Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Novas metodologias de análise molecular voltadas para estudos populacionais, clínicos, evolutivos, da biodiversidade e identificação forense foram desenvolvidas com base em marcadores microssátelites ou STR Short Tandem Repeats. Os marcadores STR, que estão amplamente espalhados nos genomas e se caracterizam por apresentar alto grau de polimorfismo, podem ser analisados a partir da amplificação por PCR (Reação em Cadeia da polimerase). A análise foi facilitada a partir do desenvolvimento de sistemas de amplificação simultânea de múltiplos STR (multiplex STR) e com a detecção automatizada dos produtos de amplificação marcados por fluorescência. Recentemente, o uso de marcadores STR do cromossomo X (X-STR) tornou-se significativo na prática forense. Devido ao seu modo de transmissão, os X-STR são úteis em situações particulares de investigação de relações de parentesco, apresentando vantagens sobre o uso de STR autossômicos. Este estudo teve como principal objetivo o desenvolvimento e validação de sistema multiplex, denominado LDD (X-STR) Decaplex, capaz de amplificar dez loci X-STR (DXS7133, DXS7424, DXS8378, DXS6807, DXS7132, DXS10074, DXS7423, DXS8377, GATA172D05 e DXS10101) para aplicação em genética populacional, identificação e análises forenses. Utilizando o LDD (X-STR) Decaplex 170 indivíduos autodenominados afrodescendentes, não aparentados geneticamente, foram genotipados. As freqüências alélicas e genotípicas não apresentaram desvio do equilíbrio de Hardy-Weinberg e estão em concordância com aquelas observadas em outros estudos. Os haplótipos observados foram únicos em indivíduos de amostra masculina. A análise de desequilíbrio de ligação não revelou associação entre os marcadores X-STR. A diversidade genética foi elevada, variando entre 0,6218 para o locus DXS7133 a 0,9327 para o locus DXS8377. Os parâmetros de Probabilidade de Vinculação (PV), Índice de Vinculação (IV), Poder de Exclusão (PE), Poder de Discriminação e Razão de Verossimilhança foram também elevados, demonstraram que os dez X-STRs são altamente polimórficos e discriminativos na população estudada. A concentração mínima de DNA para a amplificação dos loci do LDD (X-STR) Decaplex é de 0,5 ng e verificamos que amplificação por PCR pode ser afetada quando são adicionados mais de 5 ng de DNA nas reações. Os percentuais de bandas stutter foram elevados para os loci DXS7132 e DXS8377. No teste de reprodutibilidade observamos consistência entre as tipagem de diferentes amostras biológicas, incluindo as de restos mortais. No teste de mistura a proporção limite em que observamos a coexistência de duas espécies biológicas foi de 2,5:1ng (feminino-masculino). Os resultados evidenciaram que os loci do LDD (X-STR) Decaplex são altamente informativos, consistindo, em conjunto, uma ferramenta importante em estudos de identificação humana e de relações de parentesco. / New molecular analysis technologies used to evolving clinic and population studies of biodiversity and forensic identification have been developed based on microsatellite markers or STR Short Tandem Repeats. These STR markers, which are widely spread on genomes and characterized by their high degree of polymorphism, can be analyzed by PCR (Polymerase Chain Reaction) amplification technology. This analysis was facilitated from the development of simultaneous amplification system of multiples STR (multiplex STR) and automatic detection of amplification products by fluorescence. Lately, the use of STR markers of chromosome X (X-STR) has become significant in forensic practice. Due to their transmission ways, X-STR are useful at especial kinship investigation, showing advantages in relation to the use of STR autosomal. This study main focus was the development and validation of the multiplex system called LDD (X-STR) Decaplex, which was able to amplify ten X-STR loci (DXS7133, DXS7424, DXS8378, DXS6807, DXS7132, DXS10074, DXS7423, DXS8377, GATA172D05 and DXS10101) in order to be used in population genetics application, identification and forensic analysis. By using LDD (X-STR) Decaplex 170, individuals, who were self-appointed Africandescendants and genetically unrelated, were genotyped. Allele and genotype frequencies have not shown Hardy-Weinberg equilibrium deviation and are in agreement with the ones observed in other studies. The observed haplotypes were unique in male individual samples. The linkage disequilibrium analysis has not shown any association among the X-STR markers. The genetic diversity was high, ranging from 0.6218 (DXS7133 locus) to 0.9327 (DXS8377 locus). The parameters of Probability of Relatedness (PR), Avuncular Index (AI), Power of Exclusion (PE), Power of Discrimination and Likelihood Ratios were also high, showing that these ten X-STRs are highly polymorphous and discriminating on the studied population. The minimum DNA concentration for the amplification of the LDD (X-STR) Decaplex loci is 0.5 ng and it was also verified that the PCR amplification can be affected when more than 5 ng of DNA are added to the reactions. The "stutter" bands percentages were high to DXS7132 and DXS8377 loci. At the reproducing test, consistency among typing of different biological samples was observed, including the ones from remains. At the mixing test, the limit proportion in which two biological species coexisted was 2.5:1 ng (female-male). The results point out that LDD (X-STR) Decaplex loci are able to provide many important piece of information which, as a whole, are a very important tool at human identification and of kinship studies.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Novas metodologias de análise molecular voltadas para estudos populacionais, clínicos, evolutivos, da biodiversidade e identificação forense foram desenvolvidas com base em marcadores microssátelites ou STR Short Tandem Repeats. Os marcadores STR, que estão amplamente espalhados nos genomas e se caracterizam por apresentar alto grau de polimorfismo, podem ser analisados a partir da amplificação por PCR (Reação em Cadeia da polimerase). A análise foi facilitada a partir do desenvolvimento de sistemas de amplificação simultânea de múltiplos STR (multiplex STR) e com a detecção automatizada dos produtos de amplificação marcados por fluorescência. Recentemente, o uso de marcadores STR do cromossomo X (X-STR) tornou-se significativo na prática forense. Devido ao seu modo de transmissão, os X-STR são úteis em situações particulares de investigação de relações de parentesco, apresentando vantagens sobre o uso de STR autossômicos. Este estudo teve como principal objetivo o desenvolvimento e validação de sistema multiplex, denominado LDD (X-STR) Decaplex, capaz de amplificar dez loci X-STR (DXS7133, DXS7424, DXS8378, DXS6807, DXS7132, DXS10074, DXS7423, DXS8377, GATA172D05 e DXS10101) para aplicação em genética populacional, identificação e análises forenses. Utilizando o LDD (X-STR) Decaplex 170 indivíduos autodenominados afrodescendentes, não aparentados geneticamente, foram genotipados. As freqüências alélicas e genotípicas não apresentaram desvio do equilíbrio de Hardy-Weinberg e estão em concordância com aquelas observadas em outros estudos. Os haplótipos observados foram únicos em indivíduos de amostra masculina. A análise de desequilíbrio de ligação não revelou associação entre os marcadores X-STR. A diversidade genética foi elevada, variando entre 0,6218 para o locus DXS7133 a 0,9327 para o locus DXS8377. Os parâmetros de Probabilidade de Vinculação (PV), Índice de Vinculação (IV), Poder de Exclusão (PE), Poder de Discriminação e Razão de Verossimilhança foram também elevados, demonstraram que os dez X-STRs são altamente polimórficos e discriminativos na população estudada. A concentração mínima de DNA para a amplificação dos loci do LDD (X-STR) Decaplex é de 0,5 ng e verificamos que amplificação por PCR pode ser afetada quando são adicionados mais de 5 ng de DNA nas reações. Os percentuais de bandas stutter foram elevados para os loci DXS7132 e DXS8377. No teste de reprodutibilidade observamos consistência entre as tipagem de diferentes amostras biológicas, incluindo as de restos mortais. No teste de mistura a proporção limite em que observamos a coexistência de duas espécies biológicas foi de 2,5:1ng (feminino-masculino). Os resultados evidenciaram que os loci do LDD (X-STR) Decaplex são altamente informativos, consistindo, em conjunto, uma ferramenta importante em estudos de identificação humana e de relações de parentesco. / New molecular analysis technologies used to evolving clinic and population studies of biodiversity and forensic identification have been developed based on microsatellite markers or STR Short Tandem Repeats. These STR markers, which are widely spread on genomes and characterized by their high degree of polymorphism, can be analyzed by PCR (Polymerase Chain Reaction) amplification technology. This analysis was facilitated from the development of simultaneous amplification system of multiples STR (multiplex STR) and automatic detection of amplification products by fluorescence. Lately, the use of STR markers of chromosome X (X-STR) has become significant in forensic practice. Due to their transmission ways, X-STR are useful at especial kinship investigation, showing advantages in relation to the use of STR autosomal. This study main focus was the development and validation of the multiplex system called LDD (X-STR) Decaplex, which was able to amplify ten X-STR loci (DXS7133, DXS7424, DXS8378, DXS6807, DXS7132, DXS10074, DXS7423, DXS8377, GATA172D05 and DXS10101) in order to be used in population genetics application, identification and forensic analysis. By using LDD (X-STR) Decaplex 170, individuals, who were self-appointed Africandescendants and genetically unrelated, were genotyped. Allele and genotype frequencies have not shown Hardy-Weinberg equilibrium deviation and are in agreement with the ones observed in other studies. The observed haplotypes were unique in male individual samples. The linkage disequilibrium analysis has not shown any association among the X-STR markers. The genetic diversity was high, ranging from 0.6218 (DXS7133 locus) to 0.9327 (DXS8377 locus). The parameters of Probability of Relatedness (PR), Avuncular Index (AI), Power of Exclusion (PE), Power of Discrimination and Likelihood Ratios were also high, showing that these ten X-STRs are highly polymorphous and discriminating on the studied population. The minimum DNA concentration for the amplification of the LDD (X-STR) Decaplex loci is 0.5 ng and it was also verified that the PCR amplification can be affected when more than 5 ng of DNA are added to the reactions. The "stutter" bands percentages were high to DXS7132 and DXS8377 loci. At the reproducing test, consistency among typing of different biological samples was observed, including the ones from remains. At the mixing test, the limit proportion in which two biological species coexisted was 2.5:1 ng (female-male). The results point out that LDD (X-STR) Decaplex loci are able to provide many important piece of information which, as a whole, are a very important tool at human identification and of kinship studies.

Entre as muitas aplicações das tecnologias de identificação biológica humana, estão as finalidades forenses. O objetivo desta pesquisa foi verificar frequências alélicas de Short Tandem Repeat (STR) e os parâmetros estatísticos de interesse em genética de populações e forense para desenvolver o primeiro banco de dados populacional de DNA na Faculdade de Odontologia de Bauru, Universidade de São Paulo, (FOB/USP) para futuros usos forenses. Frequências alélicas de 15 locos autossômicos e do marcador de gênero amelogenina foram determinadas utilizando amostras de 200 μL de saliva doados por 296 alunos de graduação da FOB/USP, com idade ≥ 18 anos, após aprovação ética. Os testes laboratoriais foram feitos com kits comerciais. Resultados e parâmetros estatísticos foram obtidos por meio de programas clássicos: GeneMapper-ID-X, MS Excel 2002 versão 10.6871.6870, GenAlEx 6.5 e Arlequin 3.5, comparando quatro populações (brasileira, portuguesa, norte-americana e a população deste estudo). Os locos mais polimórficos foram D18S51 (17 alelos) e FGA (15 alelos), seguidos pelo D21S11 (13 alelos) e os menos polimórficos foram D16S539 e TH01 (7 alelos cada). A análise comparativa com amostra da população brasileira proveniente de estudos anteriores (n > 100.000) pelo teste goodness of fit X2 index não mostrou diferenças significativas entre estes grupos (p = 0,9999). Outros parâmetros estatísticos foram calculados comparando as populações: local (deste estudo), portuguesa e norte-americana. A análise de variância molecular (AMOVA) entre as três populações, entre as pessoas da mesma população e para cada pessoa de cada população mostrou que existe uma elevada variância individual (99%), que esta variância é mantida uniformemente entre as pessoas da mesma amostra/região (1%) e entre as três populações estudadas (0%). O estudo confirmou o elevado grau de polimorfismo e a alta heterozigosidade (96,5%) da população. Houve diferença significativa quanto ao gênero (79,7% mulheres) quando comparado à população brasileira em geral (50,4%), explicada pelas características do corpo discente da FOB/USP composto por 80,6% de pessoas do gênero feminino. Interessante foi a observação de uma microvariante alélica no loco D18S51, fora da escada padrão e da escala de abrangência do kit, correspondente ao alelo 29, ainda não definida na base de dados internacional (STRBase, atualizada em 07/08/2015). Esta microvariante deverá ser confirmada por testes familiares e sequenciamento de DNA para verificar a possibilidade de outra ocorrência familiar ou duplicação de nucleotídeos. No futuro, os dados obtidos neste estudo devem ser incorporados ao banco de dados da população brasileira e podem ser considerados como referência genética da população regional, ajudando a elucidar casos forenses. Após a confirmação, a potencial nova microvariante alélica contribuirá para a base de dados internacional STRBase. / There are many ways of applying biological human identification technologies, among these are forensic applications. The objective of this study was to verify allele frequencies for 15 autosomal short tandem repeat (STR) loci and develop the first human DNA population database at the Bauru School of Dentistry, University of São Paulo (FOB/USP), for future forensic uses. Allele frequencies for these STR loci and an amelogenin gender marker were determined using 200 μL samples of saliva donated by 296 undergraduates from FOB/USP who were ≥ 18 years old at the time of the sample collect after signing a consent form with ethical approval. For laboratory tests, commercial kits were used. Results and statistical parameters were obtained using the following software: GeneMapper IDX (version 1.5), MS Excel 2002 (version 10.6871.6870), GenAlEx (version 6.5) and Arlequin (version 3.5) to compare four populations (Brazil, Portugal, U.S. and this study population). Results indicated that the most polymorphic loci were D18S51 (17 alleles) and FGA (15 alleles), followed by D21S11 (13 alleles); the least polymorphic loci were D16S539 and TH01 (7 alleles each). Various Brazilian populations (n > 100,000) from other studies were compared with this studys Brazilian population using a goodness-of-fit chi-squared test, and no significant differences in these frequencies were observed between these two population groups (p = 0.9999). Other forensic and population genetic statistical parameters were calculated comparing this studys population with Portugal and U.S. populations. For example, an analysis of molecular variance (AMOVA) among all populations, among people of the same population and for each person for each population, showed that people have high individual variance (99%) and that this variance is maintained evenly between people of the same sample/region (1%) and among the three populations studied (0%). This study reinforced the conclusion of other allele frequency population studies for the 15 autosomal STR loci tested, confirming high polymorphic grade and high heterozygosity (96.5%). There were significantly more women in the study (79.7%) when compared to the general Brazilian population (50.4%) since the student body of FOB/USP is 80.6% female. Interestingly, an off-ladder D18S51 allele micro-variance corresponding to the allele 29, not yet identified, was found which should be confirmed using paternity and sequencing tests to verify the possibility of either familial occurrence or nucleotide duplication. In the future, due to the small differences found, the parameters obtained in this study should be incorporated into the Brazilian population database and be considered for a regional population genetic prototype database potentially aiding forensic cases with comparisons and calculations. After confirmation, the potentially new micro variant allele found could be included in the international STRBase.

Magister Scientiae - MSc / The objective of the study was to conduct an internal validation on miniSTR systems and apply it to cases received from the South African Missing Persons Task Team (SAMPTT). This was prompted by the fact that miniSTR systems have been shown to out perform some of the commercial kits available in the time of the study and provide an alternative to mtDNA when analysing degraded DNA from skeletal remains and that the DNA extracted from skeletal remains received from the SAMPTT would be degraded due to the remains generally being fragmented or charred and buried for many years. The miniSTR loci chosen for validation comprised the Combined DNA Index System (CODIS) thirteen core loci and were arranged into four triplexes and one uniplex. / South Africa

Roseate spoonbills are colonial nesting birds with breeding grounds extending from the United States Gulf coast to the pampas of Argentina. The U.S. population suffered a severe bottleneck from 1890 to 1920. The population's recovery was slow and partially credited to migrations from Mexican rookeries, but a gene pool reduction would be expected. Five polymorphic Spoonbill autosomal short tandem repeat (STR) loci [three (GAT)n, one (AAAG)n and one (GT)n] and one Z/W-linked microsatellite exhibiting sex-specific dimorphism were isolated and characterized. The Z/W-linked STR locus accurately confirmed the sex of each bird. Allelic profiles for 51 spoonbills obtained from Dallas (Texas), Fort Worth (Texas) and Sedgwick County (Kansas) zoos revealed a non-continuous distribution of allele frequencies, consistent with the effects of a population bottleneck. Allelic frequencies also differed significantly between the isolated zoo populations. Although extra-pair copulations were suspected and difficult to document, zoos commonly used observational studies of mating pairs to determine familial relationships among adults and offspring. STR parentage analysis of recorded family relationships excluded one or both parents in 10/25 cases studied and it was further possible to identify alternative likely parents in each case. Mistaken familial relationships quickly lead to the loss of genetic variability in captive populations. Here, a decreased heterozygosity (HO) in 2nd generation captive-bred birds was observed at 3 out of 4 loci evaluated. Although these results could not be statistically validated because of the small number of individuals available for study (15 wild birds with no offspring vs. eight 2nd generation captive birds), they are considered biologically important, as decreased HO is an indicator of inbreeding and this apparent decrease occurred within two generations of removal from the wild. Collectively, the evidence obtained from this study suggests that captive spoonbill populations are experiencing rapid loss of diversity from an already depleted wild gene pool.

Degenerate Oligonucleotide Primed-PCR (DOP-PCR) can potentially enhance analysis of low copy number DNA samples. Theoretically, this procedure replicates fragments of the genome that can then be used for downstream multiplex STR analysis. The objective of this study is to optimize DOP-PCR by examining ramplelongation times and cycle numbers in the non-specific amplification portion of DOP-PCR, and by modifying the degenerate primer. Additionally, other methods such as Multiple Displacement Amplification (MDA) and Low Copy Number PCR (LCN PCR) were examined for their ability to create accurate DNA profiles from low DNA input amounts. Increasing the ramplelongation times showed no effect on downstream STR amplification success. An increase of cycle number increased DNA yield, but STR amplification success was undetermined. Although modifying the degenerate primer to one with a higher degeneracy decreased DNA yield, it ultimately improved STR amplification success. In comparison studies, LCN PCR produced higher STR amplification success than MDA.

Y chromosome is due to its special characteristics the ideal tool of archaeogenetic studies. Its diversity is influenced by several factors and I analysed two of them (geographical location and subsistence). I generated SNPs and STRs data from several loci of samples from Slovakia (156 samples, 5 regions) and sub-Saharan Africa, where I analysed samples of sedentary farmers (481 samples, 18 regions) and nomadic pastorals (405 samples, 16 regions). Slovakia is situated at the meeting point of two migration ways. First of them was spread from the east to the west and is associated with enlargement of haplogroup R1a in Europe. The second came from the Iberian Peninsula eastward and is associated with enlargement of haplogroup R1b. Results of MDS graphs replicate the geographical map of Europe. Slovakia is situated in the middle of Russian, Balkanian and Iberian samples. Correlation between genetics and geographic distances is indicated by hierarchical AMOVA analysis and Mantel tests. Populations in sub-Saharan Africa differ from each other by the subsistence pattern. Different life style influence the diversity of the Y chromosome. Nomadic pastoralists and sedentary farmers share different haplogroups, for example, while haplogroup R1b was detected only in nomadic pastoral groups, sedentary farmers...

Det finns idag ingen konsensus om den optimala rehabiliteringen efter en främre korsbandsrekonstruktion, vi vet heller inte idag huruvida rehabiliteringen ska specificeras efter parametrar som kön och ålder. Studien var en korrelerande tvärsnittsstudie med syfte att ta reda på om det förelåg någon skillnad i postoperativ lårmuskelstyrka 4-7 månader efter en korsbandsrekonstruktion med avseende på kön, ålder, skada och typ av operativt ingrepp. Av de 330 aktuella försökspersonerna var det endast 74 personer som godkänt deltagande och uppfyllde kraven för inklusionskriterierna. Försökspersonerna testades i en isokinetisk dynamometer(Biodex) för maximal styrka(60gr/s) och uthållig styrka(180gr/s). Resultaten presenterades genom ett Limb Symmetry Index. Studien fann inga signifikanta skillnader i de undersökta parametrarna mellan försökspersonerna i uppmätt lårmuskelstyrka mellan opererade och icke opererade benet. Detta tyder på att ålder och kön inte är avgörande vid initial rehabilitering avseende post operativ lårmuskelstyrka.

Modern DNA analysis is possible due to the discovery of repeating microsatellite regions in DNA and successful implementation of the polymerase chain reaction (PCR) in laboratories. PCR amplification chemistries that contain short tandem repeat (STR) loci are sensitive. As a result, the discrimination power within human identification sciences has increased in recent years. Despite these advances, cellular admixtures are commonly collected, and the resultant “DNA mixture profile” is difficult to interpret as it is often encumbered by low-signals and allele drop-out. Regularly detected PCR artifacts can further complicate interpretation. One commonly encountered artifact is stutter, the result of strand slippage during PCR. Stutter can be of two types: forward and reverse. Reverse stutter (or back stutter) is the most prevalent and is one repeat unit shorter (n - 1) than the template strand. In contrast, forward stutter is one repeat unit longer (n + 1). If a reverse stutter amplicon is produced there is the distinct possibility that a stutter product of stutter may occur. This artifact is usually referred to as double-back stutter (DBS) or n - 2 stutter. Recently there has been renewed interest in examining signal approaching baseline levels. As the sensitivity of the process improves, so does the probability of detecting DBS. Therefore, studies that examine the peak height distributions, rarity, stutter signal-to-noise distances and the general impact of DBS on the signal are warranted. Models simulating PCR, and the entire forensic DNA process, have been created by this laboratory. The work presented herein builds upon a preexisting model; specifically, the dynamic model was extended such that DNA profiles consisting of 21 autosomal STRs, consistent with the GlobalFilerTM multiplex, are simulated. Furthermore, this expansion incorporated a three-type Galton-Watson branching process allowing DBS to be added to the simulated electropherogram (EPG). The in silico model was used to simulate the amplification of a 1:43 and 1:73 mixture at a total DNA concentration of 0.3 and 0.5 ng, respectively. We chose these extreme mixture ratios because the signal from these minor contributors would be most susceptible to DBS effects from the major contributor. A total of 1200 alleles from each contributor were simulated at each target, and effects of DBS on the signal from the minor contributor were characterized. At 0.3 and 0.5 ng both the noise and stutter signal histograms are right-skewed and a Kolmogorov-Smirnov (KS) test indicates that the noise and DBS were significantly different (p-value < 4x10-6). The average peak height of DBS for all loci in both scenarios were less than 50 RFU (Relative Fluorescence Units), and the DBS ratios ranged from 0.29 to 2.15% of the main allele, with the median ratios less than 0.5%. A per locus analytical threshold (AT) was calculated for both the 0.3 and 0.5 ng targets using two k-values: 3 and 4. The k-value is chosen based on the Type I risk assessment, wherein increasing the k-value increases AT. The percentage of DBS peaks greater than AT when k = 3 for the mixtures amplified at 0.3 and 0.5 ng ranged from 0 to 7.08% and 0 to 10.50%, respectively. Interestingly, when k = 4 the percentage of DBS peaks greater than AT for 0.3 and 0.5 ng reduced to 0 to 1.08% and 0 to 0.17%, respectively. This suggests that modeling DBS in continuous systems may not be necessary if the laboratory continues to rely on a system that requires an AT of sufficient strength. However, with the advent of Bayesian or machine learning-based approaches to analyzing EPGs, thus removing AT in its entirety, a complete understanding of the prevalence of DBS is necessary. This work shows that DBS from an extreme major using our laboratory protocols is not likely to be in the same signal regime as the signal from alleles; however, it does show that signal from DBS is significantly different from noise. Therefore, the software expert pair should be carefully considered during the validation stage and laboratories should consider DBS during interpretation, especially if enhanced post-PCR parameters are implemented into the forensic laboratory process.

Methods to determine genetic diversity and relatedness within populations are essential tools for proper wildlife management. Today the approach of choice is polymerase chain reaction-based microsatellite analysis. Seven new polymorphic loci were isolated from a microsatellite-enriched Caribbean flamingo genomic library and used to characterize survey populations of Caribbean and African greater flamingos. In addition, four of these loci were used to verify parentage relationships within a captive-breeding population of African greater flamingos. Parentage predictions based upon gamekeeper observations of breeding and nesting did not always agree with genetic-based parentage analyses of the nine suggested family groups. Four family groups were supported (groups I, II, III and VI) by there results. However, an analysis of the remaining five suggested groups, with a total of eight offspring/dam and eight offspring/sire suggested relationships, yielded seven exclusions of the suggested dam and six exclusions of the suggested sire. This put the overall suggested dam exclusion rate at 35% and exclusion rate for suggested sires at 29%. Although the keeper observation data for our family groups must be considered a variable of concern at this time, these findings are certainly suggestive that more carefully controlled studies may reveal that flamingos are not monogamous as long accepted, but rather socially monogamous or even promiscuous. Thus we have now been able to both characterize and demonstrate the utility of our polymorphic microsatellite loci. We hope these results will interest additional wildlife facilities in further parentage and behavioral studies that will collectively aid to improve monitoring and maintenance of genetic diversity, and as provide better insight into breeding habits of both wild and captive populations.