Microsatellite polymorphism in closely related dogs.

The effectiveness of microsatellites in parentage testing and individual identification has been proven in many species, including dogs. However, the use of these markers has not been extended to control for pedigrees in large populations of closely related animals. We have analyzed polymorphism in a set of 10 microsatellites over three generations of 360 pedigree rottweilers. Results were compared with two pure-bred populations of unrelated animals and with one population constituted by unrelated dogs of mixed breeds to measure polymorphism variation. We optimized this set of microsatellites to be analyzed by a semiautomated capillary electrophoresis method after amplification in two multiplex polymerase chain reactions (PCRs). The mean polymorphism information content (PIC) value in the rottweiler pedigree is 0.401 and the combined paternity exclusion probability (CPE) is 95.6%. These values are similar to those obtained in pure-bred populations of unrelated animals, and although polymorphism is reduced in relation to the pool population, we solved all paternity exclusions. In only a few cases did we have to use two additional microsatellites to solve individual identification of full-sib dogs.     

A standard panel of microsatellites for Asian seabass (Lates calcarifer)

Microsatellites are the most popular markers for parentage assignment and population genetic studies. To meet the demand for international comparability for genetic studies of Asian seabass, a standard panel of 28 microsatellites has been selected and characterized using the DNA of 24 individuals from Thailand, Malaysia, Indonesia and Australia. The average allele number of these markers was 10.82 ± 0.71 (range: 6–19), and the expected heterozygosity averaged 0.76 ± 0.02 (range: 0.63–1.00). All microsatellites showed Mendelian inheritance. In addition, eight standard size controls have been developed by cloning a set of microsatellite alleles into a pGEM‐T vector to calibrate allele sizes determined by different laboratories, and are available upon request. Seven multiplex PCRs, each amplifying 3–5 markers, were optimized to accurately and rapidly genotype microsatellites. Parentage assignment using 10 microsatellites in two crosses (10 × 10 and 20 × 20) demonstrated a high power of these markers for revealing parent‐sibling connections. This standard set of microsatellites will standardize genetic diversity studies of Asian seabass, and the multiplex PCR sets will facilitate parentage assignment.     

Multiplex genotyping of novel tetranucleotide microsatellites from a marine foodfish species crimson red snapper (Lutjanus erythropterus)

Crimson red snapper (Lutjanus erythropterus) is an important marine foodfish species in Asia with great potentials for aquaculture. We isolated nine polymorphic microsatellites, among which eight were tetranucleotide repeats, and one was CA‐microsatellite. The average allele number present in 48 individuals was 11.1/locus, ranging from three to 33. The expected heterozygosity ranged from 0.49 to 0.97 with an average of 0.74. Six of the nine markers conformed to the Hardy–Weinberg expectations. No pairwise markers showed the possibility of linkage. Protocols for two multiplex polymerase chain reactions (PCRs), each amplifying two and seven markers, respectively, were presented. The developed microsatellites and optimized multiplex PCRs will be useful for studying population structure of wild stocks and parentage assignment for cultured populations.     

微卫星标记在种群生物学研究中的应用

微卫星是以几个碱基 (一般为 1～ 6个 )为重复单位组成的简单的串联重复序列 ,具有丰度高、多态性高、共显性标记、选择中性、可自动检测等优点。本文着重介绍了微卫星在种群生物学研究中的应用。微卫星位点可以提供具高分辨率的遗传信息 ,这一特点使微卫星既适合于个体水平上的研究 ,又适合于种群水平上的研究。在个体水平上包括个体识别、交配系统和亲本分析、基因流等研究。微卫星是常用的个体识别手段 ,但在克隆植物遗传结构研究方面的应用还很有限 ;微卫星提高了交配系统和亲本分析、基因流等研究的准确性。在种群水平上微卫星可用于遗传结构、有效种群大小、种群的系统发育重建等研究。微卫星在很多物种 (包括珍稀物种 )的遗传结构研究中得到应用 ;利用微卫星标记确定有效种群大小、检测有效种群大小的波动可以促使我们正确理解种群遗传结构动态和种群进化过程 ;微卫星在种群的系统发育重建研究方面有很大的应用潜力。然而微卫星并不是研究所有问题的唯一选择。文中还讨论了在实际工作中应如何正确利用分子标记等问题     

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.     

Genetic diversity and assessment of 23 microsatellite markers for parentage testing of Santa Inês hair sheep in Brazil

Santa Inês is the most common hair sheep breed in Brazil and probably has the highest genetic diversity among sheep breeds in this country. Successful breeding programs for Brazilian sheep breeds are not common for various reasons, including a lack of control of parentage in the flocks. We developed an allele frequency database for 23 STR loci for the Santa Inês breed based on 285 animals sampled from five populations distributed across the central-western and north-eastern regions of Brazil. The marker set included seven microsatellites used in the 2011 International Society for Animal Genetics sheep genotyping comparison tests and all eight microsatellites currently approved by the Brazilian Agricultural Ministry laboratory accreditation guidelines for sheep identification. The microsatellites had an average of 10 alleles and a mean expected heterozygosity of 0.745. Combined paternity exclusion probabilities when no parent or one parent was known were >99.99%. A small proportion (5.8%) of the existing genetic variation was found to be among the Santa Inês populations, possibly derived from genetic drift and selection. We found that the marker panel proposed by the Agricultural Ministry, although generally useful, should be enhanced by including more markers for improved exclusionary power in parentage testing. This database provides a useful tool for parentage testing of this major Brazilian breed, contributing to improved management and breeding of existing herds.     

Determination of the minimum number of microsatellite markers for individual genotyping in wild boar (Sus scrofa) using a test with close relatives

In the context of developing a noninvasive, practicable method for population size estimation in wild boar, we present a stepwise procedure to reduce the number of required microsatellite markers for individual genotyping. Step1: an initial marker set of 12 microsatellite loci was tested for species specificity with nontarget DNA and resulted in an exclusion of two markers. Step 2: a variability test regarding heterozygosity and deviations from Hardy–Weinberg equilibrium led to the rejection of two further markers. Step 3: the remaining eight markers were tested for transferability across populations with three separate wild boar sample sets. Step 4: on the basis of probability of identity values, a reduction from eight to five markers was possible. Step 5: a novel test using tissue samples from female wild boars and their embryos provided evidence that four variable microsatellite markers and one sex marker are sufficient for individual identification of close relatives. Step 6: feces samples were finally used to estimate PCR (PS) and genotyping success (GS). In conclusion, we recommend a specific four-marker combination with both PS and GS >50% for a reliable individual identification in noninvasive population size estimation of wild boar.     

Multiplexed microsatellites for rapid identification and characterization of individuals and populations of Cercopithecidae

Cross-amplification of 15 human microsatellites was performed successfully in cynomolgus (Macaca fascicularis) and rhesus (M. mulatta) macaques and 11 other Cercopithecidae species of biomedical and conservation relevance. To allow for quick, efficient, and high-throughput genotyping to assess intra- and interspecific genetic variation, we performed three multiplex sets, each comprised of five markers from different parts of the genome (i.e., autosomes, the MHC region, and the X-chromosome). These multiplex sets are likely to reveal allelic divergence between taxa, which could be used for their discrimination. Population studies on three regional populations of M. fascicularis and one of M. mulatta revealed that most of the loci, with the exception of one monomorphic locus, displayed polymorphisms (the expected heterozygosities were 0.48-0.91 for M. fascicularis, and 0.61-0.93 for M. mulatta), which makes them useful for population genetics. For the multiplex set M1, including the nonlinked autosomal markers, low probabilities of identity were observed: P(ID) values ranged from 8 x 10(-7) to 3 x 10(-5). This multiplex set is reliable for forensic applications, such as individual identification, parentage testing, and kinship analysis, in wild and captive populations.     

p-loci: a computer program for choosing the most efficient set of loci for parentage assignment

Determining how many and which codominant marker loci are required for accurate parentage assignment is not straightforward because levels of marker polymorphism, linkage, allelic distributions among potential parents and other factors produce differences in the discriminatory power of individual markers and sets of markers. p-loci software identifies the most efficient set of codominant markers for assigning parentage at a user-defined level of success, using either simulated or actual offspring genotypes of known parentage. Simulations can incorporate linkage among markers, mating design and frequencies of null alleles and/or genotyping errors. p-loci is available for windows systems at http://marineresearch.oregonstate.edu/genetics/ploci.htm.     

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

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

Genotyping‐free parentage assignment using RAD‐seq reads

Parentage assignment is defined as the identification of the true parents of one focal offspring among a list of candidates and has been commonly used in zoological, ecological, and agricultural studies. Although likelihood‐based parentage assignment is the preferred method in most cases, it requires genotyping a predefined set of DNA markers and providing their population allele frequencies. In the present study, we proposed an alternative method of parentage assignment that does not depend on genotype data and prior information of allele frequencies. Our method employs the restriction site‐associated DNA sequencing (RAD‐seq) reads for clustering into the overlapped RAD loci among the compared individuals, following which the likelihood ratio of parentage assignment could be directly calculated using two parameters—the genome heterozygosity and error rate of sequencing reads. This method was validated on one simulated and two real data sets with the accurate assignment of true parents to focal offspring. However, our method could not provide a statistical confidence to conclude that the first ranked candidate is a true parent.     

Genetic structure of northwestern Spanish brown trout (Salmo trutta L.) populations, differences between microsatellite and allozyme loci

Genetic variation in nine wild brown trout (Salmo trutta L.) populations was studied by means of allozyme and microsatellite markers. All brown trout populations were clearly separated into two clusters that represented the Sil and Duero basins. Although both markers revealed a strong genetic differentiation between basins, microsatellite loci resulted much more accurate when population structure at the intrabasin level was analysed. Also pairwise multilocus FST estimates and assignment tests of individual fish to the set of sampled populations demonstrated a much higher efficiency of microsatellites compared to allozymes. The analysis of both markers provides new insights in defining the conservation units at this local area and confirms the existence of a recognized sub-lineage in the Duero basin. The management implications of these findings are discussed and changes in trout release activity are recommended to avoid mixing of trout gene pools mainly in the Sil basin.     

Microsatellites and single nucleotide polymorphisms in avian hybrid identification: a comparative case study

The correct identification of hybrids is essential in avian hybridisation studies, but selection of the appropriate set of genetic markers for this purpose is at times complicated. Microsatellites and single nucleotide polymorphisms (SNPs) are currently the most commonly used markers in this field. We compare the efficiency of these two marker types, and their combination, in the identification of the threatened avian species, the greater spotted eagle and the lesser spotted eagle, as well as hybrids between the two species. We developed novel SNP markers from genome-wide distributed 122 candidate introns using only sympatric samples, and tested these markers successfully in 60 sympatric and allopatric spotted eagles using Bayesian model-based approaches. Comparatively, only one out of twelve previously described avian nuclear intron markers showed significant species-specific allele frequency difference, thus stressing the importance of selecting the proper markers. Twenty microsatellites outperformed selected nine SNPs in species identification, but were poorer in hybrid detection, whereas the resolution power of ten microsatellites remained too low for correct assignment. A combination of SNPs and microsatellites resulted in the most efficient and accurate identification of all individuals. Our study shows that the use of various sets of markers could lead to strikingly different assignment results, hybridisation studies may have been affected by too low a resolution power of used markers, and that an appropriate set of markers is essential for successful hybrid identification.     

Comparative analysis of STR and SNP polymorphism in the populations of sockeye salmon (Oncorhynchus nerka) from Eastern and Western Kamchatka

Sockeye salmon samples from five largest lacustrine-riverine systems of Kamchatka Peninsula were tested for polymorphism at six microsatellite (STR) and five single nucleotide polymorphism (SNP) loci. Statistically significant genetic differentiation among local populations from this part of the species range examined was demonstrated. The data presented point to pronounced genetic divergence of the populations from two geographical regions, Eastern and Western Kamchatka. For sockeye salmon, the individual identification test accuracy was higher for microsatellites compared to similar number of SNP markers. Pooling of the STR and SNP allele frequency data sets provided the highest accuracy of the individual fish population assignment.     

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.     

Comparative analysis of STR and SNP polymorphism in the populations of sockeye salmon (<Emphasis Type="Italic">Oncorhynchus nerka</Emphasis>) from Eastern and Western Kamchatka

Sockeye salmon samples from five largest lacustrine-riverine systems of Kamchatka Peninsula were tested for polymorphism at six microsatellite (STR) and five single nucleotide polymorphism (SNP) loci. Statistically significant genetic differentiation among local populations from this part of the species range examined was demonstrated. The data presented point to pronounced genetic divergence of the populations from two geographical regions, Eastern and Western Kamchatka. For sockeye salmon, the individual identification test accuracy was higher for microsatellites compared to similar number of SNP markers. Pooling of the STR and SNP allele frequency data sets provided the highest accuracy of the individual fish population assignment.     

Evaluation and selection of microsatellite markers for an identification and parentage test of Asian elephants (<Emphasis Type="Italic">Elephas maximus</Emphasis>)

Numbers of the Asian elephants (Elephas maximus) population are declining due to poaching, human-elephant conflicts, capture of wild calves for tourism and export and habitat destruction, which also may cause inbreeding in fragmented populations. In order to contribute to a reversal of this trend, we have developed an identification and parentage test by evaluation and selection of markers from 43 microsatellite loci that have been previously described for Asian or African elephants. Testing these markers on a panel of 169 Asian elephants comprising the 23 mother-offspring, 13 father-offspring and 13 parents-offspring pairs yielded 26 polymorphic markers. However, only 14 of these were found to be suitable for an analysis of molecular diversity, 12 of which will be implemented for an identification and parentage test to control the capture of wild calves in Thailand and neighboring countries.     

An empirical comparison of SNPs and microsatellites for parentage and kinship assignment in a wild sockeye salmon (Oncorhynchus nerka) population

Because of their high variability, microsatellites are still considered the marker of choice for studies on parentage and kinship in wild populations. Nevertheless, single nucleotide polymorphisms (SNPs) are becoming increasing popular in many areas of molecular ecology, owing to their high-throughput, easy transferability between laboratories and low genotyping error. An ongoing discussion concerns the relative power of SNPs compared to microsatellites-that is, how many SNP loci are needed to replace a panel of microsatellites? Here, we evaluate the assignment power of 80 SNPs (H(E) = 0.30, 80 independent alleles) and 11 microsatellites (H(E) = 0.85, 192 independent alleles) in a wild population of about 400 sockeye salmon with two commonly used software packages (Cervus3, Colony2) and, for SNPs only, a newly developed software (SNPPIT). Assignment success was higher for SNPs than for microsatellites, especially for parent pairs, irrespective of the method used. Colony2 assigned a larger proportion of offspring to at least one parent than the other methods, although Cervus and SNPPIT detected more parent pairs. Identification of full-sib groups without parental information from relatedness measures was possible using both marker systems, although explicit reconstruction of such groups in Colony2 was impossible for SNPs because of computation time. Our results confirm the applicability of SNPs for parentage analyses and refute the predictability of assignment success from the number of independent alleles.     

AFLP utility for population assignment studies: analytical investigation and empirical comparison with microsatellites

Individual-based population assignment tests have thus far mainly relied on the use of microsatellite loci. However, the logistic difficulty of screening large numbers of loci required to reach sufficient statistical power hampers the usefulness of microsatellites in situations of weak population structuring. Amplified fragment length polymorphisms (AFLP) represents an alternative for overcoming this logistical issue as the technique allows the user to characterize a much larger number of loci with a comparable analytical effort. In this study, an assignment test based on maximum likelihood for dominant markers was used to investigate the potential usefulness of AFLP for population assignment. We also compared assignment success achieved with AFLP with that obtained using microsatellites in a case study of low population differentiation involving whitefish (Coregonus clupeaformis) sympatric ecotypes. The analytical investigation showed that the minimum number of AFLP loci required to reach an assignment success of 95% stood within values that are easily achievable in many situations. This also showed how assignment success varied according to the number of AFLP loci used, their absolute frequency and their frequency differential and sampling errors, as well as the number of putative source populations. The case study showed that given a comparable analytical effort in the laboratory, AFLP were much more efficient than the microsatellite loci in discriminating the source of an individual among putative populations. AFLP resulted in higher assignment success at all levels of stringency and the log-likelihood differences between populations obtained with AFLP for each individual were much larger than those obtained with microsatellites. These results indicate that research involving individual-based population assignment methods should benefit importantly from the use of AFLP markers, especially in systems characterized by weak population structuring.