 loci (Hdd114B and Hdd229) showed deviation from Mendelian segregation in at least one family even though null alleles were considered. These results indicated the need to test the inheritance pattern for microsatellite markers in abalones before using them for population genetic or parentage analysis.     

Sixteen novel microsatellite loci developed for the giant panda (Ailuropoda melanoleuca)

Sixteen novel microsatellite DNA loci were developed from the giant panda (Ailuropoda melanoleuca) using a magnetic-bead capture method. A total of 115 alleles were obtained for these markers, ranging from 4 to 12 alleles per locus (average 7.188). These loci exhibited high levels of polymorphic information content and expected heterozygosity, 0.558–0.855 (average 0.729) and 0.628–0.885 (average 0.778), respectively. Therefore, the allelic polymorphism and heterozygosity show that the giant pandas raised in China Research and Conservation Center possess abundant genetic variation. In addition, if the three markers showing null alleles were excluded, the remaining 13 microsatellite loci still presented extremely low non-exclusion probabilities of parentage (0.002), paternity (0.000) and identity (0.000). As a result, this new suit of microsatellite markers would be a very informative tool for the genetic and conservation studies of giant pandas.     

An evaluation of the International Society for Animal Genetics recommended parentage and identification panel for the domestic pigeon (Columba livia domestica)

In this study, the International Society for Animal Genetics (ISAG) recommended panel for the identification of the domestic pigeon (Columba livia domestica) is characterized based on commonly used statistical parameters. The marker panel is based on 16 short tandem repeat (STR) loci (PIGN15, PIGN10, PIGN57, PIGN26, CliμD16, CliμD19, PIGN12, CliμD17, CliμT17, PIGN04, CliμD01, CliμD11, CliμD35, CliμT02, CliμT13, CliμT43). The alleles of the 16 loci consist of a mixture of tri‐, tetra‐, penta‐ and hexameric repeat patterns. A sex determination marker was included in the multiplex for quality control. The repeat sequence of the PIGN markers was previously unpublished and therefore sequenced to reveal the sequence pattern. In total, 1421 pigeons were genotyped on 16 STR loci to generate allele frequency data for each locus. For all 16 markers combined, a PE1 (combined non‐exclusion probability, first parent) of 0.9986 and PE2 (combined non‐exclusion probability, second parent) of >0.9999 was observed. Comparing the alleged father and mother, a PE value of >0.9999 was observed. Two of the markers, CliμD19 and PIGN12, were found to have relatively high Hardy–Weinberg equilibrium and F(null) values. Therefore these markers may be considered to be replaced by other STRs. Another point of discussion may be to add a gender identification marker to the recommended ISAG panel. Not only can this serve as an extra identification marker, but this can also confirm the sex of a sample, because it is challenging to determine the sex based on phenotypical characteristics, especially for chicks. In conclusion, the set of 16 STR markers can be used in routine parentage verification and the identification of individuals.     

Characterization of 29 polymorphic artiodactyl microsatellite markers for the mountain goat (Oreamnos americanus)

We report the results of a cross‐species amplification test of 156 bovine, ovine and cervid microsatellite markers in a wild population of mountain goats, Oreamnos americanus, inhabiting Caw Ridge, Alberta, Canada. Twenty‐nine markers were found to be low to moderately polymorphic with between two to nine alleles per locus. Observed heterozygosity ranged from 0.14 to 0.85 for a sample of 215 mountain goats. This set of markers will be used in parentage analyses to construct the pedigree of the long‐term studied population and to investigate the effects of individual genetic variability on life‐history traits.     

Twelve novel microsatellite markers for the Great Plains toad,Bufo cognatus

Here I report on 12 microsatellite loci designed for populations of the Great Plains toad (Bufo cognatus) in the deserts of the southwestern USA. Diversity at these loci measured for 134 individuals from four breeding aggregations was relatively high with seven to 34 alleles per locus (mean = 17.8). Observed heterozygosities ranged from 0.444 to 0.949 and expected heterozygosities ranged from 0.597 to 0.951. These markers will be useful for studies of population genetic structure, parentage and relatedness in this explosively breeding amphibian.     

Implementation of a parentage control system in Portuguese beef-cattle with a panel of microsatellite markers

A study was conducted to assess the feasibility of applying a panel of 10 microsatellite markers in parentage control of beef cattle in Portugal. In the first stage, DNA samples were collected from 475 randomly selected animals of the Charolais, Limousin and Preta breeds. Across breeds and genetic markers, means for average number of alleles, effective number of alleles, expected heterozygosity and polymorphic information content, were 8.20, 4.43, 0.733 and 0.70, respectively. Enlightenment from the various markers differed among breeds, but the set of 10 markers resulted in a combined probability above 0.9995 in the ability to exclude a random putative parent. The marker-set thus developed was later used for parentage control in a group of 140 calves from several breeds, where there was the suspicion of possible faulty parentage recording. Overall, 76.4% of the calves in this group were compatible with the recorded parents, with most incompatibilities due to misidentification of the dam. Efforts must be made to improve the quality of pedigree information, with particular emphasis on information recorded at the calf's birth.     

Development and characterization of 12 compound microsatellite markers in <Emphasis Type="Italic">Platypus </Emphasis><Emphasis Type="Italic">quercivorus</Emphasis> (Murayama) (Coleoptera: Platypodidae)

Twelve compound microsatellite loci were isolated from the ambrosia beetle Platypus quercivorus. Every locus was polymorphic among 50 individuals from two localities, with two to six alleles per locus, without linkage disequilibrium. The observed and expected heterozygosities ranged from 0.140 to 0.740 and from 0.255 to 0.731, respectively. These markers will be available for population genetic studies and parentage analysis of this beetle.     

Assessment of microsatellite and SNP markers for parentage assignment in ex situ African Penguin (Spheniscus demersus) populations

Captive management of ex situ populations of endangered species is traditionally based on pedigree information derived from studbook data. However, molecular methods could provide a powerful set of complementary tools to verify studbook records and also contribute to improving the understanding of the genetic status of captive populations. Here, we compare the utility of single nucleotide polymorphisms (SNPs) and microsatellites (MS) and two analytical methods for assigning parentage in ten families of captive African penguins held in South African facilities. We found that SNPs performed better than microsatellites under both analytical frameworks, but a combination of all markers was most informative. A subset of combined SNP (n = 14) and MS loci (n = 10) provided robust assessments of parentage. Captive or supportive breeding programs will play an important role in future African penguin conservation efforts as a source of individuals for reintroduction. Cooperation among these captive facilities is essential to facilitate this process and improve management. This study provided us with a useful set of SNP and MS markers for parentage and relatedness testing among these captive populations. Further assessment of the utility of these markers over multiple (>3) generations and the incorporation of a larger variety of relationships among individuals (e.g., half‐siblings or cousins) is strongly suggested.