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.     

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.     

Can microsatellite markers replace PIT tags in rohu (Labeo rohita Hamilton, 1822) selective breeding programmes?

This study was undertaken to assess the feasibility of microsatellite markers for resolving parentage in Labeo rohita (rohu) selective breeding programmes in lieu of conventional PIT tag marking. Ten microsatellite markers were used to assign offspring parentage. A total of 160 individuals were tested from eight full‐sibling families. Eight highly polymorphic loci with allele numbers ranging from 2 to 8 and polymorphism information content (PIC) values in the 0.47–0.82 range were utilized. With the use of a real data set from a breeding programme, all but 12 individuals out of 160 could be assigned exclusively to their correct parental pairs. The matching rate was 92.5%, which was lower than in a simulated study (98.44%, Excl‐2). Discrepancies between simulations and real data sets may be due to several factors such as genetic relatedness among candidate parents (half‐sib and full‐sib), presence of null alleles, and sampling variance of the parents. The results of this study suggest that a marker panel probably with a wider genome coverage may be necessary for near‐100% accuracy in assigning parentage in rohu selective breeding programmes.     

Development of hypervariable microsatellite loci for use in eastern phoebes (Sayornis phoebe) and related Tyrannids

In order to complement on‐going research on reproductive strategies and dispersal in the eastern phoebe (Sayornis phoebe), we developed five polymorphic microsatellite loci which would permit us to assign parentage to offspring and to evaluate population genetic structure. The number of alleles per locus ranged from 3 to 15; probability of exclusion, with a known maternal genotype, was 0.995. Preliminary screening revealed that primers were polymorphic in other Tyrannidae.     

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.     

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.     

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     

Genetic Variation in Captive Koalas (Phascolarctos cinereus): Parentage Determination and Individual Identification

Highly repeatable randomly amplified polymorphicDNA (RAPD) markers were developed for parentage studiesin the koala (Phascolarctos cinereus). Of the 25 RAPDprimers screened, 5 (20.0%) produced 32 repeatable polymorphic RAPD bands (average/primer = 6.4± 4.2). A high level of polymorphism was observedfor each group of koalas (Featherdale, 71.9%; Lone Pine,84.4%). All 25 koalas could be uniquely identified using either RAPD or microsatellite markers. Of the32 RAPD markers generated in koalas, 25 were informativefor parentage analyses. These RAPD markers successfullydetermined both parents to three offspring and a male parent to a fourth offspring.Paternity analysis (where the female parent is known)succeeded in assigning the correct male parent to sevenoffspring. Our RAPD–PCR method generatesinformative genetic markers that are useful for parentagedetermination and individual identification of captivekoalas. This would provide genetic analysis to zoos andwildlife parks as a low-cost alternative to the more expensive microsatellite markers.     

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.     

Single nucleotide polymorphisms for DNA typing in the domestic horse

Genetic markers are important resources for individual identification and parentage assessment. Although short tandem repeats (STRs) have been the traditional DNA marker, technological advances have led to single nucleotide polymorphisms (SNPs) becoming an attractive alternative. SNPs can be highly multiplexed and automatically scored, which allows for easier standardization and sharing among laboratories. Equine parentage is currently assessed using STRs. We obtained a publicly available SNP dataset of 729 horses representing 32 diverse breeds. A proposed set of 101 SNPs was analyzed for DNA typing suitability. The overall minor allele frequency of the panel was 0.376 (range 0.304–0.419), with per breed probability of identities ranging from 5.6 × 10^?35 to 1.86 × 10^?42. When one parent was available, exclusion probabilities ranged from 0.9998 to 0.999996, although when both parents were available, all breeds had exclusion probabilities greater than 0.9999999. A set of 388 horses from 35 breeds was genotyped to evaluate marker performance on known families. The set included 107 parent–offspring pairs and 101 full trios. No horses shared identical genotypes across all markers, indicating that the selected set was sufficient for individual identification. All pairwise comparisons were classified using ISAG rules, with one or two excluding markers considered an accepted parent–offspring pair, two or three excluding markers considered doubtful and four or more excluding markers rejecting parentage. The panel had an overall accuracy of 99.9% for identifying true parent–offspring pairs. Our developed marker set is both present on current generation SNP chips and can be highly multiplexed in standalone panels and thus is a promising resource for SNP‐based DNA typing.     

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.     

采用微卫星标记分析10个双峰驼群体的遗传变异和群体间的遗传关系

为从分子水平上对我国双峰驼(Camelus bactrianus)群体的遗传多样性、群体间遗传关系、群体遗传分化及近交情况进行全面、系统地研究,为双峰驼种质资源保护和新品种培育提供基础数据,本文利用18对微卫星引物,分析了我国9个双峰驼群体和1个蒙古双峰驼群体的遗传多样性和遗传关系。结果显示:10个群体均具有较高的遗传多样性,共检测到242个等位基因,平均等位基因数为13.44,平均有效等位基因数为4.18,平均观察杂合度(Ho)为0.5528。10个群体间存在显著的遗传分化,有9.6%的遗传变异来自群体间,90.4%的遗传变异来自群体内部的个体间。聚类分析、主成分分析和群体遗传结构分析结果都表明10个群体被分成2个明显的分支,新疆4个群体单独聚为一类,剩下的6个群体聚为一类。这一结果可能与它们的地理分布和群体间的地理屏障有关。     

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.     

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.     

Population genetic analysis of the domestic Bactrian camel in China by RAD‐seq

Restriction site‐associated DNA sequencing (RAD‐seq) is one of the most effective high‐throughput sequencing technologies for SNP development and utilization and has been applied to studying the origin and evolution of various species. The domestic Bactrian camels play an important role in economic trade and cultural construction. They are precious species resources and indispensable animals in China's agricultural production. Recently, the rapid development of modern transportation and agriculture, and the deterioration of the environment have led to a sharp decline in the number of camels. Although there have been some reports on the evolution history of the domestic Bactrian camel in China, the origin, evolutionary relationship, and genetic diversity of the camels are unclear due to the limitations of sample size and sequencing technology. Therefore, 47 samples of seven domestic Bactrian camel species from four regions (Inner Mongolia, Gansu, Qinghai, and Xinjiang) were prepared for RAD‐seq analysis to study the evolutionary relationship and genetic diversity. In addition, seven domestic Bactrian camel species are located in different ecological zones, forming different characteristics and having potential development value. A total of 6,487,849 SNPs were genotyped. On the one hand, the filtered SNP information was used to conduct polymorphism mapping construction, LD attenuation analysis, and nucleotide diversity analysis. The results showed that the number of SNPs in Dongjiang camel was the highest, the LD coefficient decayed the fastest, and the nucleotide diversity was the highest. It indicates that Dongjiang camel has the highest genetic diversity. On the other hand, the filtered SNPs information was used to construct the phylogenetic tree, and F_ST analysis, inbreeding coefficient analysis, principal component analysis, and population structure analysis were carried out. The results showed that Nanjiang camel and Beijiang camels grouped together, and the other five Bactrian camel populations gathered into another branch. It may be because the mountains in the northern part of Xinjiang and the desert in the middle isolate the two groups from the other five groups.     

Difficulties in parentage analysis: the probability that an offspring and parent have the same heterozygous genotype.

Parentage studies often estimate the number of parents contributing to half-sib progeny arrays by counting the number of alleles attributed to unshared parents. This approach is compromised when an offspring has the same heterozygous genotype as the shared parent, for then the contribution of the unshared parent cannot be unambiguously deduced. To determine how often such cases occur, formulae for co-dominant markers with n alleles are derived here for Ph, the probability that a given heterozygous parent has an offspring with the same heterozygous genotype, and Pa, the probability that a randomly chosen offspring has the same heterozygous genotype as the shared parent. These formulae have been derived assuming Mendelian segregation with either (1) an arbitrary mating system, (2) random mating or (3) mixed mating. The maximum value of Pa under random mating is 0.25 and occurs with any two alleles each at a frequency of 0.5. The behaviour with partial selfing (where reproduction is by selfing with probability s, and random mating otherwise) is more complex. For n < or = 3 alleles, the maximum value of Pa occurs with any two alleles each at a frequency of 0.5 if s < 0.25, and with three equally frequent alleles otherwise. Numerically, the maximum value of Pa for n > or = 4 alleles occurs with n* < or = n alleles at equal frequencies, where the maximizing number of alleles n* is an increasing function of the selfing rate. Analytically, the maximum occurs with all n alleles present and equally frequent if s > or = 2/3. In addition, the potential applicability of these formulae for evolutionary studies is briefly discussed.     

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.     

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.     

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。