Made available in DSpace on 2016-06-02T20:20:34Z (GMT). No. of bitstreams: 1 4932.pdf: 1990172 bytes, checksum: 7025c9dd589a8c2a6771bc24539895b5 (MD5) Previous issue date: 2013-03-05 / Universidade Federal de Sao Carlos / The genetic diversity of the cattle egret (Bubulcus ibis) was analyzed to investigate reproductive behavior and characterize Brazilian populations of the species. Genotypes at seven microsatellite loci were used to investigate the occurrence of more than one female laying eggs in the same nest, characterizing the occurrence of multiple maternity. DNA was extracted from swabs collected from the outer surface of eggs and sexed; males were excluded. Forty-eight clutches from two breeding seasons (2010 and 2011) were genetically analyzed. Thirty-nine eggs laid by a second or third female were recorded. In five nests, the first egg of the clutch was from a different female, the laying happening prior to that of the incubating female. Suggesting nest takeover by another pair of egrets that kept the pre-existing eggs together with its own clutch. In the other 43 nests, the hypothesis of brood parasitism was posed to explain why one or two additional females were found laying eggs in a nest. A 463-bp fragment of the mitochondrial DNA control region Domain I was amplified for 148 individuals from seven Brazilian populations to investigate genetic-population and demographic parameters. Genetic diversity indices, the population structure tests Fst and AMOVA, a haplotype network, mismatch distribution and neutrality tests (Tajima s D, Fu s Fs, Fu and Li s D* and F*, Ramos-Onsins and Rozas R2) revealed the following: i) a high level of diversity was recorded for the cattle egret in Brazil in comparison to other closely related species studied in the country; ii) genetic diversity levels determined for the Brazilian regions are similar; iii) genetic structuring was not observed between the seven studied populations; and iv) the different tests performed to determine demographic expansion revealed no significant results. This is the first genetic characterization study for Bubulcus ibis to date and the findings indicate a high degree of plasticity in reproductive behavior and confirm a marked dispersion behavior of the species, leading to the homogenization of Brazilian populations. / A diversidade genética da garça-vaqueira (Bubulcus ibis) foi utilizada para se investigar o comportamento reprodutivo e para se caracterizar populações brasileiras da espécie. Genótipos em locos de microssatélites foram utilizados na detecção da presença de mais de uma fêmea ovipositando em um mesmo ninho, o que pode caracterizar a ocorrência de maternidade múltipla. O DNA extraído dos swabs coletados na superfície externa dos ovos foi sexado e eventuais amostras de machos foram excluídas. Quarenta e oito ninhadas, de duas temporadas reprodutivas (2010 e 2011) foram analisadas geneticamente. Foram registrados 39 ovos ovipositados por uma segunda ou terceira fêmea. Em 33 ninhos foram encontrados genótipos distintos de duas fêmeas e em seis ninhos genótipos de três fêmeas. Em cinco ninhos o primeiro ovo na sequência de oviposição mostrou-se ser de uma fêmea diferente, tendo a oviposição acontecido previamente àquela da fêmea incubante. Esse achado foi explicado supondo a tomada de ninho por um segundo casal de garças, com a manutenção do ovo pré-existente juntamente com os da própria ninhada. Nos outros 43 ninhos, a presença das fêmeas extras foi explicada hipotetizando a ocorrência de parasitismo de ninho intraespecífico. Um fragmento de 463 pb do Domínio I da região controladora do DNA mitocondrial foi amplificado para 148 indivíduos para a investigação de parâmetros genéticopopulacionais e processos demográficos nas sete populações estudadas. A estimativa de índices de diversidade genética, testes de estruturação populacional Fst e AMOVA, a construção de uma rede de relação entre haplótipos, a análise de mudanças no tamanho populacional pela mismatch distribution e a realização de testes de neutralidade (D de Tajima, Fs de Fu, D* e F* de Fu e Li, R2 de Ramos-Onsins e Rozas) permitiram identificar: i) um nível alto de diversidade genética para B. ibis no Brasil, quando comparado a espécies proximamente relacionadas estudadas no país; ii) níveis semelhantes de diversidade genética determinados para as regiões brasileiras; iii) ausência de estruturação genética entre as sete populações estudadas; e iv) ausência de sinais de expansão demográfica pelos testes realizados. Os resultados aqui apresentados são os primeiros resultados genéticos na espécie até o momento e apontam para uma alta plasticidade no comportamento reprodutivo e confirmam a dispersão bastante acentuada da espécie, levando a homogeneização das populações brasileiras.

Prenatal assessment of fetal health is routinely offered throughout pregnancy to ensure that the most effective management can be provided to maintain fetal and maternal well-being. Currently, invasive testing is used for definitive diagnosis of fetal aneuploidy, which is associated with a 1% risk of iatrogenic fetal loss. Developing non-invasive prenatal testing (NIPT) is a key area of research and methods to increase the level of cell-free fetal DNA (cffDNA) within the maternal circulation have been discussed to improve accuracy of such tests. In this study, three strategies; co-amplification at lower denaturation temperature polymerase chain reaction (COLD-PCR), inverse-PCR and Pippin Prep™ gel electrophoresis, were analysed to identify a novel approach to selectively enrich shorter cffDNA fragments from larger maternal cell-free DNA (cfDNA). The sensitivity of droplet digital PCR (ddPCR) against real-time PCR (qPCR) was compared for fetal sex and RHD genotyping. In addition RHD zygosity testing was carried out for non-maternal samples. Consequently, Pippin Prep™ gel electrophoresis was combined with ddPCR analysis for the NIPD of Down Syndrome (DS) in pseudo-maternal samples. The results revealed that the Pippin Prep™ gel electrophoresis enrichment approach successfully demonstrated 2-fold to 5-fold increases in the cffDNA fraction. However, further optimisation assays of COLD-PCR and inverse-PCR using actual maternal samples were required. The spike experiments for DS detection revealed that with the present assay IV overrepresentation of the chromosome 21 target could be significantly detected for samples with ≥15% ‘cffDNA fraction’. In conjunction with the Pippin Prep™ enrichment method, this would have enabled assessment of all 10 maternal samples. Alternatively, fetal sex and RHD genotyping results determined that ddPCR provides a more sensitive platform compared to qPCR approaches, particularly for samples that express low cffDNA fractions (<2%). The ddPCR platform also proved to be a rapid and accurate system for the determination of RHD zygosity. This study highlights that ddPCR could be used as opposed to qPCR for accurate determination of fetal sex and RHD status. While sequencing approaches currently provide the most sensitive platforms for NIPT of fetal aneuploidy, high costs (>£400) prevent universal application. The combination of cffDNA enrichment with ddPCR analysis could provide a cheaper and more widely available platform for NIPD. However, further large scale validation studies using actual maternal samples are required.