Isolation and characterization of 149 novel microsatellite DNA markers for striped bass,Morone saxatilis,and cross‐species amplification in white bass,Morone chrysops,and their hybrid

To support detailed genetic analysis of striped bass (Morone saxatilis) and white bass (Morone chrysops), we isolated 153 microsatellite loci from repeat‐enriched striped bass DNA libraries. Of these, 147 markers amplified in striped bass (average 4.7 alleles per locus) and 133 in white bass (average 2.2 alleles per locus). One hundred twenty‐two markers amplified in their hybrid. Development of new microsatellite markers will facilitate evaluations of genetic structure in wild populations and will support pedigree analysis and linkage mapping for selective breeding.     

饲养东北虎的微卫星变异研究

东北虎是世界上濒危动物之一,具有极其重要的研究价值和保护意义。该研究利用10个在东北虎基因组中表现多态性的微卫星基因座（Fca005, Fca075, Fca094, Fca152, Fca161, Fca294, Pti002, Pti003, Pti007和Pti010）对113只饲养东北虎进行了遗传多样性检测。用非变性聚丙烯酰胺凝胶电泳检测微卫星的PCR扩增产物,计算了10个微卫星基因座的等位基因频率、基因杂合度、多态信息含量和有效等位基因数。在113只东北虎样品中,10个基因座的等位基因数为3～6个,其中Fca152最多;等位基因频率处于0.009～0.767之间。基因杂合度值在0.385～0.707间,平均为0.616,多态信息含量值在0.353～0.658间,平均为0.558,有效等位基因数处于1.629～3.409之间,平均为2.784,表明所选用的10个微卫星基因座在研究样品中均为中高度多态性基因座,具有比较明显的遗传变异。113只样品中包括75只毛发样品,23只血液样品和15只组织样品,不同样品的结果比较表明,毛发、血液和组织样品均可以得到清晰的扩增结果。所以,微卫星基因座与非损伤性DNA分析方法可以成功地应用于濒危珍稀动物的遗传多样性研究。 Abstract:. The tiger is one of the most threatened wildlife species since the abundance and distribution of tiger have decreased dramatically in the last century. The wild Amur tiger (Panthera tigris altaica) only distributed in northeast China, the far east area of Russia and the north Korea and its size of wild population is about 450 in the world and 20 in China. Several hundred captive populations of Amur tigers are the main source to protect gene library of tiger and the source of recovering the wild populations. The Breeding Center for Felidae at Hengdaohezi and Ha’erbin Tiger Park in Heilongjiang Province is the biggest captive breeding base in China. How to make clear the genetic pedigree and establish reasonable breeding system is the urgent issues. So we use the microsatellite DNA markers and non-invasive technology to research on the genetic diversity of captive Amur tiger in this study. Ten microsatellite loci (Fca005, Fca075, Fca094, Fca152, Fca161, Fca294, Pti002, Pti003, Pti007 and Pti010), highly variable nuclear markers, were studied their genetic diversity in 113 captive Amur tigers. The PCR amplified products of microsatellite loci were detected by non-denatured polyacry lamide gel electrophoresis. Allele numbers, allelic frequency, gene heterozygosity(He), polymorphism information content(PIC) and effective number of allele(Ne) were calculated. 41 alleles were found and their size were ranged from 110bp to 250bp in ten microsatellite loci, Fca152 had 6 alleles, Fca075, Fca094 and Fca294 had 5 alleles, Fca005 and Pti002 had 4 alleles and the others had 3 alleles in all tiger samples, respectively. The allelic frequencies were from 0.009 to 0.767; The He ranged from 0.385 to 0.707, and Fca294 and Pti010 locus had the highest and lowest value; the PIC were from 0.353 to 0.658, Fca294 and Pti010 locus had the highest and lowest value; and Ne were from 1.626 to 3.409, Fca294 and Pti010 locus had the highest and lowest value, which showed the ten microsatellie loci had high or medium polymorphism in these Amur tigers and had high genetic diversity. At the same time, we only found even bases variability which showed the even bases repeat sequence (CA/GT) maybe the basic unit for length variability of microsatellite in all loci. In this study, the samples were made up of 75 hair specimens, 23 blood specimens and 15 tissue specimens, we obtained the genome DNA from hairs using the non-invasive DNA technology and demonstrated that DNA derived from hair samples is as good as that obtained from blood samples for the analaysis of microsatellite polymorphism. These results imply that microsatellite DNA markers and non-invasive DNA technology can help study the genetic diversity of Amur tiger. This method could be used in the captive management of other endangered species.     

Molecular paternity determination in captive bonobos and the impact of inbreeding on infant mortality

Inbreeding and the loss of genetic diversity may lower fitness and reduce the potential for a population to adapt to changing environments. In small populations, for example in captive populations or populations of endangered species, this can have considerable consequences for their survival. We investigated the effects of inbreeding on infant mortality in the world captive population of bonobos Pan paniscus . Using a combination of studbook data and high-quality pedigree data from genotyped individuals, inbreeding information was available for 142 captive-born individuals. For the determination of paternities that were unresolved in the studbook, nuclear microsatellite DNA was amplified from hair and blood samples using the Great Ape Kit and PowerPlex^® 16 System. In total, 54 bonobos (17 offspring and their putative parents) were genotyped at eight tetranucleotide repeat microsatellite loci. Inbreeding coefficients were calculated for each individual for whom paternity was confirmed by either studbook data or DNA analysis. We found significantly higher infant mortality in inbred offspring compared with non-inbred offspring, suggesting that inbreeding reduces infant survival in captive bonobos. In addition, we argue that the total magnitude of inbreeding depression is probably underestimated in this captive population. In conclusion, even though the breeding programme of captive bonobos is aimed at avoiding inbreeding, closely related individuals do occasionally produce offspring that do show inbreeding depression. There is, however, no indication that this currently threatens the long-time survival of the captive population of bonobos.     

Isolation and mapping of microsatellite markers specific for the D genome of bread wheat.

The potential of Aegilops tauschii, the diploid progenitor of the D genome of wheat, as a source of microsatellite markers for hexaploid bread wheat was investigated. By screening lambda phage and plasmid libraries of Ae. tauschii genomic DNA, dinucleotide microsatellites containing GA and GT motifs were isolated and a total of 65 functional microsatellite markers were developed. All primer pairs that were functional in Ae. tauschii amplified well in hexaploid wheat. Fifty-five loci amplified by 48 primer sets were placed onto a genetic framework map of the reference population of the International Triticeae Mapping Initiative (ITMI) 'Opata 85' x 'W7984'. The majority of microsatellite markers could be assigned to the chromosomes of the D genome of wheat. The distribution of the markers along the chromosomes is random. Chromosomal location of 22 loci nonpolymorphic in the reference population was determined using nullitetrasomic lines of Triticum aestivum 'Chinese Spring'. The results of this study demonstrate the value of microsatellite markers isolated from Ae. tauschii for the study of bread wheat. The microsatellite markers developed improve the existing wheat microsatellite map and can be used in a wide range of genetic studies and breeding programs.     

Genetic assessment of the Iberian wolf Canis lupus signatus captive breeding program

The main goal of ex situ conservation programs is to improve the chances of long term survival of natural populations by founding and managing captive colonies that can serve as a source of individuals for future reintroductions or to reinforce existing populations. The degree in which a captive breeding program has captured the genetic diversity existing in the source wild population has seldom been evaluated. In this study we evaluate the genetic diversity in wild and captive populations of the Iberian wolf, Canis lupus signatus, in order to assess how much genetic diversity is being preserved in the ongoing ex situ conservation program for this subspecies. A sample of domestic dogs was also included in the analysis for comparison. Seventy-four wolves and 135 dogs were genotyped at 13 unlinked microsatellite loci. The results show that genetic diversity in Iberian wolves is comparable in magnitude to that of other wild populations of gray wolf. Both the wild and the captive Iberian wolf populations have a similarly high genetic diversity indicating that no substantial loss of diversity has occurred in the captive-breeding program. The effective number of founders of the program was estimated as ∼ ∼16, suggesting that all founders in the studbook pedigree were genetically independent. Our results emphasize also the genetic divergence between wolves and domestic dogs and indicate that our set of 13 microsatellite loci provide a powerful diagnostic test to distinguish wolves, dogs and their hybrids.     

Evaluation of different sources of DNA for use in genome wide studies and forensic application

In the field of epidemiology, Genome-Wide Association Studies (GWAS) are commonly used to identify genetic predispositions of many human diseases. Large repositories housing biological specimens for clinical and genetic investigations have been established to store material and data for these studies. The logistics of specimen collection and sample storage can be onerous, and new strategies have to be explored. This study examines three different DNA sources (namely, degraded genomic DNA, amplified degraded genomic DNA and amplified extracted DNA from FTA card) for GWAS using the Illumina platform. No significant difference in call rate was detected between amplified degraded genomic DNA extracted from whole blood and amplified DNA retrieved from FTA™ cards. However, using unamplified–degraded genomic DNA reduced the call rate to a mean of 42.6% compared to amplified DNA extracted from FTA card (mean of 96.6%). This study establishes the utility of FTA™ cards as a viable storage matrix for cells from which DNA can be extracted to perform GWAS analysis.     

Evaluation of the genetic management of the endangered black‐footed ferret (Mustela nigripes)

Empirical support for the genetic management strategies employed by captive breeding and reintroduction programs is scarce. We evaluated the genetic management plan for the highly endangered black‐footed ferret (Mustela nigripes) developed by the American Zoo and Aquarium Associations (AZA) as a part of the species survival plan (SSP). We contrasted data collected from five microsatellite loci to predictions from a pedigree‐based kinship matrix analysis of the captive black‐footed ferret population. We compared genetic diversity among captive populations managed for continued captive breeding or reintroduction, and among wild‐born individuals from two reintroduced populations. Microsatellite data gave an accurate but only moderately precise estimate of heterozygosity. Genetic diversity was similar in captive populations maintained for breeding and release, and it appears that the recovery program will achieve its goal of maintaining 80% of the genetic diversity of the founder population over 25 years. Wild‐born individuals from reintroduced populations maintained genetic diversity and avoided close inbreeding. We detected small but measurable genetic differentiation between the reintroduced populations. The model of random mating predicted only slightly lower levels of heterozygosity retention compared to the SSP strategy. The random mating strategy may be a viable alternative for managing large, stable, captive populations such as that of the black‐footed ferret. Zoo Biol 22:287–298, 2003. © 2003 Wiley‐Liss, Inc.     

Development of a multiplex PCR assay for fine-scale population genetic analysis of the Komodo monitor Varanus komodoensis based on 18 polymorphic microsatellite loci

Multiplex PCR assays for the coamplification of microsatellite loci allow rapid and cost-effective genetic analyses and the production of efficient screening protocols for international breeding programs. We constructed a partial genomic library enriched for di-nucleotide repeats and characterized 14 new microsatellite loci for the Komodo monitor (or Komodo dragon, Varanus komodoensis). Using these novel microsatellites and four previously described loci, we developed multiplex PCR assays that may be loaded on a genetic analyser in three separate panels. We tested the novel set of microsatellites for polymorphism using 69 individuals from three island populations and evaluated the resolving power of the entire panel of 18 loci by conducting (i) a preliminary assignment test to determine population(s) of origin and (ii) a parentage analysis for 43 captive Komodo monitors. This panel of polymorphic loci proved useful for both purposes and thus can be exploited for fine-scale population genetic analyses and as part of international captive breeding programs directed at maintaining genetically viable ex situ populations and reintroductions.     

Isolation and characterization of polymorphic microsatellites from red coral grouper (Plectropomus maculatus)

The red coral grouper, Plectropomus maculatus, is a high value marine food fish species. We isolated the first set of 10 polymorphic microsatellites and examined their polymorphism in two related species: the Malabar grouper Epinephelus malabaricus and the brown‐marbled grouper Epinephelus fuscoguttatus. The average allele number of these 10 microsatellite DNA loci is 9.1/locus with a range of four to 17, whereas the expected heterozygosity ranged from 0.18 to 0.94, averaging 0.76. Five of the 10 markers amplified polymorphic and specific products in E. malabaricus, whereas only four were amplified in E. fuscoguttatus. These microsatellites could be applicable to captive breeding and to the studies on genetic diversity and population structure of grouper wild stocks.     

Captive ring‐tailed lemur breeding in semi‐free ranging conditions and genetic parentage analysis

The ring‐tailed lemur (Lemur catta), one of the most iconic and widely recognized primates in the world, is threatened in its native range and classified as endangered by the International Union for Conservation of Nature. The global conservation strategy for the species includes ex situ conservation efforts led by zoological institutions in the framework of regional captive breeding programs. To maximize the conservation of genetic diversity and optimize breeding programs, an accurate pedigree must be established in captive populations. Our study documents the formation and subsequent monitoring of a large ring‐tailed lemur group in a French zoological institution. Based on staff's behavioral observations and DNA analyses using 11 microsatellite markers, we were able to document the survival and reproduction rates of the lemurs in this situation and reconstruct the pedigree of infants born in the institution. In total, 22 of the 28 imported animals as well as 4 locally born individuals gave birth to 58 infants of which 28 survived. The consistent genotypes obtained from the 53 sampled animals allowed us to identify 25 of the sires and 27 of the dams for the 28 surviving infants. A total of 12 different females and 14 different males produced the 28 surviving infants, suggesting that no individual dominates reproduction. This pioneering study has practical applications for the captive management of the species, which can be raised to the individual level instead of the group level.     

Microsatellite DNA markers in Populus tremuloides.

Markers for eight new microsatellite DNA or simple sequence repeat (SSR) loci were developed and characterized in trembling aspen (Populus tremuloides) from a partial genomic library. Informativeness of these microsatellite DNA markers was examined by determining polymorphisms in 38 P. tremuloides individuals. Inheritance of selected markers was tested in progenies of controlled crosses. Six characterized SSR loci were of dinucleotide repeats (two perfect and four imperfect), and one each of trinucleotide and tetranucleotide repeats. The monomorphic SSR locus (PTR15) was of a compound imperfect dinucleotide repeat. The primers of one highly polymorphic SSR locus (PTR7) amplified two loci, and alleles could not be assigned to a specific locus. At the other six polymorphic loci, 25 alleles were detected in 38 P. tremuloides individuals; the number of alleles ranged from 2 to 7, with an average of 4.2 alleles per locus, and the observed heterozygosity ranged from 0.05 to 0.61, with an average of 0.36 per locus. The two perfect dinucleotide and one trinucleotide microsatellite DNA loci were the most informative. Microsatellite DNA variants of four SSR loci characterized previously followed a single-locus Mendelian inheritance pattern, whereas those of PTR7 from the present study showed a two-locus Mendelian inheritance pattern in controlled crosses. The microsatellite DNA markers developed and reported here could be used for assisting various genetic, breeding, biotechnology, genome mapping, conservation, and sustainable forest management programs in poplars.     

Integrating microsatellite and pedigree analyses to facilitate the captive management of the endangered Mississippi sandhill crane (Grus canadensis pulla)

The minimization of kinship in captive populations is usually achieved through the use of pedigree information. However, pedigree knowledge alone is not sufficient if pedigree information is missing, questionable, or when the founders of the captive population are related to one another. If this is the case, higher levels of inbreeding and lower levels of genetic diversity may be present in a captive population than those calculated by pedigree analyses alone. In this study, the genetic status of the critically endangered Mississippi sandhill crane (MSC) (Grus canadensis pulla) was analyzed using studbook data from the U.S. Fish and Wildlife Service managed captive breeding program as well as microsatellite DNA data. These analyses provided information on shared founder genotypes, allowing for refined analysis of genetic variation in the population, and the development of a new DNA-based studbook pedigree that will assist in the genetic management of the MSC population.     

Genetic structure analysis of a highly inbred captive population of the African antelope Addax nasomaculatus. Conservation and management implications

The African antelope Addax nasomaculatus is a rare mammal at high risk of extinction, with no more than 300 individuals in the wild and 1,700 captive animals distributed in zoos around the world. In this work, we combine genetic data and genealogical information to assess the structure and genetic diversity of a captive population located at Parque Lecocq Zoo (N=27), originated from only two founders. We amplified 39 microsatellites previously described in other Artiodactyls but new to this species. Seventeen markers were polymorphic, with 2–4 alleles per locus (mean=2.71). Mean expected heterozygosity (He) per locus was between 0.050 (marker ETH3) and 0.650 (marker D5S2), with a global He of 0.43. The mean inbreeding coefficient of the population computed from pedigree records of all registered individuals (N=53) was 0.222. The mean coancestry of the population was 0.298 and F_IS index was ?0.108. These results reflect the importance of an adequate breeding management on a severely bottlenecked captive population, which would benefit by the incorporation of unrelated individuals. Thanks to the successful amplification of a large number of microsatellites commonly used in domestic bovids, this study will provide useful information for the management of this population and serve as future reference for similar studies in other captive populations of this species. Zoo Biol 30:399–411, 2011. © 2010 Wiley‐Liss, Inc.     

Identification of Hybrids of Painted and Milky Storks Using FTA Card-Collected Blood, Molecular Markers, and Morphologies

Suspicious hybrids of painted storks and milky storks were found in a Malaysian zoo. Blood of these birds was sampled on FTA cards for DNA fingerprinting. Of 44 optimized primers, 6 produced diagnostic markers that could identify hybrids. The markers were based on simple, direct PCR-generated multilocus banding patterns that provided two sets of genetic data, one for each of the two stork species and another for the hybrids. It also revealed that large DNA fragments (3,000 bp) could be amplified from blood collected on FTA cards. When the results of each individual bird’s DNA fingerprint were compared with plumage characters, the hybrids were found to express a range of intermediate phenotypic traits of the pure breeds with no dominant plumage characteristic from either parental species.     

Pedigree Analysis of the Sika Deer (Cervus nippon) using Microsatellite Markers

The usefulness of microsatellite markers in pedigree analysis of the sika deer (Cervus nippon) was tested in a herd in which the maternal lineages were recorded. Eighteen sets of microsatellite primers originally designed for bovine, ovine, and cervine loci successfully amplified polymorphic DNA in the deer. The numbers of alleles per locus ranged from two to seven, and the observed heterozygosity ranged from 0.350 to 0.900. The resolution power of the markers in paternity testing was then determined by calculating exclusion probabilities and paternity indices. Parentages of the study population were efficiently discriminated by genotyping 17 microsatellite loci. The microsatellite data were also used to calculate the genetic relatedness between individuals, which significantly correlated with coancestry coefficients for the pairs. Our results demonstrate that the microsatellite markers are efficient tools in studying the social structure and behavior of the sika deer, as well as in monitoring the inbreeding status.     

适用于种群遗传管理的小熊猫微卫星亲子鉴定体系的建立

由于标记缺失、生产记录不详、亲权关系不明及部分个体来源不清等历史问题,中国小熊猫圈养种群存在谱系错漏、近亲繁殖等风险。近年来,随着小熊猫种群规模不断扩大,管理者们对谱系的准确性提出了更高的需求,亲子鉴定工作也成为了研究的重点。本文采用26个微卫星标记,对国内3个小熊猫圈养种群进行了亲缘关系运算,完成了相关谱系的查错与整理。26个位点多态性与稳定性良好,联合非亲排除概率达到0.9999以上,可解决国内小熊猫圈养种群的各类亲子鉴定需求。在单亲未知或双亲未知的情况下,8或11个位点组合可实现亲子鉴定。5个位点组合可进行个体识别。在小熊猫圈养管理过程中,应用一套亲子鉴定体系对小熊猫圈养的谱系进行查漏补缺,有利于制定科学的配对计划、避免近亲繁殖,对小熊猫种群保护有着重要意义。     

Microsatellite markers isolated from polyploid wood‐sorrel Oxalis alpina (Oxalidaceae)

Twelve polymorphic microsatellite loci were isolated from polyploid alpine wood‐sorrel, Oxalis alpina (Oxalidaceae), and optimized for future studies of its breeding system. The loci were screened for variability among 72 individuals from Pinos Altos, New Mexico. The primers amplified loci with allele number ranging from two to 17 per locus and with estimates of Nei's genetic diversity varying from 0.10 to 0.99. These primers provide an opportunity to use polymorphic DNA markers to study the causes of breeding system variability in this species.     

Non-invasive genotyping of primates

Using DNA amplified from shed or plucked hair follicles it is now possible to genotype individual primates at many nuclear and mitochondrial gene loci. Sequence specific primers and the polymerase chain reaction permit the rapid production of sufficient DNA from a single hair for numerous analyses. The direct sequencing of relatively conservative mtDNA sequences like cytochromeb is proving useful in establishing species and subspecies-level relationships. More variable sequences (e.g. the mtDNA control region or D-loop) are useful at the population and social community levels. Paternity exclusion, pedigree relationships, and community structure can be determined using simple sequence length polymorphisms (SSLPs) of multiple hypervariable nuclear microsatellite or simple sequence repeat (SSR) loci. Studies involving captive and free-ranging chimpanzees, gibbons, and macaques illustrate the resolving power of these new non-invasive molecular genetic genotyping techniques.     

Development of a set of highly polymorphic genomic microsatellites (gSSRs) in Sitka spruce (Picea sitchensis (Bong.) Carr.)

Robust, polymorphic microsatellite DNA markers (simple sequence repeats—SSRs) are valuable tools for a range of tree conservation and breeding applications. SSRs are routinely used in the study of population genetic structure and diversity, pedigree reconstruction and genetic linkage mapping. Their abundance in the genome, co-dominant inheritance and potential for cross-species amplification make microsatellites highly prized markers. This paper characterises 22 novel genomic polymorphic microsatellite loci for Sitka spruce (Picea sitchensis (Bong.) Carr.). Amplification of DNA from Sitka spruce material was carried out both with a set of unrelated trees to obtain diversity statistics for each locus, and with the progeny of a full-sib family to test simple Mendelian inheritance. Observed heterozygosity ranged from 0.38 to 0.91 and allele number per locus ranged from 6 to 21, with a mean of 12.2. In addition, the primer pairs were tested with DNA from Norway spruce (P. abies) and white spruce (P. glauca) to investigate their potential for cross-species amplification and ten loci amplified in all three species. The results from these genomic microsatellites are compared to data generated from microsatellites derived from Picea EST libraries. In summary, this novel, highly polymorphic markers represent a significant addition to the rapidly expanding Picea genomics tool-box. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Parentage verification of a mountain zebra (Equus zebra hartmannae) using polymorphic microsatellite markers

The Hartmann's mountain zebra (Equus zebra hartmannae) has been classified by the IUCN as a vulnerable species. Approximately 300 individuals, maintained in zoos throughout Europe and the United States of America, are being managed as part of a captive breeding program. An International Studbook is maintained for the Hartmann's mountain zebra at Marwell Zoological Park, UK. Despite the use of a variety of means to identify each individual in a captive herd, confusion sometimes occurs, resulting in the misidentification of an animal. Here we report the first application of DNA typing, using polymorphic microsatellite loci, to resolve a misidentification involving two female Hartmann's mountain zebra. This case demonstrates the way in which genetic tests derived from a related domesticated species may be used as an effective tool for captive management. Further, this case highlights the need to be able to conclusively identify captive individuals and to maintain accurate pedigree information for successful captive management. © 1995 Wiley-Liss, Inc.