Twenty-one novel tri- and tetranucleotide microsatellite loci for the Amur tiger (Panthera tigris altaica)

Amur tiger is the largest subspecies of tiger in the world and his conservation has also received much attention. In this study, we isolated and characterized twenty-one tri- and tetranucleotide microsatellite markers from this species. The number of alleles for each locus ranged from two to nine in a group of 60 individuals and the observed and expected heterozygosities were 0.333–0.917 and 0.302–0.822, respectively. The overall discrimination power and exclusion probabilities in parentage and paternity testing for these markers were 1.00, 0.9947 and 0.9999, respectively, indicating high-resolution power of microsatellite markers.     

Analysis of microsatellites and parentage testing in saltwater crocodiles

Fifteen microsatellite loci were evaluated in farmed saltwater crocodiles for use in parentage testing. One marker (C391) could not be amplied. For the remaining 14, the number of alleles per locus ranged from two to 16, and the observed heterozygosities ranged from 0.219 to 0.875. The cumulative exclusion probability for all 14 loci was .9988. the 11 loci that showed the greatest level of polymorphism were used for parentage testing, with an exclusion probability of .9980. With these 11 markers on 107 juveniles from 16 known breeding pairs, a 5.6% pedigree error rate was detected. This level of pedigree error, if consistent, could have an impact on the accuracy of gentic parameter and breeding value estimation. The usefulness of these markers was also evaluated for assigning parentage in situations where maternity, paternity, or both may not be known. In these situations, a 2% error in parentage assignment was predicted. It is therefore recommended that more micro-satellite markers be used in these situations. The use of these microsatellite markers will broaden the scope of a breeding program, allowing progeny to be tested from adults maintained in large breeding lagoons for selection as future breeding animals.     

北京地区汉族群体D1S1612和D18S535基因座的遗传多态性

采用扩增片段长度多态性（Amp-FLP）分型技术,调查中国北京地区汉族群体D1S1612、D18S535 基因座的遗传多态性,获得等位基因频率分布。结果显示, D1S1612检出9个等位基因,25种基因型, D18S535检出9个等位基因,27种基因型。两个STR基因座的杂和度（H）分别为0.779、0.887;个人识别率（Dp）分别为0.901、0.927;非父排除率（PE）分别为0.564、0.770;多态信息容量（PIC）分别为0.723、0.796,卡方检验表明两个STR 基因座基因型频率分布符合Hardy-Weinberg平衡 (P>0.01 )。D1S1612和D18S535 基因座均属高杂合度、高识别能力的遗传标记,可用于法庭科学亲子鉴定和个人识别。 Abstract: To investigate the genetic polymorphism of D1S1612 and D18S535 in Han population of Beijing. Amp-FLP method was used. 9 alleles, 25 genotypes were observed for D1S1612 locus; and 9 alleles and 27 genotypes for D18S535 locus. All allele frequencies, heterozygosity (H), discrimination power (Dp), exclusion of paternity probability (PE) and polymorphism information content (PIC) were calculated. The allele distributions of the two loci were conformed to Hardy-Weinberg equilibrium (P>0.01). According to the results obtained in this study, it is suggested that both D1S1612 and D18S535 are useful genetic markers for individual identification and paternity testing in forensic science practice as well for genetic study.     

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.     

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.     

Polymorphism profile of nine short tandem repeat Loci in the Han chinese

Nine short tandem repeat (STR) markers (D3S1358, VWA, FGA, THO1, TPOX, CSFIPO, D5S818, D13S317, and D7S820) and a sex-identification marker (Amel-ogenin locus) were amplified with multiplex PCR and were genotyped with a four-color fluorescence method in samples from 174 unrelated Han individuals in North China. The allele frequencies, genotype frequencies, heterozygosity, probability of discrimination powers, probability of paternity exclusion and Hardy-Weinberg equilibrium expectations were determined. The results demonstrated that the genotypes at all these STR loci in Han population conform to Hardy-Weinberg equilibrium expectations. The combined discrimination power (DP) was 1.05×10-10 within nine STR loci analyzed and the probability of paternity exclusion (EPP) was 0.9998. The results indicate that these nine STR loci and the Amelo-genin locus are useful markers for human identification, paternity and maternity testing and sex determination in forensic sciences.     

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.     

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.     

Microsatellite primers for Sorbus torminalis and related species

This study reports the cloning and characterization of nine microsatellite primer pairs in a scattered woody species (Sorbus torminalis), and shows their potential for further use in 36 species of the Maloideae, a Rosaceae subfamily containing important fruit crop and ornamental species. These primers were designed from a microsatellite library constructed from genomic DNA of S. torminalis and enriched for CA and GA repeats. Genotyping 48 S. torminalis of a natural population with the six best markers yielded a mean of 10.7 alleles per locus, and an expectation of exclusion probability for paternity analysis greater than 0.993.     

Development and characterization of 15 microsatellite loci for Cariniana estrellensis and transferability to Cariniana legalis, two endangered tropical tree species

From a genomic library enriched for GA/CA repeats, 15 highly polymorphic microsatellite markers were developed for Cariniana estrellensis, a tropical forest tree. The microsatellite loci were screened in 49 mature trees found between Pardo river and Mogi-Guaçu river basins, in the state of São Paulo, Brazil. A total of 140 alleles were detected with an average of 9.33 alleles per locus. The expected heterozygosity ranged from 0.37 to 0.88. These loci showed a high probability of paternity exclusion. Additionally, 12 loci were effectively transferred to Cariniana legalis. High levels of polymorphism make the present SSR markers useful for population genetic studies.     

Characterization of microsatellites in wild and sweet cherry (Prunus avium L.)--markers for individual identification and reproductive processes.

Nuclear microsatellites were characterized in Prunus avium and validated as markers for individual and cultivar identification, as well as for studies of pollen- and seed-mediated gene flow. We used 20 primer pairs from a simple sequence repeat (SSR) library of Prunus persica and identified 7 loci harboring polymorphic microsatellite sequences in P. avium. In a natural population of 75 wild cherry trees, the number of alleles per locus ranged from 4 to 9 and expected heterozygosity from 0.39 to 0.77. The variability of the SSR markers allowed an unambiguous identification of individual trees and potential root suckers. Additionally, we analyzed 13 sweet cherry cultivars and differentiated 12 of them. An exclusion probability of 0.984 was calculated, which indicates that the seven loci are suitable markers for paternity analysis. The woody endocarp was successfully used for resolution of all microsatellite loci and exhibited the same multilocus genotype as the mother tree, as shown in a single seed progeny. Hence, SSR fingerprinting of the purely maternal endocarp was also successful in this Prunus species, allowing the identification of the mother tree of the dispersed seeds. The linkage of microsatellite loci with PCR-amplified alleles of the self-incompatibility locus was tested in two full-sib families of sweet cherry cultivars. From low recombination frequencies, we inferred that two loci are linked with the S locus. The present study provides markers that will significantly facilitate studies of spatial genetic variation and gene flow in wild cherry, as well as breeding programs in sweet cherry.     

Polymorphic microsatellites in the Reeves's pheasant developed by cross-species amplification

Cross-species microsatellite amplification is an effective way of obtaining microsatellite loci for closely related taxa in bird species. The Reeves's pheasant, Syrmaticus reevesii, is a vulnerable species endemic to China. To improve population genetics and parentage analysis studies in this species, we obtained nine polymorphic microsatellite markers, in addition to the nine markers previously isolated, from the cross-species amplification of 52 markers. The number of alleles per locus varied between two and 12 with expected heterozygosity ranging from 0.298 to 0.714 (n = 107). The success rates of simulated paternity tests using CERVUS software improved at different confidence levels after adding these markers to the previous ones.     

Development and characterization of microsatellite markers for the tropical tree species Tabebuia aurea (Bignoniaceae)

In this study, we report the development and characterization of 21 polymorphic microsatellite loci for Tabebuia aurea, using genomic library enrichment. Number of alleles per locus and expected heterozygosity ranged from nine to 26 and from 0.808 to 0.955. The high combined probability of genetic identity (1.03 × 10^?37) and probability of paternity exclusion (0.9889) showed that multilocus genotypes are likely to be unique and will allow detailed parentage studies in natural populations of T. aurea. Additionally, a high percentage of transferability was achieved for the four species of the same genus studied.     

Exclusion probabilities for pedigree testing farm animals

Pedigree testing, using genetic markers, may be undertaken for a variety of situations, of which the classical paternity testing is only one. This has not always been made clear in the literature. Exclusion probabilities associated with various testing situations, including the use of autosomal or X-linked codominant marker systems with any number of alleles, are presented. These formulae can be used to determine the appropriate exclusion probability for the situation being investigated. One such situation is where sire groups of progeny are to be verified without knowledge of the dams' genotypes, in which case the classical paternity exclusion probability is too high, and if used may result in an optimistic declaration about the progeny that have not been excluded. On the other hand, if mating pairs are known then incorrect progeny can be excluded at a higher rate than suggested by paternity exclusion calculations. The formulae also assist in determining the usefulness of X-linked markers, particularly if the pedigree checks involve progeny of only one sex. A system of notation that is useful for the algebraic manipulation of genetic probabilities, including exclusion probabilities as presented here, is also given.     

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

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

Microsatellite analysis of genetic diversity in the Chinese alligator (<Emphasis Type="Italic">Alligator sinensis</Emphasis>) Changxing captive population

Chinese alligator (Alligator sinensis) is a critically endangered species endemic to China. In this study, the extent of genetic variation in the captive alligators of the Changxing Reserve Center was investigated using microsatellite markers derived from American alligators. Out of 22 loci employed, 21 were successfully amplified in the Chinese alligator. Sequence analysis showed loci in American alligators had a bigger average size than that of the Chinese alligators and the longest allele of an individual locus almost always existed in the species with longer stretch of repeat units. Eight of the 22 loci were found to be polymorphic with a total of 26 alleles present among 32 animals scored, yielding an average of 3.25 alleles per polymorphic locus. The expected heterozygosity (H _E) ranged at a moderate level from 0.4385 to 0.7163 in this population. Compared to that in the American alligators, a lower level of microsatellite diversity existed in the Changxing population as revealed by about 46% fewer alleles per locus and smaller H _E at the homologous loci. The average exclusion power and the ability to detect shared genotypes and multiple paternity were evaluated for those markers. Results suggested that when the polymorphic loci were combined, they could be sensitive markers in genetic diversity study and relatedness inference within the Chinese alligator populations. The level of genetic diversity present in the current Changxing population indicated an important resource to complement reintroductions based on the individuals from the other population. In addition, the microsatellite markers and their associated diversity characterized in this population could be utilized to further investigate the genetic status of this species.     

Individual Identification and Paternity Determination in Chimpanzees (Pan troglodytes) Using Human Short Tandem Repeat (STR) Markers

We studied 155 human short tandem repeat (STR) DNA markers in chimpanzees (Pan troglodytes) via the polymerase chain reaction (PCR). There is no difference in number of alleles per locus among STRs of different motif length (di-, tri-, or tetranucleotide repeats). We investigated 42 of the most informative STRs in greater detail using DNA isolated from a panel of 41 African-born, captive-housed chimpanzees. They reveal a wealth of genetic variability in chimpanzees, with an average of six alleles and 70.6% heterozygosity. The average paternity exclusion probability is 51.6%, and the best three STRs jointly provide >95% mean exclusion probability. Used in combination to define a multiple-locus genotype, the five most informative focal STRs can potentially uniquely identify every chimpanzee alive in the world. Although the subjects are of unknown geographical origin, homozygosity tests indicate little evidence for population subdivision. These markers represent the basis of a powerful battery of genetic tests, including individual identification, e.g., in poaching, paternity testing, or reconstruction of pedigrees among captive and wild chimpanzee breeding populations.     

Characterization of 22 microsatellites loci from the endangered Houbara bustard (Chlamydotis undulata undulata)

Here we present a new set of 22 microsatellite loci isolated from Chlamydotis undulata undulata, an endangered Houbara bustard found across North Africa. The number of alleles per locus ranged from one to nine, and heterozygosities ranged from 0.167 to 0.944. Total exclusionary probabilities using these loci for the first and the second parent were 0.992932 and 0.999915, respectively. Successful cross‐amplification was observed in eight other Otididae species (12–22 of the 22 loci). These microsatellite markers are powerful tools for genetic identification, paternity assignment and population genetic studies.     

Genetic characterization of 12 heterologous microsatellite markers for the giant tropical tree Cariniana legalis (Lecythidaceae)

Twelve microsatellite loci previously developed in the tropical tree Cariniana estrellensis were genetically characterized in Cariniana legalis. Polymorphisms were assessed in 28 C. legalis individuals found between the Pardo and Mogi-Guaçu River basins in the state of São Paulo, Brazil. Of the 12 loci, 10 were polymorphic and exhibited Mendelian inheritance. The allelic richness at each locus ranged from 2-11, with an average of 7 alleles per locus, and the expected heterozygosity ranged from 0.07-0.88. These loci showed a high probability of paternity exclusion. The characteristics of these heterologous microsatellite markers indicate that they are suitable tools for investigating questions concerning population genetics in C. legalis.     

Isolation and characterization of novel microsatellites from the critically endangered hawksbill sea turtle (Eretmochelys imbricata)

We isolated and characterized 12 microsatellite loci from the hawksbill sea turtle (Eretmochelys imbricata). The loci exhibited a variable number of alleles that ranged from three to 14 with an average observed heterozygosity of 0.70 (SD 0.18) across 40 hawksbill turtles from the Caribbean. The polymorphism exhibited individually and in combination makes them suitable for fine-scale genetic studies. In particular, the low probability of identity and high paternity exclusion of these markers makes them highly useful for parentage and relatedness studies. These new markers greatly increase the power of genetic studies directed towards the conservation of this endangered species.