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.     

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.     

Estimating levels of inbreeding using AFLP markers

In the absence of detailed pedigree records, researchers have attempted to estimate individuals' levels of inbreeding using molecular markers, generally making use of heterozygosity measures based on microsatellite markers. Here we report and validate a method for estimating an individual's inbreeding coefficient, f, using amplified fragment length polymorphism (AFLP) markers. We use simulations to confirm that our measure scales appropriately with f when allele frequencies can be estimated from a subset of outbred individuals. We also present an approach for obtaining satisfactory estimates even in the absence of an independent set of known outbred individuals from which to estimate allele frequencies. We then test our method against empirical data from 179 wild and captive-bred old-field mice, Peromyscus polionotus subgriseus, comprising pedigree-based estimates of f, along with genetic data from 94 AFLP markers and 12 microsatellites. Inbreeding estimates based on both AFLP and microsatellite markers were found to correlate strongly with pedigree-based inbreeding coefficients. Owing to their ease of amplification in any species, AFLP markers may prove to be a valuable new tool for estimating f in natural populations and for examining correlations between heterozygosity and fitness.     

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.     

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.     

中国对虾微卫星家系鉴定的模拟分析与应用

本研究基于中国对虾群体所获的微卫星标记等位基因频率进行了计算机模拟分析,并选择5个微卫星标记,就单独养殖家系群体微卫星标记家系鉴定的准确性及混养家系群体微卫星标记家系鉴定的应用价值做了研究.模拟分析表明4个微卫星标记可以鉴定95%的后裔.而单独养殖的家系鉴定准确率达到92.9%,在30个可能的父母对,215尾中国对虾组成的混养家系群体中,90.7%的后裔可以鉴定其父母.本研究结果表明微卫星分子标记可以应用于中国对虾的家系鉴定.模拟分析与实际应用的差异及父母与子代间的错配部分原因是由于无效等位基因的出现,基因分型错误也是一个重要原因.基于父母LOD值的分析可以降低错配的几率.     

EXAMINATION OF POPULATION STRUCTURE IN RED-COCKADED WOODPECKERS USING DNA PROFILES

The degree to which DNA similarity is related to kinship and population structure in natural populations was investigated for a small population of cooperatively-breeding Red-cockaded Woodpeckers (Picoides borealis) in the western Piedmont region of South Carolina. An independent pedigree was established from records of color-banded individuals. Results of DNA profiles were then examined relative to this pedigree. DNA similarity among unrelated woodpeckers averaged 0.55 ± 0.01 (SE). The mean number of DNA bands scored and similarity did not significantly differ between founders and the current population. Examination of parentage in 10 families indicated that multiple paternity did not occur when band by band comparisons or similarity values were compared among parents, helpers, and offspring. Thus, Red-cockaded Woodpeckers were monogamous in this population. DNA similarity among all individuals ranged from 0.32-0.78. Distribution of these similarity values by kinship resulted in some overlap with other kin values. Therefore, specific similarity values could not be assigned a kinship value without knowledge of the pedigree. However, least-squares linear regression indicated that similarity was significantly related to kinship (P < 0.05). These results indicate that use of DNA profiles may be important in quantifying population structure, however, they must be used in conjunction with a known pedigree before any assessment of kinship among individuals is made. Band by band comparisons remain a viable technique for examination of parentage when all putative parents have been sampled.     

A standardized microsatellite marker panel for parentage and kinship analyses in channel catfish,Ictalurus punctatus

This research was designed to produce a standardized set of microsatellite loci for parentage and kinship analyses in channel catfish, the leading species of US aquaculture. Three panels of five to six markers each were developed that contained a total of two dinucleotide‐, eight trinucleotide‐ and seven tetranucleotide‐microsatellite loci respectively. The loci had a range of nine to 31 alleles per locus in an outbred population. Based on the allele frequencies measured in commercial randomly bred broodstock, the combined probability of non‐exclusion of an unrelated candidate parent pair was 5.36e‐18. The combined probability of non‐exclusion of unrelated identical genotypes was 2.58e‐08. The microsatellite panels were validated by parentage and kinship evaluation in three populations. A total of 697 spawns were collected from matings of outbred broodstock over three spawning seasons, and parents were determined unambiguously for all but three spawns. Genotype analysis also enabled the identification of half‐sibling and full‐sibling families produced by pond spawning. In a second experiment, parentage was unambiguously determined in nine spawns from a population consisting of broodstock derived from only four families. A third experiment demonstrated that all but one of 374 individuals from 10 full‐sibling families could be assigned to a family after coculture in an earthen pond for 1 year. The standardized microsatellite panels enable the development of pedigreed catfish populations and large‐scale performance evaluations in common environments to support the genetic improvement of cultured catfish through selective breeding.     

Microsatellite DNA in fishes

For the last 30 years, attempts have been made to discriminate among fish populations by using molecular markers. Although some techniques have proved successful in certain circumstances, the consistent trend to newer markers among fishery geneticists highlights the general lack of resolving power observed with older technologies. The last decade has seen the increasing use of satellite DNA in investigations of genetic variability and divergence. Applications to fish and fisheries-related issues initially concentrated on minisatellite single-locus probes. Although minisatellites have successfully addressed a number of fishery-related questions, this class of satellite DNA has not been widely adopted by fishery geneticists. Most of the current research effort is concentrated on another class of satellite DNA called microsatellites. The large interest in microsatellite loci is largely due to the very high levels of variability that have been observed and the ability to investigate this variation using PCR technology. The isolation and application of microsatellites to research fields as diverse as population genetics, parentage analyses and genome mapping are reviewed. Despite the undisputed advantages that the marker possesses, there are a number of potential problems associated with investigating variation at microsatellite loci. Statistical considerations (e.g. appropriate sample sizes, number of loci and the mutation model assumptions on which the estimate is based) have not been considered in detail yet and the problems are often exacerbated in fish species, as some species show very large numbers of alleles at microsatellite loci. These issues and others, e.g. null alleles, are reviewed and possible solutions are proposed     

Analysis of microsatellites and parentage testing in saltwater crocodiles

Fifteen microsatellite loci were evaluated in farmed saltwater crocodiles for use in parentage testing. One marker (C391) could not be amplied. For the remaining 14, the number of alleles per locus ranged from two to 16, and the observed heterozygosities ranged from 0.219 to 0.875. The cumulative exclusion probability for all 14 loci was .9988. the 11 loci that showed the greatest level of polymorphism were used for parentage testing, with an exclusion probability of .9980. With these 11 markers on 107 juveniles from 16 known breeding pairs, a 5.6% pedigree error rate was detected. This level of pedigree error, if consistent, could have an impact on the accuracy of gentic parameter and breeding value estimation. The usefulness of these markers was also evaluated for assigning parentage in situations where maternity, paternity, or both may not be known. In these situations, a 2% error in parentage assignment was predicted. It is therefore recommended that more micro-satellite markers be used in these situations. The use of these microsatellite markers will broaden the scope of a breeding program, allowing progeny to be tested from adults maintained in large breeding lagoons for selection as future breeding animals.     

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.     

Genetic base of tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis> L.) cultivars in Sri Lanka as revealed by pedigree analysis

An understanding of genetic diversity and relationships among breeding materials is a prerequisite for crop improvement. Coefficient of parentage (COP) can be used to measure the genetic diversity among genotypes on the basis of pedigree information. In the present study, COP was estimated for 56 cultivars, including commercial tea cultivars developed by the Tea Research Institute of Sri Lanka and their parental lines. Mean COP of the 56 accessions studied was 0.097 and the value was raised up to 0.272 when non-related pair-wise comparisons were excluded. A single cultivar (Assam/Cambod introduction) was the nucleus of the commercial cultivars. Group mean COP of the cultivars derived from Assam/Cambod parentage was 0.17. Thirty-three percent of the pair-wise comparisons had 0.00 COP, highlighting that many cultivars were unrelated. Within the pedigree, 2 major COP clusters were identified: Assam/Cambod open-pollinated half-sib progenies, and full-sib progenies derived from crosses between Assam/Cambod and other parental lines. The elite groups within the pedigree, where Assam/Cambod parentage was concentrated, were also identified. Information generated in this study should be useful for effective utilization of available diversity in future breeding programmes as well as for proper conservation of genetic diversity in the adapted germplasm. This is the first report on estimates of genetic diversity based on COP in a woody perennial crop, such as tea.     

Non-Mendelian transmission of alleles at microsatellite loci: an example in Ixodes ricinus, the vector of Lyme disease

Microsatellite loci are usually considered to be neutral co-dominant and Mendelian markers. We undertook to study the inheritance of five microsatellite loci in the European Lyme disease vector, the tick Ixodes ricinus. Only two loci appeared fully Mendelian while the three others displayed non-Mendelian patterns that highly frequent null alleles could not fully explain. At one locus, IR27, some phenomenon seems to hinder the PCR amplification of one allele, depending on its origin (maternal imprinting) and/or its size (short allele dominance). DNA methylation, which appeared to be a possible explanation of this amplification bias, was rejected by a specific test comparing the amplification efficiency that did not differ between unmethylated and experimentally methylated DNA. The role of allele size in heterozygous individuals was then revealed from the data available on field collected ticks and consistent with the results of a theoretical approach. These observations highlight the need for prudence while inferring reproductive systems (selfing rates), parentage or even allelic frequencies from microsatellite markers, in particular for parasitic organisms for which molecular approaches often represent the only way for population biology inferences.     

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.     

Evaluation of DNA pooling for the estimation of microsatellite allele frequencies: a case study using striped bass (Morone saxatilis)

Using striped bass (Morone saxatilis) and six multiplexed microsatellite markers, we evaluated procedures for estimating allele frequencies by pooling DNA from multiple individuals, a method suggested as cost-effective relative to individual genotyping. Using moment-based estimators, we estimated allele frequencies in experimental DNA pools and found that the three primary laboratory steps, DNA quantitation and pooling, PCR amplification, and electrophoresis, accounted for 23, 48, and 29%, respectively, of the technical variance of estimates in pools containing DNA from 2-24 individuals. Exact allele-frequency estimates could be made for pools of sizes 2-8, depending on the locus, by using an integer-valued estimator. Larger pools of size 12 and 24 tended to yield biased estimates; however, replicates of these estimates detected allele frequency differences among pools with different allelic compositions. We also derive an unbiased estimator of Hardy-Weinberg disequilibrium coefficients that uses multiple DNA pools and analyze the cost-efficiency of DNA pooling. DNA pooling yields the most potential cost savings when a large number of loci are employed using a large number of individuals, a situation becoming increasingly common as microsatellite loci are developed in increasing numbers of taxa.     

Assignment of the gene for porcine insulin-like growth factor 1 (IGF1) to chromosome 5 by linkage mapping

Investigation of published sequence data from the porcine insulin-like growth factor 1 (IGF1) gene, resulted in the detection of a microsatellite in the first intron of the gene. Polymerase chain reaction (PCR) primers flanking the (CA)₁₉ repeat were constructed. Polymorphism and Mendelian segregation were documented in a three-generation pedigree and allele frequencies were determined in 74 unrelated animals from four different breeds. Seven alleles were encountered. Linkage analysis was performed in a large pedigree established for gene mapping. Linkage between the IGF1 microsatellite and an anonymous microsatellite marker, S0005, was detected. Furthermore, IGF1 and S0005 was found to be linked to the porcine submaxillary gland mucin (MUC) gene, previously assigned to chromosome 5. The results presented here extend the linkage group on pig chromosome 5 and are in accordance with conserved synteny between human chromosome 12, cattle chromosome 5, mouse chromosome 10 and pig chromosome 5.     

Microsatellite analysis as a tool for discriminating an interfamily hybrid between olive flounder and starry flounder

An interspecific artificial hybrid was produced between two economically important aquaculture flatfish: olive flounder (Paralichthys olivaceus) and starry flounder (P. stellatus). This hybrid displays the rapid growth characteristic of the former and tolerance to low temperatures and low salinity of the latter, but the genetics of inheritance in this hybrid have not been elucidated. Polymorphic microsatellite markers developed for P. olivaceus and P. stellatus were tested to determine if these markers can be used for analysis of parentage and genetic inheritance. Multiplex PCR using two primer sets that were specific to each species produced PCR products of different sizes; these could be used for the identification of interspecific hybrids. Among the 192 primers derived from olive flounder, 25.5% of the primer sets successfully amplified genomic DNA from starry flounder, and 23% of the 56 primer sets originating from starry flounder amplified DNA from olive flounder. Analysis of genetic inheritance in the hybrid using seven of the 62 microsatellite markers common to both species demonstrated classic Mendelian inheritance of these markers in the hybrid progeny, with the exception of one locus identified as a null allele in the hybrid. These results demonstrate that cross-specific microsatellite markers can be used tools for parentage analysis of hybrid flatfish, for mapping quantitative trait loci, for marker-assisted selective breeding, and for studies of the evolution of fish.     

Genetic relationships among wheat genotypes, as revealed by microsatellite markers and pedigree analysis

Genetic relationships among 20 elite wheat genotypes were studied using microsatellite markers and pedigree analysis. A total of 93 polymorphic bands were obtained with 25 microsatellite primer pairs. Coefficient of parentage (COP) values were calculated using parentage information at the expansion level of 5. The pedigree-based similarity (mean 0.115, range 0.00-0.53) was lower than the similarity assessed using microsatellite markers (mean 0.70, range 0.47-0.91). Similarity estimates were used to construct dendrograms by using the unweighted pair-group method with arithmetic averages (UPGMA). Clustering of genotypes in respect of marker-based similarity revealed two groups. Genotype PBW442 diverged and appeared as distinct from all other genotypes in both marker-based and pedigree-based analysis. The correlation of COP values with genetic similarity values based on microsatellite markers is low (r = 0.285, p < 0.05). The results indicate a need to develop wheat varieties with a diverse genetic background and to incorporate new variability into the existing wheat gene pool.     

Estimation and adjustment of microsatellite null alleles in nonequilibrium populations

Nonamplified (null) alleles are a common feature of microsatellite genotyping and can bias estimates of allele and genotype frequencies, thereby hindering population genetic analyses. The frequency of microsatellite null alleles in diploid populations can be estimated for populations that are in Hardy–Weinberg equilibrium. However, many microsatellite data sets are from nonequilibrium populations, often with known inbreeding coefficients (F) or fixation indices (F_IS or F_ST). Here, we propose a novel null allele estimator that can be used to estimate the null allele frequency and adjust visible allele frequencies in populations for which independent estimates of F, F_IS or F_ST are available. The algorithm is currently available as an Excel macro that can be downloaded at no cost from http://www.microchecker.hull.ac.uk/ and will be incorporated into the software micro ‐checker .