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.     

Effectiveness of bovine microsatellites in resolving paternity cases in American bison, Bison bison L.

A set of 33 cattle microsatellite primer pairs was tested with the DNA of American bison from a captive population in Belgium and evaluated for usefulness in parentage testing. Two primer sets did not amplify and three were monomorphic. Among the polymorphic markers, the number of alleles ranged from two to nine. Heterozygosity, polymorphism information content (PIC) and probability of exclusion (PE) values were low by comparison with those obtained with the same markers in cattle. Two methods of estimating PE were used, one which assumed equal allele frequencies between parental sexes and another which took into account differences in allele frequencies between parental sexes. An internationally accepted set of nine microsatellites gives cumulative PE values of 0·98 and 0·97, respectively, for the two methods. The potential of this marker set to identify bison × cattle hybrids is discussed. Because bison and cattle have a common ancestor, these microsatellites are a useful way to establish genetic distances and can lead to the construction of phylogenetic trees.     

Parentage assignment of the mud crab (Scylla paramamosain) based on microsatellite markers

In this study, microsatellite markers were employed to identify the parentage relationship in Scylla paramamosain. The exclusion probability of loci was found to be related with the level of their heterozygosity. When no parent information or only one parent information was available, the exclusion probability ranged from 22.0% to 56.6% and from 41.2% to 73.1%, with the combined exclusion probability for ten loci being 97.0% and 99.8%, respectively. The cumulative assignment success rate was 100% when no parent information was available using seven most informative microsatellite markers. Moreover, the power of the seven microsatellite markers for parentage assignment was tested by a double-blind test, which indicated that 95% of the progeny can be correctly assigned to their parents. This study provided a microsatellite-based approach for parentage assignment in S. paramamosain that will be useful for investigation of genetic background and molecular marker-assisted selective breeding in this important crab species.     

DNA microsatellite analysis for parentage control in Austrian pigs.

We present an efficient parentage control for pigs based on ten polymorphic microsatellite markers analyzed in a single PCR reaction. Assuming one known parent ("paternity control"), combined exclusion probabilities (CEPs) ranged from 99.18% (Landrace), 99.74% (Piétrain) to 99.76% (Large White) for the most important Austrian breeds. Assuming a known parent-pair ("parentage control", e.g. a substituted offspring), the CEP of the 10-plex PCR increased to 99.97% (Landrace) and 99.99% (Piétrain and Large White). We developed an additional standby battery of 5 markers, which might be applied in those cases, where the CEP of the 10-plex PCR is not sufficient. Therefore an automated, cost and time reduced genotype analysis for pigs is available.     

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.     

Eighteen novel polymorphic microsatellite loci developed from the Père David’s deer (<Emphasis Type="Italic">Elaphurus davidianus</Emphasis>)

Père David’s deer is a severely bottlenecked species but without showing inbreeding depression, making it essential to develop molecular markers to explore her genetic mechanism of population recovery. In this study, we isolated 18 novel polymorphic microsatellite loci from a dinucleotide-enriched library. This suit of markers presented 2–3 alleles for each locus and their observed and expected heterozygosities were 0.057–0.610 and 0.056–0.598, respectively. These new microsatellite loci had an average of 2.12 alleles and thus contributed to relatively low exclusion probabilities of parentage and paternity testing (0.768 and 0.921). However, when these loci were examined in combination with previous microsatellite markers, overall probabilities of parentage and paternity exclusion went up to 0.905 and 0.990, respectively, showing that these 26 microsatellite loci should be adopted together in future genetic analyses for this highly inbred species.     

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.     

Selection and implementation of single nucleotide polymorphism markers for parentage analysis in crossbred cattle population

Crossbreeding is an essential way of improving herd performance. However, frequent parentage record errors appear, which results in the lower accuracy of genetic parameter estimation and genetic evaluation. This study aims to build a single nucleotide polymorphism (SNP) panel with sufficient power for parentage testing in the crossbred population of Simmental and Holstein cattle. The direct sequencing technique in PCR products of pooling DNA along with matrix-assisted laser desorption/ionization time-of-flight MS method for genotyping the individuals was applied. A panel comprising 50 highly informative SNPs for parentage analysis was developed in the crossbred population. The average minor allele frequency for SNPs was 0.43, and the cumulative probability of exclusion for single-parent and both-parent inference met 0.99797 and 0.999999, respectively. The maker-set for parentage verification was then used in a group of 81 trios with aid of the likelihood-based parentage-assignment program of Cervus software. Reconfirmation with on-farm records showed that this 50-SNP system could provide sufficient and reliable information for parentage testing with the parental errors for mother–offspring and sire–offspring being 8.6 and 18.5%, respectively. In conclusion, a set of low-cost and efficient SNPs for the paternity testing in the Simmental and Holstein crossbred population are provided.     

Using blocks of linked single nucleotide polymorphisms as highly polymorphic genetic markers for parentage analysis

Single nucleotide polymorphisms (SNPs) are plentiful in most genomes and amenable to high throughput genotyping, but they are not yet popular for parentage or paternity analysis. The markers are bi-allelic, so individually they contain little information about parentage, and in nonmodel organisms the process of identifying large numbers of unlinked SNPs can be daunting. We explore the possibility of using blocks of between three and 26 linked SNPs as highly polymorphic molecular markers for reconstructing male genotypes in polyandrous organisms with moderate (five offspring) to large (25 offspring) clutches of offspring. Haplotypes are inferred for each block of linked SNPs using the programs Haplore and Phase 2.1. Each multi-SNP haplotype is then treated as a separate allele, producing a highly polymorphic, 'microsatellite-like' marker. A simulation study is performed using haplotype frequencies derived from empirical data sets from Drosophila melanogaster and Mus musculus populations. We find that the markers produced are competitive with microsatellite loci in terms of single parent exclusion probabilities, particularly when using six or more linked SNPs to form a haplotype. These markers contain only modest rates of missing data and genotyping or phasing errors and thus should be seriously considered as molecular markers for parentage analysis, particularly when the study is interested in the functional significance of polymorphisms across the genome.     

Polymorphic microsatellite loci for assigning parentage in least flycatchers (Empidonax minimus)

Least flycatchers (Empidonax minimus) are socially monogamous birds that form tight territorial aggregations on the breeding grounds. We designed five polymorphic microsatellite loci for assigning parentage to offspring within least flycatcher clusters. The number of alleles per locus ranged from 7 to 18. Mean polymorphic information content was 83.8%; the probability of exclusion with known maternal genotype was 0.999. These microsatellites are powerful DNA markers for identifying extra‐pair paternity in this species. Preliminary data also suggest that these loci may be useful for other members of this genus.     

Isolation and characterization of microsatellite loci in Acer opalus (Aceraceae), a sexually-polymorphic tree, through an enriched genomic library

An enrichment protocol was used to isolate and characterize microsatellite loci in Acer opalus, a Mediterranean tree species. Highly polymorphic microsatellite loci were required for paternity analyses in a population of this species. Eight microsatellite loci were amplified and a total of 87 alleles were detected in a sample of 142 individuals from one population, allowing the identification of each individual with a unique multilocus genotype. The paternity exclusion probabilities varied from 0.261 for locus Aop820 to 0.806 for locus Aop450, and the parent-pair exclusion probabilities varied from 0.433 for Aop820 to 0.940 for Aop450. The cumulative probabilities of exclusion for paternity and parentage of the eight loci were both higher than 0.999, supporting the usefulness of these microsatellite loci for future paternity and parentage analyses in A. opalus. Cross-species transferability was also assayed, supporting their potential use in other eight Acer species.     

中国美利奴(新疆军垦型)绵羊9个微卫星基因座多态性研究

利用PCR技术和复合电泳银染技术检测中国美利奴(新疆军垦型)绵羊第1号染色体上BM6506,BM1824,BM6438, ILSTS004和OarDB6 等5个基因座和第6号染色体上 BM4621,OarHH55,BM143和OarJMP8 等4个基因座,共9个基因座的基因频率（Pi）、个体鉴别力（DP）、杂合度（H）、多态信息含量（PIC）、和非父排除概率（PE）。结果显示：9个微卫星基因座的基因型分布符合Hardy-Weinberg平衡,绵羊中9个微卫星基因座中BM4621 基因座的DP、H、PIC和PE都为最高。9个微卫星基因座的累积个体鉴别力(CDP)为0.99999,累积非父排除能力(CPE)为0.99915。结果显示9个微卫星基因座适用于中国美利奴(新疆军垦型)绵羊的遗传连锁分析、个体识别和亲权鉴定等研究领域。     

Comparison of microsatellite and single nucleotide polymorphism markers for the genetic analysis of a Galloway cattle population

Highly informative genetic markers are essential for efficient management of cattle populations, as well as for food safety. After a decade of domination by microsatellite markers, a new type of genetic marker, single nucleotide polymorphism (SNP), has recently appeared on the scene. In the present study, the exclusion power of both kinds of markers with regards to individual identification and parental analysis was directly compared in a Galloway cattle population. Seventeen bovine microsatellites were distributed in three incremental marker sets (10, 14 and 17 microsatellite markers) and used for cattle genotyping. A set of 43 bovine SNP was used for genotyping the same cattle population. The accuracy of both kinds of markers in individual identification was evaluated using probability of identity estimations. These were 2.4 x 10(-8) for the 10 microsatellite set, 2.3 x 10(-11) for the 14 microsatellite set, and 1.4 x 10(-13) for the 17 microsatellite marker set. For the 43 SNP markers, the estimated probability of identity was 5.3 x 10(-11). The exclusion power of both kinds of markers in parental analysis was evaluated using paternity exclusion estimations, and, in addition to this, by estimation of the parental exclusion probability in 18 Galloway family trios. Paternity exclusion was estimated to be over 99% for microsatellites, and approx. 98% for SNP. Both, microsatellite and SNP sets of markers showed similar parental exclusion probabilities.     

鳙基于10个微卫星标记的亲子鉴定分析

为开展鳙(Hypophthalmichthys nobilis)家系选育工作,本研究进行了基于微卫星标记的亲子鉴定研究。试验中筛选了10个扩增效率较高的微卫星标记,通过引物荧光修饰,引物结合毛细管电泳分型技术,对鳙48尾亲本及384尾子代进行了基因分型,并计算了等位基因频率和模拟分析和亲子鉴定等分析。结果发现,各位点的等位基因数介于4~13之间,其中9个位点均具有较高的多态性和杂合度(PIC>0.5,He>0.5),研究发现位点的多态性信息含量(PIC)与亲本对排除率(E-PP)存在显著正相关(p<0.01)。模拟分析结果显示,该10个标记预计可用于已知性别的50组亲本(100尾)或未知性别的50尾亲本的鉴定分析(鉴别成功率>95%)。亲子鉴定发现,对试验中2个交配组(每组12对亲本)的鉴别成功率分别为98.96%和100%;且父母本对子代的贡献率存在极显著差异(p<0.01)。通过累积位点的鉴定分析发现,当标记数为7个和9个时分别能满足试验中12组和24组亲本对应子代的鉴定分析(鉴别率>95%),模拟分析和亲子鉴定分析成功率趋势基本符合。本研究所开发的亲子鉴定技术可为鳙家系选育提供技术支持。     

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.     

基于微卫星标记的圆口铜鱼亲子鉴定技术

为快速有效地鉴别不同的圆口铜鱼家系及来源, 研究从已发表的40个微卫星标记中筛选出20个多态性较高且稳定扩增的微卫星位点, 通过对8个圆口铜鱼家系339尾个体进行微卫星基因分型检测, 建立了圆口铜鱼荧光微卫星标记与多重毛细管电泳相结合的亲子鉴定技术。遗传多样性分析结果显示, 圆口铜鱼8个家系群体的平均等位基因数(N_a)为9个, 平均多态信息含量(PIC)为0.616, 平均期望杂合度(H_e)为0.659, 平均观测杂合度(H_o)为0.691, 其中子一代群体的遗传多样性水平明显低于亲本群体。亲子鉴定分析结果显示, 当双亲基因型未知时其单亲累积排除概率(CE-1P)为0.99954473, 当单亲基因型已知时其累积排除概率(CE-2P)为0.99999825, 当双亲基因型未知时其双亲累积排除概率(CE-PP)为1.00000000, 当使用20个微卫星位点进行亲子鉴定时, 297尾子一代均能正确找到其父母本, 亲子鉴定准确率为100%。由此可见, 研究建立的圆口铜鱼亲子鉴定技术是可靠的, 能为圆口铜鱼的家系管理、种群遗传管理和增殖放流效果评估提供科学依据     

Using molecular markers for pedigree reconstruction of the greater amberjack (Seriola dumerili) in the absence of parental information

Ensuring appropriate levels of genetic diversity in captive populations is essential to avoid inbreeding and loss of rare alleles by genetic drift. Pedigree reconstruction and parentage analysis in the absence of parental genotypes can be a challenging task that relies in the assignment of sibship relationships among the offspring. Here, we used eight highly variable microsatellite markers and three different assignment methods to reconstruct the most likely genotypes of a parental group of wild Seriola dumerili fish based on the genotypes of six cohorts of their offspring, to assess their relative contributions to the offspring. We found that a combination of the four most variable microsatellites was enough to identify the number of parents and their contribution to the offspring, suggesting that the variability of the markers can be more critical than the number of markers. Estimated effective population sizes were lower than the number of breeders and variable among years. The results suggest unequal parental contribution that should be accounted for breeding programs in the future.     

Single nucleotide polymorphisms for pig identification and parentage exclusion

Single nucleotide polymorphisms (SNPs) have become an important type of marker for commercial diagnostic and parentage genotyping applications as automated genotyping systems have been developed that yield accurate genotypes. Unfortunately, allele frequencies for public SNP markers in commercial pig populations have not been available. To fulfil this need, SNP markers previously mapped in the USMARC swine reference population were tested in a panel of 155 boars that were representative of US purebred Duroc, Hampshire, Landrace and Yorkshire populations. Multiplex assay groups of 5-7 SNP assays/group were designed and genotypes were determined using Sequenom's massarray system. Of 80 SNPs that were evaluated, 60 SNPs with minor allele frequencies >0.15 were selected for the final panel of markers. Overall identity power across breeds was 4.6 x 10(-23), but within-breed values ranged from 4.3 x 10(-14) (Hampshire) to 2.6 x 10(-22) (Yorkshire). Parentage exclusion probability with only one sampled parent was 0.9974 (all data) and ranged from 0.9594 (Hampshire) to 0.9963 (Yorkshire) within breeds. Sire exclusion probability when the dam's genotype was known was 0.99998 (all data) and ranged from 0.99868 (Hampshire) to 0.99997 (Yorkshire) within breeds. Power of exclusion was compared between the 60 SNP and 10 microsatellite markers. The parental exclusion probabilities for SNP and microsatellite marker panels were similar, but the SNP panel was much more sensitive for individual identification. This panel of SNP markers is theoretically sufficient for individual identification of any pig in the world and is publicly available.     

Exclusion probabilities obtainable by biochemical polymorphisms in dogs

General formulae are given to calculate the exclusion probabilities in false paternity and parentage cases by means of gene loci with an arbitrary number of alleles whereas in paternity cases an arbitrary number of offspring per litter is considered additionally.
By aid of these formulae and on the basis of the allele frequencies of four blood protein and enzyme systems the probabilities of excluding incorrect paternity and parentage are calculated in seven German dog breeds. The results are tabulated and discussed.
It can be shown that the exclusion probability in false paternity cases increases distinctly with an increasing number of offspring per litter and its maximum is nearly attained if 5 offspring are examined. Therefore it is of value to consider entire litters in paternity controls in dogs.