Genetic bottlenecks driven by population disconnection.

Connectivity among populations plays a crucial role in maintaining genetic variation at a local scale, especially in small populations affected strongly by genetic drift. The negative consequences of population disconnection on allelic richness and gene diversity (heterozygosity) are well recognized and empirically established. It is not well recognized, however, that a sudden drop in local effective population size induced by such disconnection produces a temporary disequilibrium in allelic frequency distributions that is akin to the genetic signature of a demographic bottleneck. To document this effect, we used individual-based simulations and empirical data on allelic richness and gene diversity in six pairs of isolated versus well-connected (core) populations of European tree frogs. In our simulations, population disconnection depressed allelic richness more than heterozygosity and thus resulted in a temporary excess in gene diversity relative to mutation drift equilibrium (i.e., signature of a genetic bottleneck). We observed a similar excess in gene diversity in isolated populations of tree frogs. Our results show that population disconnection can create a genetic bottleneck in the absence of demographic collapse.

Characterization of thirty two microsatellite loci for three Atlanto-Mediterranean echinoderm species

Thirty two microsatellites were optimized from 454 pyrosequencing libraries for three Atlanto-Mediterranean echinoderms: Coscinasterias tenuispina, Echinaster sepositus and Arbacia lixula. We observed different frequency of microsatellite types (di-, tri-, tetra- and pentanucleotide) throughout the genome of the species, but no significant differences were observed in allele richness among different microsatellite repeats. No loci showed linkage disequilibrium. Heterozygosity deficit and departure from Hardy Weinberg equilibrium were observed for some loci, in two species, probably due to high levels of inbreeding. Heterozygosity excess observed in C. tenuispina could be explained by selection against homozygotes and/or outcrossing.

Morphological and molecular diversity among cassava genotypes

The objective of this work was to characterize morphologically and molecularly the genetic diversity of cassava accessions, collected from different regions in Brazil. A descriptive analysis was made for 12 morphological traits in 419 accessions. Data was transformed into binary data for cluster analysis and analysis of molecular variance. A higher proportion of white or cream (71%) root cortex color was found, while flesh colors were predominantly white (49%) and cream (42%). Four accession groups were classified by the cluster analysis, but they were not grouped according to their origin, which indicates that diversity is not structured in space. The variation was greater within regions (95.6%). Sixty genotypes were also evaluated using 14 polymorphic microsatellite markers. Molecular results corroborated the morphological ones, showing the same random distribution of genotypes, with no grouping according to origin. Diversity indices were high for each region, and a greater diversity was found within regions, with: a mean number of alleles per locus of 3.530; observed and expected heterozygosity of 0.499 and 0.642, respectively; and Shannon index of 1.03. The absence of spatial structure among cassava genotypes according to their origins shows the anthropic influence in the distribution and movement of germplasm, both within and among regions.

Within- and among-population impact of genetic erosion on adult fitness-related traits in the European tree frog Hyla arborea.

Assessing in wild populations how fitness is impacted by inbreeding and genetic drift is a major goal for conservation biology. An approach to measure the detrimental effects of inbreeding on fitness is to estimate correlations between molecular variation and phenotypic performances within and among populations. Our study investigated the effect of individual multilocus heterozygosity on body size, body condition and reproductive investment of males (that is, chorus attendance) and females (that is, clutch mass and egg size) in both small fragmented and large non-fragmented populations of European tree frog (Hyla arborea). Because adult size and/or condition and reproductive investment are usually related, genetic erosion may have detrimental effects directly on reproductive investment, and also on individual body size and condition that in turn may affect reproductive investment. We confirmed that the reproductive investment was highly size-dependent for both sexes. Larger females invested more in offspring production, and larger males attended the chorus in the pond more often. Our results did not provide evidence for a decline in body size, condition and reproductive effort with decreased multilocus heterozygosity both within and among populations. We showed that the lack of heterozygosity-fitness correlations within populations probably resulted from low inbreeding levels (inferior to ca. 20% full-sib mating rate), even in the small fragmented populations. The detrimental effects of fixation load were either low in adults or hidden by environmental variation among populations. These findings will be useful to design specific management actions to improve population persistence.

Genetic aberrations in pediatric follicular lymphoma

Background: Pediatric follicular lymphoma (FL) is a rare disease that differs from its adult counterpart both genetically and clinically. Excluding pediatric FL with IRF4-translocation, the genetic events associated with pediatric FL have not yet been defined. Objectives: The aim of this study was to perform a complete genetic characterization of IRF4-translocation negative pediatric follicular lymphomas to elucidate the genetic profile of these rare pediatric cases and determine common genetic alterations that could be associated to this phenotype. Design/Methods: We applied array-comparative genomic hybridization and molecular inversion probe assay adapted to formalin-fixed paraffin-embedded tissues from 18 patients aged £18 years diagnosed with FL. With the exception of one case with only focal involvement by lymphoma, the tumor cell content exceeded 50% in the evaluable samples. Eleven of 18 patients were treated according to NHL-BFM group multicenter trials whereas the remaining according to different protocols. All lacked t(14;18) translocation. Mutational analysis of TNFRSF14 gene was performed in 17 cases. Results: Only six pediatric cases displayed chromosomal imbalances, with gain/amplification of 6pter-p24.3 (including IRF4) and deletion/ copy number neutral-loss of heterozygosity in 1p36 (including TNFRSF14) being the most frequent alterations. Sequencing of the candidate gene TNFRSF14 at 1p36.32 showed nine mutations in seven cases. Conclusion: Combination of molecular and genetic features differentiated a recurrent pattern of genomic imbalances as well as of TNFRSF14 mutations in pediatric FL which together with other genetic alterations distinguishes two subsets of pediatric follicular lymphomas. The first group shows genomic aberrations and is associated with more aggressive histopathologic and clinical features. The second group lacks genetic alterations detectable with the present approaches and is associated with a more limited disease. Despite the absence of genomic aberrations, these cases resembled FL by their histopathological features.

Global Diversity Lines-A Five-Continent Reference Panel of Sequenced Drosophila melanogaster Strains.

Reference collections of multiple Drosophila lines with accumulating collections of "omics" data have proven especially valuable for the study of population genetics and complex trait genetics. Here we present a description of a resource collection of 84 strains of Drosophila melanogaster whose genome sequences were obtained after 12 generations of full-sib inbreeding. The initial rationale for this resource was to foster development of a systems biology platform for modeling metabolic regulation by the use of natural polymorphisms as perturbations. As reference lines, they are amenable to repeated phenotypic measurements, and already a large collection of metabolic traits have been assayed. Another key feature of these strains is their widespread geographic origin, coming from Beijing, Ithaca, Netherlands, Tasmania, and Zimbabwe. After obtaining 12.5× coverage of paired-end Illumina sequence reads, SNP and indel calls were made with the GATK platform. Thorough quality control was enabled by deep sequencing one line to >100×, and single-nucleotide polymorphisms and indels were validated using ddRAD-sequencing as an orthogonal platform. In addition, a series of preliminary population genetic tests were performed with these single-nucleotide polymorphism data for assessment of data quality. We found 83 segregating inversions among the lines, and as expected these were especially abundant in the African sample. We anticipate that this will make a useful addition to the set of reference D. melanogaster strains, thanks to its geographic structuring and unusually high level of genetic diversity.

Diversity and genetic struture in cajá tree (spondias mombin l.) populations in northeastern Brazil

Spondias mombin L. is a fruit tree from the American continent from the Anacardiaceae family. In Brazil it is common in different vegetation types but is more frequent in the Atlantic and Amazonian rainforests. It is economically important because of its fruits, which are widely consumed raw or processed as fruit jellies, juices and ice creams. The leaves have great importance in the pharmaceutical industry because of their antibacterial properties. In the state of Pernambuco, cajá tree is widely distributed in the Zona da Mata region and less frequently in the Agreste and Sertão areas. In this work diversity and genetic structure were studied in four populations of cajá tree from Pernambuco's Zona da Mata, Northeast Brazil, using isozymes polymorphism analyses from electrophoreses. The result showed 100% of polymorphism (P) for nine alleles and the average of alleles per locus s was 2.4. The expected heterozygosity ranged from 0.530 to 0.574 and the observed heterozygosity , from 0.572 to 0.735. It was not observed inbreeding and the average F IT was -0.175, whereas within population inbreeding (f) varied from -0.08 to- 0.37. The genetic divergence among the populations (F ST) ranged from 0.006 to 0.028 and the average was 0.026. The average of estimated gene flow (Nm) was high (5.27). The CG-IPA population, corresponding to the germplasm collection of IPA, showed more than 96% of genetic similarity with other populations; therefore, it is a good representative of the existent genetic diversity in the Zona da Mata region.

Diversity and genetic relatedness among genotypes of Vitis spp. using microsatellite molecular markers

The purpose of this research was to study the genetic diversity and genetic relatedness of 60 genotypes of grapevines derived from the Germplasm Bank of Embrapa Semiárido, Juazeiro, BA, Brazil. Seven previously characterized microsatellite markers were used: VVS2, VVMD5, VVMD7, VVMD27, VVMD3, ssrVrZAG79 and ssrVrZAG62. The expected heterozygosity (He) and polymorphic information content (PIC) were calculated, and the cluster analysis were processed to generate a dendrogram using the algorithm UPGMA. The He ranged from 81.8% to 88.1%, with a mean of 84.8%. The loci VrZAG79 and VVMD7 were the most informative, with a PIC of 87 and 86%, respectively, while VrZAG62 was the least informative, with a PIC value of 80%. Cluster analysis by UPGMA method allowed separation of the genotypes according to their genealogy and identification of possible parentage for the cultivars 'Dominga', 'Isaura', 'CG 26916', 'CG28467' and 'Roni Redi'.

Characterization of thirty two microsatellite loci for three Atlanto-Mediterranean echinoderm species

Thirty two microsatellites were optimized from 454 pyrosequencing libraries for three Atlanto-Mediterranean echinoderms: Coscinasterias tenuispina, Echinaster sepositus and Arbacia lixula. We observed different frequency of microsatellite types (di-, tri-, tetra- and pentanucleotide) throughout the genome of the species, but no significant differences were observed in allele richness among different microsatellite repeats. No loci showed linkage disequilibrium. Heterozygosity deficit and departure from Hardy Weinberg equilibrium were observed for some loci, in two species, probably due to high levels of inbreeding. Heterozygosity excess observed in C. tenuispina could be explained by selection against homozygotes and/or outcrossing.

Esterase polymorphism in remanant populations of Aspidosperma polyneuron Müll.Arg. (Apocynaceae)

The population genetic structure of the endangered tree species Aspidosperma polyneuron Mull.Arg. (Apocynaceae) was reported based on analysis of esterase polymorphism in two remanant populations. Allelic variation was detected at three isoesterase loci (Est-3, Est-9, and Est-10). The proportion of polymorphic loci for both populations was 30% and deviation from Hardy-Weinberg equilibrium was observed for the Est-3 locus observed in the northern population. Segregation distortion and the lower level of observed and expected heterozygosity in this population were attributed to founder genotype. The high genetic identity values for northern and northwestern populations are in accordance with the low levels of interpopulation genetic divergence demonstrated by the F(ST) (0.03) value. The F(IS) value (0.23) indicated moderate levels of inbreeding. A. polyneuron can be indicated as an example of endangered species suggesting high genetic variation in contrast to the low genetic variation reported for endangered species. The esterase isozymes may be a good genetic marker for studies of natural A. polyneuron populations.

Genetic diversity of teak (Tectona grandis L.F.) from different provenances using microsatellite markers

Teak (Tectona grandis) is one of the main timber species in the world with high economic value, famous for its beauty, strength and durability. The objective of this work was to characterize the genetic diversity of teak genotypes used in Brazilian plantations. Nine microsatellite primers were used to assess 60 teak genotypes, including 33 genotypes from seeds of plantations and 14 clones from Cáceres municipality, Mato Grosso State, Brazil, and 13 clones from Honduras, Malaysia, India, Indonesia, Ivory Coast and Solomon Islands. Two groups of genotypes were detected using the Bayesian Structure analysis: 80% were placed in group 1, represented by genotypes from Cáceres and one from Malaysia, and 20% allocated in group 2, composed of clones from India, Solomon Islands, Malaysia and Honduras and the clones from the Ivory Coast. Most of the genetic variability (73%) was concentrated within groups according to AMOVA analysis. Genetic parameters were estimated for the two groups obtained in the analysis of Structure. Moderate genetic diversity was found, with 4.1 alleles per locus, on average, and an average heterozygosity of 0.329, which was lower than the expected heterozygosity (He = 0.492). Group 1 showed the lowest values for these parameters. Suggestions were made concerning the identification of contrasting genotypes to be used as parents in breeding programs.

Genetic structure and diversity of Copaifera langsdorffii Desf. in Cerrado fragments of the São Paulo State, Brazil

The loss of large areas of Cerrado (Brazilian savanna) in Brazil can lead to reduced biodiversity and to the extinction of species. Therefore, the present study aimed to investigate the genetic fragility of populations of Copaifera langsdorffii Desf exposed to different anthropic conditions in fragments of Cerrado in the state of São Paulo. The study was carried out in two Experimental Stations operated by the Forest Institute (Assis and Itirapina), in one fully protected conservation unit (Pedregulho) and in one private property (Brotas). Analyses were conducted using leaf samples from 353 adult specimens and eight pairs of microsatellite loci. The number of alleles per locus ranged from 13 to 15 in all populations, but the mean number of effective alleles was approximately half this value (7.2 to 9-1). Observed heterozygosity was significant and lower than the expected in all populations. Consequently, all populations deviated from Hardy-Weinberg expected frequencies. Fixation indexes were significant for all populations, with the Pedregulho population having the lowest value (0.189) and Itirapina having the highest (0.283). The analysis of spatial genetic structure detected family structures at distance classes of 20 to 65 m in the populations studied. No clones were detected in the populations. Estimates of effective population size were low, but the area occupied by each population studied was large enough for conservation, medium and long term. Recent reductions or bottlenecks were detected in all four populations. Mean Gst’ (genetic divergence) indicated that most of the variation was within populations. Cluster structure analysis based on the genotypes detected K= 4 clusters with distinct allele frequencies patterns. The genetic differentiation observed among populations is consistent with the hypothesis of genetic and geographic isolation. Therefore, it is essential to adopt conservation strategies that raise the gene flow between fragments.

Pluripotency and genetic stability of human pluripotent stem cells

Pluripotent cells have the potential to differentiate into all somatic cell types. As the adult human body is unable to regenerate various tissues, pluripotent cells provide an attractive source for regenerative medicine. Human embryonic stem cells (hESCs) can be isolated from blastocyst stage embryos and cultured in the laboratory environment. However, their use in regenerative medicine is restricted due to problems with immunosuppression by the host and ethical legislation. Recently, a new source of pluripotent cells was established via the direct reprogramming of somatic cells. These human induced pluripotent stem cells (hiPSCs) enable the production of patient specific cell types. However, numerous challenges, such as efficient reprogramming, optimal culture, directed differentiation, genetic stability and tumor risk need to be solved before the launch of therapeutic applications. The main objective of this thesis was to understand the unique properties of human pluripotent stem cells. The specific aims were to identify novel factors involved in maintaining pluripotency, characterize the effects of low oxygen culture on hESCs, and determine the high resolution changes in hESCs and hiPSCs during culture and reprogramming. As a result, the previously uncharacterized protein L1TD1 was determined to be specific for pluripotent cells and essential for the maintenance of pluripotency. The low oxygen culture supported undifferentiated growth and affected expression of stem cell associated transcripts. High resolution screening of hESCs identified a number of culture induced copy number variations and loss of heterozygosity changes. Further, screening of hiPSCs revealed that reprogramming induces high resolution alterations. The results obtained in this thesis have important implications for stem cell and cancer biology and the therapeutic potential of pluripotent cells.

Genetic polymorphism of fifteen microsatellite loci in Brazilian (blue-egg Caipira) chickens

The purpose of this study was to investigate the genetic polymorphism of fifteen microsatellites loci in Brazilian (blue-egg Caipira) chickens. Samples were collected from 100 blue eggs of Caipira chickens from rural properties in the city of Dois Lajeados, RS. After DNA extraction, the fragments related to molecular markers LEI0248, LEI0221, LEI0214, LEI0192, LEI0217, LEI0254, LEI0194, LEI0212, MCW0371, ADL0278, LEI0234, MCW0183, MCW0216, MCW0330 and MCW0081 were obtained by polymerase chain reaction (PCR). The statistical analysis were carried out with the softwares ARLEQUIN 3.5 version and CERVUS 3.0.3 version. The allelic and genotypic frequencies, deviations from Hardy-Weinberg equilibrium, estimates of observed (HO) and expected (HE) heterozygosity and polymorphic information content (PIC) were obtained for each marker locus. A total of 186 alleles from 15 loci were obtained, with sizes ranging of 83 to 490 base pairs. The medium number of alleles was 12.4, the HE was 0.76±0.14 and HO was 0.49±0.21 and PIC was 0.706. The first conclusion is that the microsatellites used are polymorphic and can be used to genetic studies in chickens. The second is that the "Caipira" chicken (blue eggs) population investigated has a great genic variability, which makes than an important source of genetic resources for future animal breeding programs.

The genetics of behaviour and other adaptive traits in nine-spined sticklebacks (Pungitius pungitius)

One of the main goals in current evolutionary biology research is to identify genes behind adaptive phenotypic variations. The advances in genomic technologies have made it possible to identify genetic loci behind these variations, also concerning non-model species. This thesis investigates the genetics of the behaviour and other adaptive traits of the nine-spined stickleback (Pungitius pungitius) through the application of different genetic approaches. Fennoscandian nine-spined stickleback populations express large phenotypical differences especially in behaviour, life –history traits and morphology. However the underlying genetic bases for these phenotypical differences have not been studied in detail. The results of the project will lay the foundation for further genetics studies and provide valuable information for our understanding of the genetics of the adaptive divergence of the nine-spined stickleback. A candidate gene approach was used to develop microsatellite markers situating close to candidate genes for behaviour in the nine-spined stickleback. Altogether 13 markers were developed and these markers were used in the subsequent studies with the anonymous random markers and physiologically important gene markers which are already currently available for nine-spined sticklebacks. It was shown that heterozygosity correlated with behaviour in one of the marine nine-spined stickleback populations but with contrasting effects: correlations with behaviour were negative when using physiological gene markers and positive with random markers. No correlation was found between behavioural markers and behaviour. From the physiological gene markers, a strong correlation was found between osmoregulation-related gene markers and behaviour. These results indicate that both local (physiological) and general (random) effects are important in the shaping of behaviour and that heterozygosity– behaviour correlations are population dependent. In this thesis a second linkage map for nine-spined sticklebacks was constructed. Compared to the earlier nine-spined stickleback linkage map, genomic rearrangements were observed between autosomal (LG7) and sex-determing (LG12) linkage groups. This newly constructed map was used in QTL mapping studies in order to locate genomic regions associated with pelvic structures, behaviour and body size/growth. One major QTL was found for pelvic structures and Pitx1 gene was related to these traits as was predicted from three-spined stickleback studies, but this was in contrast to earlier nine-spined stickleback study. The QTL studies also revealed that behaviour and body size/growth were genetically more complex by having more QTL than pelvic traits. However, in many cases, pelvic structure, body size/growth and behaviour were linked to similar map locations indicating possible pleiotropic effects of genes locating in these QTL regions. Many of the gene related markers resided in the QTL area. In the future, studying these possible candidate genes in depth might reveal the underlying mechanism behind the measured traits.