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Sex chromosome microsatellite markers from an Australian marsupial: development, application and evolutionMacDonald, Anna Jayne, n/a January 2008 (has links)
Microsatellites are simple repetitive DNA sequences that are used as genetic markers
throughout the biological sciences. The high levels of variation observed at microsatellite loci
contribute to their utility in studies at the population and individual levels. This variation is a
consequence of mutations that change the length of microsatellite repeat tracts. Current
understanding suggests that most mutations are caused by polymerase slippage during DNA
replication and lead to changes of a single repeat unit in length, but some changes involving
multiple repeats can also occur. Despite this simplistic overview, there is evidence for
considerable heterogeneity in mutation processes between species, loci and alleles. Such
complex patterns suggest that other mechanisms, including those associated with DNA
recombination, are also involved in the generation of microsatellite mutations. Understanding
which mutational mechanisms are responsible for variation at microsatellite markers is
essential to enable accurate data interpretation in genotyping projects, as many commonly
used statistics assume specific mutation models.
I developed microsatellite markers specific to the X and Y chromosomes and an autosome in
the tammar wallaby, Macropus eugenii, and investigated their evolutionary properties using
two approaches: indirectly, as inferred from population data, and directly, from observation of
mutation events. First, I found that allelic richness increased with repeat length and that two
popular mutation models, the stepwise mutation model and the infinite allele model, were
poor at predicting the number of alleles per locus, particularly when gene diversity was high.
These results suggest that neither model can account for all mutations at tammar wallaby
microsatellites and hint at the involvement of more complex mechanisms than replication
slippage. I also determined levels of variation at each locus in two tammar wallaby
populations. I found that allelic richness was highest for chromosome 2, intermediate for the
X chromosome and lowest for the Y chromosome in both populations. Thus, allelic richness
varied between chromosomes in the manner predicted by their relative exposure to
recombination, although these results may also be explained by the relative effective
population sizes of the chromosomes studied. Second, I used small-pool PCR from sperm
DNA to observe de novo mutation events at three of the most polymorphic autosomal
markers. To determine the reliability of my observations I developed and applied strict criteria
for scoring alleles and mutations at microsatellite loci. I observed mutations at all three
markers, with rate variation between loci. Single step mutations could not be distinguished
because of the limitations of the approach, but 24 multi-step mutations, involving changes of
up to 35 repeat units, were recorded. Many of these mutations involved changes that could not
be explained by the gain or loss of whole repeat units. These results imply that a large number
of mutations at tammar wallaby microsatellites are caused by mechanisms other than
replication slippage and are consistent with a role for recombination in the mutation process.
Taken as a whole, my results provide evidence for complex mutation processes at tammar
wallaby microsatellites. I conclude that careful characterisation of microsatellite mutation
properties should be conducted on a case-by-case basis to determine the most appropriate
mutation models and analysis tools for each locus. In addition, my work has provided a set of
chromosome-specific markers for use in macropod genetic studies, which includes the first
marsupial Y chromosome microsatellites. Sex chromosome microsatellites open a new range
of possibilities for population studies, as they provide opportunities to investigate gene flow
in a male context, to complement data from autosomal and maternally-inherited mitochondrial
markers.
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Sexagem de embriões bovinos produzidos in vitro com sêmen selecionado por PERCOLL ou SWIM-UP / Sexing in vitro produced bovine embryos with semen selected by PERCOLL or SWIM-UPWolf, Caroline Antoniazzi 27 February 2007 (has links)
Conselho Nacional de Desenvolvimento Científico e Tecnológico / Preimplantation genetic diagnosis (PGD) is becoming a current issue in animal reproduction biotechnology due to economical reasons. Predetermining the sex of
offspring is one example of PGD. This study aimed to determine the percentage of male and female bovine embryos in vitro produced after oocyte fertilization with
Percoll density gradient centrifugation or with self-migration (swim-up) selected semen. In experiment 1, sperm selection was performed by 90%-45% discontinuous
Percoll density gradient centrifugation (T1) and swim-up (T2). In experiment 2, along side the discontinuous gradient, a 67.5% continuous density gradient, and
centrifugation time of 5 and 10 minutes were used. A total of 4 treatment groups was defined (TI = continuous, 5 minutes, TII = discontinuous, 5 minutes, TIII = continuous,
10 minutes and TIV = discontinuous, 10 minutes). Polymerase chain reaction (PCR) was used to determine the sex of the embryos. T1 (n=185) resulted in 48.65% (n=90)
male embryos and 51.35% (n=95) female embryos and T2 (n=142) in 58.45% (n=83) male and 41.55% (n=59) female embryos. In experiment 2, the percentages of male
and female embryos obtained in TI (n=93), TII (n=70), TIII (n=82) and TIV (n=82) were 49.46% (n=46) and 50.54% (n=47), 57.14% (n=40) and 42.86% (n=30), 36.59%
(n=30) and 63.41% (n=52) and 48.78% (n=40) and 51.22% (n=42), respectively. There was no difference on the percentage of males and females in all treatment
groups from experiments 1 and 2 when these were individually compared to the expected percentage of 50% of each sex. There was also no difference in male and
female embryo percentage between treatment groups in experiments 1 and 2. / O diagnóstico genético pré-implantação (DGP) vem se destacando na área da biotecnologia da reprodução animal por motivos econômicos. Um exemplo de DGP é a predeterminação do sexo da prole. Neste estudo foi verificada a percentagem de embriões bovinos machos e fêmeas produzidos in vitro após a fertilização de oócitos com sêmen selecionado por centrifugação em gradiente de densidade de Percoll ou por migração ascendente (swim-up). No experimento 1 a seleção espermática foi realizada usando o gradiente descontínuo de Percoll de 90% e 45% (T1) e o swimup (T2). No experimento 2 foi utilizado, além do gradiente descontínuo, um gradiente contínuo de densidade de Percoll de 67,5%, e tempos de centrifugação de 5 e 10 minutos, totalizando 4 tratamentos (TI = contínuo 5 minutos, TII = descontínuo 5 minutos, TIII = contínuo 10 minutos e TIV = descontínuo 10 minutos). A sexagem dos embriões foi realizada através da técnica da reação em cadeia da polimerase (PCR). No T1 (n=185) foram obtidos 48,65% (n=90) de embriões masculinos e 51,35% (n=95) de femininos e no T2 (n=142) 58,45% (n=83) foram machos e 41,55% (n=59) fêmeas. No experimento 2, a percentagem de embriões masculinos e femininos no TI (n=93), TII (n=70), TIII (n=82) e TIV (n=82) foi de 49,46% (n=46) e
50,54% (n=47), 57,14% (n=40) e 42,86% (n=30), 36,59% (n=30) e 63,41% (n=52), e 48,78% (n=40) e 51,22% (n=42), respectivamente. Não houve alteração na percentagem de machos e fêmeas nos tratamentos dos experimentos 1 e 2 quando estes tratamentos foram comparados individualmente com a percentagem teoricamente esperada de 50% de cada sexo. Também não houve alteração na percentagem de machos e fêmeas na comparação entre os dois tratamentos do
experimento 1 e entre os quatro tratamentos do experimento 2.
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