Pedigree reconstruction in wild cichlid fish populations

It is common practice to use microsatellites to detect parents and their offspring in wild and captive populations, in order to reconstruct a pedigree. However, correct inference is often constrained by a number of factors, including the absence of demographic data and ignorance regarding the completeness of parental sampling. Here we present a new Bayesian estimator that simultaneously estimates the pedigree and the size of the unsampled population. The method is robust to genotyping error, and can estimate pedigrees in the absence of demographic data. Using a large-scale microsatellite assay in four wild cichlid fish populations of Lake Tanganyika (1000 individuals in total), we assess the performance of the Bayesian estimator against the most popular assignment program, Cervus. We found small but significant pedigrees in each of the tested populations using the Bayesian procedure, but Cervus had very high type I error rates when the size of the unsampled population was assumed to be lower than what it was. The need of pedigree relationships to infer adaptive processes in natural populations places strong constraints on sampling design and identification of multigenerational pedigrees in natural populations.     

Evidence of adoption,monozygotic twinning,and low inbreeding rates in a large genetic pedigree of polar bears

Multigenerational pedigrees have been developed for free-ranging populations of many species, are frequently used to describe mating systems, and are used in studies of quantitative genetics. Here, we document the development of a 4449-individual pedigree for the Western Hudson Bay subpopulation of polar bears (Ursus maritimus), created from relationships inferred from field and genetic data collected over six generations of bears sampled between 1966 and 2011. Microsatellite genotypes for 22–25 loci were obtained for 2945 individuals, and parentage analysis was performed using the program FRANz, including additional offspring–dam associations known only from capture data. Parentage assignments for a subset of 859 individuals were confirmed using an independent medium-density set of single nucleotide polymorphisms. To account for unsampled males in our population, we performed half-sib–full-sib analysis to reconstruct males using the program COLONY, resulting in a final pedigree containing 2957 assigned maternities and 1861 assigned paternities with only one observed case of inbreeding between close relatives. During genotyping, we identified two independently captured 2-year-old males with identical genotypes at all 25 loci, showing—for the first time—a case of monozygotic twinning among polar bears. In addition, we documented six new cases of cub adoption, which we attribute to cub misidentification or misdirected maternal care by a female bereaved of her young. Importantly, none of these adoptions could be attributed to reduced female vigilance caused by immobilization to facilitate scientific handling, as has previously been suggested.     

The aunt and uncle effect: an empirical evaluation of the confounding influence of full sibs of parents on pedigree reconstruction.

This study used simulations and a known two-generation pedigree of chinook salmon (Oncorhynchus tshawytscha) to evaluate the effect of full sibs of parents on pedigree reconstruction. Parentage analysis was conducted on 100 parent pair-offspring relationships from pedigrees with unrelated (simulation) and related (chinook salmon) candidate parents. Parentage assignment success for the chinook salmon was lower than in the simulated populations. For example, the six most variable loci (mean H(E) = 0.87) provided a mean of 97% unambiguous assignments in the simulated population and 67% unambiguous assignments for the chinook salmon. Estimates of the pairwise relatedness coefficient ((xy)) for most nonexcluded false parents and true parents of chinook salmon offspring exceeded 0.50. These results support the conclusion that closely related candidate parents decrease the power of genetic markers for pedigree reconstruction based on exclusion. Ambiguous parentage may be resolved using single parent- and parent pair-offspring likelihood analysis, however, these methods should be used with caution and they are not replacements for using more loci when many candidate parents are full sibs.     

Relationship type affects the reliability of dispersal distance estimated using pedigree inferences in partially sampled populations: A case study involving invasive American mink in Scotland

Estimating dispersal—a key parameter for population ecology and management—is notoriously difficult. The use of pedigree assignments, aided by likelihood‐based software, has become popular to estimate dispersal rate and distance. However, the partial sampling of populations may produce false assignments. Further, it is unknown how the accuracy of assignment is affected by the genealogical relationships of individuals and is reflected by software‐derived assignment probabilities. Inspired by a project managing invasive American mink (Neovison vison), we estimated individual dispersal distances using inferred pairwise relationships of culled individuals. Additionally, we simulated scenarios to investigate the accuracy of pairwise inferences. Estimates of dispersal distance varied greatly when derived from different inferred pairwise relationships, with mother–offspring relationship being the shortest (average = 21 km) and the most accurate. Pairs assigned as maternal half‐siblings were inaccurate, with 64%–97% falsely assigned, implying that estimates for these relationships in the wild population were unreliable. The false assignment rate was unrelated to the software‐derived assignment probabilities at high dispersal rates. Assignments were more accurate when the inferred parents were older and immigrants and when dispersal rates between subpopulations were low (1% and 2%). Using 30 instead of 15 loci increased pairwise reliability, but half‐sibling assignments were still inaccurate (>59% falsely assigned). The most reliable approach when using inferred pairwise relationships in polygamous species would be not to use half‐sibling relationship types. Our simulation approach provides guidance for the application of pedigree inferences under partial sampling and is applicable to other systems where pedigree assignments are used for ecological inference.     

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.     

On minimizing assignment errors and the trade‐off between false positives and negatives in parentage analysis

Genetic parentage analyses provide a practical means with which to identify parent–offspring relationships in the wild. In Harrison et al.'s study (2013a), we compare three methods of parentage analysis and showed that the number and diversity of microsatellite loci were the most important factors defining the accuracy of assignments. Our simulations revealed that an exclusion‐Bayes theorem method was more susceptible to false‐positive and false‐negative assignments than other methods tested. Here, we analyse and discuss the trade‐off between type I and type II errors in parentage analyses. We show that controlling for false‐positive assignments, without reporting type II errors, can be misleading. Our findings illustrate the need to estimate and report both the rate of false‐positive and false‐negative assignments in parentage analyses.     

COLONY: a program for parentage and sibship inference from multilocus genotype data

Pedigrees, depicting genealogical relationships between individuals, are important in several research areas. Molecular markers allow inference of pedigrees in wild species where relationship information is impossible to collect by observation. Marker data are analysed statistically using methods based on Mendelian inheritance rules. There are numerous computer programs available to conduct pedigree analysis, but most software is inflexible, both in terms of assumptions and data requirements. Most methods only accommodate monogamous diploid species using codominant markers without genotyping error. In addition, most commonly used methods use pairwise comparisons rather than a full-pedigree likelihood approach, which considers the likelihood of the entire pedigree structure and allows the simultaneous inference of parentage and sibship. Here, we describe colony, a computer program implementing full-pedigree likelihood methods to simultaneously infer sibship and parentage among individuals using multilocus genotype data. colony can be used for both diploid and haplodiploid species; it can use dominant and codominant markers, and can accommodate, and estimate, genotyping error at each locus. In addition, colony can carry out these inferences for both monoecious and dioecious species. The program is available as a Microsoft Windows version, which includes a graphical user interface, and a Macintosh version, which uses an R-based interface.     

Validation of microsatellite multiplexes for parentage analysis and species discrimination in two hybridizing species of coral reef fish (Plectropomus spp., Serranidae)

Microsatellites are often considered ideal markers to investigate ecological processes in animal populations. They are regularly used as genetic barcodes to identify species, individuals, and infer familial relationships. However, such applications are highly sensitive the number and diversity of microsatellite markers, which are also prone to error. Here, we propose a novel framework to assess the suitability of microsatellite datasets for parentage analysis and species discrimination in two closely related species of coral reef fish, Plectropomus leopardus and P. maculatus (Serranidae). Coral trout are important fisheries species throughout the Indo‐Pacific region and have been shown to hybridize in parts of the Great Barrier Reef, Australia. We first describe the development of 25 microsatellite loci and their integration to three multiplex PCRs that co‐amplify in both species. Using simulations, we demonstrate that the complete suite of markers provides appropriate power to discriminate between species, detect hybrid individuals, and resolve parent–offspring relationships in natural populations, with over 99.6% accuracy in parent–offspring assignments. The markers were also tested on seven additional species within the Plectropomus genus with polymorphism in 28–96% of loci. The multiplex PCRs developed here provide a reliable and cost‐effective strategy to investigate evolutionary and ecological dynamics and will be broadly applicable in studies of wild populations and aquaculture brood stocks for these closely related fish 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.     

Performance of different SNP panels for parentage testing in two East Asian cattle breeds

The International Society for Animal Genetics (ISAG) proposed a panel of single nucleotide polymorphisms (SNPs) for parentage testing in cattle (a core panel of 100 SNPs and an additional list of 100 SNPs). However, markers specific to East Asian taurine cattle breeds were not included, and no information is available as to whether the ISAG panel performs adequately for these breeds. We tested ISAG's core (100 SNP) and full (200 SNP) panels on two East Asian taurine breeds: the Korean Hanwoo and the Japanese Wagyu, the latter from the Australian herd. Even though the power of exclusion was high at 0.99 for both ISAG panels, the core panel performed poorly with 3.01% false‐positive assignments in the Hanwoo population and 3.57% in the Wagyu. The full ISAG panel identified all sire–offspring relations correctly in both populations with 0.02% of relations wrongly excluded in the Hanwoo population. Based on these results, we created and tested two population‐specific marker panels: one for the Wagyu population, which showed no false‐positive assignments with either 100 or 200 SNPs, and a second panel for the Hanwoo, which still had some false‐positive assignments with 100 SNPs but no false positives using 200 SNPs. In conclusion, for parentage assignment in East Asian cattle breeds, only the full ISAG panel is adequate for parentage testing. If fewer markers should be used, it is advisable to use population‐specific markers rather than the ISAG panel.     

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.     

Balancing population size and genetic information in parentage analysis studies

In parentage analysis studies, the parameters of interest typically are not the parent assignments themselves, but population parameters such as variance in fertility, self-pollination rate, or average dispersal distances. The precision of parameter estimates is affected by two factors: the number of offspring under consideration, and the precision with which the offspring can be assigned to parents. When assignment of parents is based on genetic information, the confidence in assignments is affected by the number and polymorphism of the loci considered, and by the number of potential parents in the population. Studying larger populations may yield higher numbers of offspring, but since larger populations contain more potential parents, more (or more highly polymorphic) loci are necessary to attain a given level of confidence in the parent assignments. This article addresses how to relate the size of the population and the number of loci when designing a study. It is shown that the number of loci needed to assign all offspring unambiguously is proportional to the logarithm of the population size. In some cases, the constant of proportionality can be determined, eliminating the need for simulation-based projections. Population-wide measures of uncertainty in parent assignments are also introduced, and it is shown that holding uncertainty "steady" as the population size increases also requires increasing the number of loci proportional to the logarithm of the population size. Data from a study of self-pollination are used to illustrate the techniques suggested.     

Relative accuracy of three common methods of parentage analysis in natural populations

Parentage studies and family reconstructions have become increasingly popular for investigating a range of evolutionary, ecological and behavioural processes in natural populations. However, a number of different assignment methods have emerged in common use and the accuracy of each may differ in relation to the number of loci examined, allelic diversity, incomplete sampling of all candidate parents and the presence of genotyping errors. Here, we examine how these factors affect the accuracy of three popular parentage inference methods (colony , famoz and an exclusion‐Bayes’ theorem approach by Christie (Molecular Ecology Resources, 2010a, 10, 115) to resolve true parent–offspring pairs using simulated data. Our findings demonstrate that accuracy increases with the number and diversity of loci. These were clearly the most important factors in obtaining accurate assignments explaining 75–90% of variance in overall accuracy across 60 simulated scenarios. Furthermore, the proportion of candidate parents sampled had a small but significant impact on the susceptibility of each method to either false‐positive or false‐negative assignments. Within the range of values simulated, colony outperformed FaMoz, which outperformed the exclusion‐Bayes’ theorem method. However, with 20 or more highly polymorphic loci, all methods could be applied with confidence. Our results show that for parentage inference in natural populations, careful consideration of the number and quality of markers will increase the accuracy of assignments and mitigate the effects of incomplete sampling of parental populations.     

CORRELATED INBREEDING AMONG RELATIVES: OCCURRENCE,MAGNITUDE, AND IMPLICATIONS

Understanding the magnitude and causes of genetic and phenotypic resemblance among relatives is key to understanding evolutionary processes. Contrary to basic expectation, individual coefficients of inbreeding ( f) were recently hypothesized to be intrinsically correlated across parents and offspring in structured populations, potentially creating an additional source of phenotypic resemblance in traits that show inbreeding depression. To test this hypothesis, we used individual‐based simulations to quantify the parent–offspring correlations in f arising under random mating in populations of different size, immigration rate, and mating system. Parent–offspring correlations in f were typically positive (median r≈ 0.2–0.4) in relatively small and isolated populations. Relatively inbred parents therefore produced relatively inbred offspring on average, although the magnitude of this effect varied considerably among replicate populations. Correlations were higher given more generations of random mating, greater variance in reproductive success, polygynous rather than monogamous mating, and for midparent–offspring rather than parent–offspring relationships. Furthermore, f was also positively correlated across half‐siblings, and closer relatives had more similar inbreeding coefficients across entire generations. Such intrinsic resemblance in f among relatives could provide an additional genetic benefit of mate choice and bias quantitative genetic analyses that do not account for correlated inbreeding depression.     

Carry-over effect of captive breeding reduces reproductive fitness of wild-born descendants in the wild

Supplementation of wild populations with captive-bred organisms is a common practice for conservation of threatened wild populations. Yet it is largely unknown whether such programmes actually help population size recovery. While a negative genetic effect of captive breeding that decreases fitness of captive-bred organisms has been detected, there is no direct evidence for a carry-over effect of captive breeding in their wild-born descendants, which would drag down the fitness of the wild population in subsequent generations. In this study, we use genetic parentage assignments to reconstruct a pedigree and estimate reproductive fitness of the wild-born descendants of captive-bred parents in a supplemented population of steelhead trout (Oncorhynchus mykiss). The estimated fitness varied among years, but overall relative reproductive fitness was only 37 per cent in wild-born fish from two captive-bred parents and 87 per cent in those from one captive-bred and one wild parent (relative to those from two wild parents). Our results suggest a significant carry-over effect of captive breeding, which has negative influence on the size of the wild population in the generation after supplementation. In this population, the population fitness could have been 8 per cent higher if there was no carry-over effect during the study period.     

Using microsatellite diversity in wild Anegada iguanas (<Emphasis Type="Italic">Cyclura pinguis</Emphasis>) to establish relatedness in a captive breeding group of this critically endangered species

Awareness of the genealogical relationships between founder animals in captive breeding programs is essential for the selection of mating pairs that maintain genetic diversity. If captive founder relationships are unknown they can be inferred using genetic data from wild populations. Here, we report the results of such an analysis for six Cyclura pinguis (Sauria: Iguanidae) acquired as adults in 1999 by the San Diego Zoo Institute for Conservation Research to begin a captive breeding program for this critically endangered species. The six founder animals were reportedly hatched in captivity from eggs collected on Anegada in 1985. No records exist, however, as to where on Anegada the eggs were collected or from how many nests they originated. To assist determination of genealogical relationships, we genotyped the six captive founders, their first six offspring, and 33 wild adult iguanas from Anegada at 23 informative microsatellite loci. With these data, we estimated allele frequencies among the wild samples and then estimated the relatedness of the captive population. Using likelihood inference, we determined that three closely related pairs exist among the six captive founders and that each pair is not closely related to the other two. In addition, we were able to assign parentage for all six of the founders’ offspring tested, one of which had been previously misdiagnosed. Using the assigned parentage and inferred relatedness of the six founders, we calculated mean kinship for each of the six founders and their five living offspring. Finally, based on the allelic diversity of the wild iguanas sampled, we conclude that the C. pinguis population on Anegada is not excessively inbred; however, further investigation is warranted.     

One-step reconstruction of multi-generation pedigree networks in apple (Malus × domestica Borkh.) and the parentage of Golden Delicious

The increasing availability of genomic tools improves our ability to investigate the patterns of genetic diversity and relatedness among individuals. The pedigrees of many apple cultivars are completely unknown, often reducing the efficiency of breeding programs. Using a multilocus simple sequence repeat dataset, we applied a novel multi-generation pedigree-network reconstruction procedure based on the software FRANz in a Malus × domestica collection (101 cultivated and 22 wild apples) with partially known pedigree relationships. The procedure produced 78 parent–offspring relationships organized into three networks and showed high power for detecting real pedigree links (98.5 %) and a low false-positive rate (9.0 %). The largest reconstructed pedigree network spanned four generations and involved 65 cultivars. The availability of detailed pedigree connections confirmed that recent genealogical relationships affect population genetic structure in apple. Finally, our analysis enabled us to confirm or discard several pedigrees known only anecdotically, among which the cultivar Grimes Golden was validated as a parent of the widely grown cultivar Golden Delicious. The pedigree reconstruction protocol here described will be of broad applicability to other collections and crop species.     

Combining noninvasive genetics and a new mammalian sex‐linked marker provides new tools to investigate population size,structure and individual behaviour: An application to bats

Monitoring wild populations is crucial for their effective management. Noninvasive genetic methods provide robust data from individual free‐ranging animals, which can be used in capture–mark–recapture (CMR) models to estimate demographic parameters without capturing or disturbing them. However, sex‐ and status‐specific behaviour, which may lead to differences in detection probabilities, is rarely considered in monitoring. Here, we investigated population size, sex ratio, sex‐ and status‐related behaviour in 19 Rhinolophus hipposideros maternity colonies (Northern France) with a noninvasive genetic CMR approach (using faeces) combined with parentage assignments. The use of the DDX3X/Y‐Mam sexual marker designed in this study, which shows inter‐ and intrachromosomal length polymorphism across placental mammals, together with eight polymorphic microsatellite markers, produced high‐quality genetic data with limited genotyping errors and allowed us to reliably distinguish different categories of individuals (males, reproductive and nonreproductive females) and to estimate population sizes. We showed that visual counts represent well‐adult female numbers and that population composition in maternity colonies changes dynamically during the summer. Before parturition, colonies mainly harbour pregnant and nonpregnant females with a few visiting males, whereas after parturition, colonies are mainly composed of mothers and their offspring with a few visiting nonmothers and males. Our approach gives deeper insight into sex‐ and status‐specific behaviour, a prerequisite for understanding population dynamics and developing effective monitoring and management strategies. Provided sufficient samples can be obtained, this approach can be readily applied to a wide range of species.     

Detection of linkage between marker loci and loci affecting quantitative traits in crosses between segregating populations

Summary By making use of pedigree information and information on marker-genotypes of the parent and F-1 individuals crossed to form an F-2 population, it is possible to carry out a linkage analysis between marker loci and loci affecting quantitative traits in a cross between segregating parent populations that are at fixation for alternative alleles at the QTL, but share the same alleles at the marker loci. For two-allele systems, depending on marker allele frequencies in the parent populations, 2–4 times as many F-2 offspring will have to be raised and scored for markers and quantitative traits in order to provide power equivalent to that obtained in a cross between fully inbred lines. Major savings in number of F-2 offspring raised can be achieved by scoring each parent pair for a large number of markers in each chromosomal region and scoring F-1 and F-2 offspring only for those markers for which the parents were homozygous for alternative alleles. For multiple allele systems, particularly when dealing with hypervariable loci, only 10%–20% additional F-2 offspring will have to be raised and scored to provide power equivalent to that obtained in a cross between inbred lines. When a resource population contains novel favorable alleles at quantitative trait loci that are not present (or rare) in a commercial population, analyses of this sort will enable the loci of interest to be identified, mapped and manipulated effectively in breeding programs.Contribution no. 2124-E, 1987 series from The Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel     

一种基于高密度遗传标记的亲子鉴定方法及其应用

系谱是人类遗传及动植物育种研究与实践的重要信息来源之一。系谱记录错误是育种生产中普遍存在的一种记录错误,影响基因定位、遗传值及表型值预测等相关研究结果的可靠性。现有的方法软件可以利用遗传标记信息对疑似亲子进行亲子鉴定,但这些软件方法操作复杂,限制标记数量,如Cervus。针对当前高密度SNP标记在人类及动植物研究中广泛应用的现状,文章提出了一种基于全基因组高密度SNP数据的亲子鉴定新方法,命名为EasyPC。对EasyPC及Cervus的运行效率进行了对比,并用中国荷斯坦牛(n=2180)和杜洛克猪(n=191)的全基因组SNP芯片数据对EasyPC进行了验证。结果表明：EasyPC运行效率高于Cervus,牛和猪群体系谱错误率分别为20%和6%,与相关研究报道相符。通过使用全基因组SNP标记对群体孟德尔错误率的经验分布进行分析,该方法不仅可以简单、快速、准确地判别系谱的正确性,而且还可以对错误系谱进行校正。EasyPC为解决全基因组研究中基因型及系谱数据前处理过程中的系谱校正问题提供了一种新的途径。