Entre os principais fatores que limitam a obtenção de altos rendimentos na cultura da soja estão as doenças. A antracnose é uma das principais doenças da cultura, sendo causada, segundo a literatura, pelo fungo Colletotrichum truncatum. No Brasil não há estudos detalhados sobre a real etiologia da doença. A identificação e a distribuição das espécies do gênero Colletotrichum nas diferentes regiões produtoras de soja do Brasil é fundamental para o entendimento da epidemiologia da doença e para o sucesso no desenvolvimento de estratégias mais eficientes de controle. Por meio de abordagem polifásica foram caracterizados 51 isolados de diferentes regiões produtoras de soja do Brasil, utilizando-se análises molecular, cultural, morfológica e patogênica. Foi realizada extração do DNA genômico e reação em cadeia da polimerase (PCR) dos genes GAPDH, HIS3 e da região ITS. Análises filogenéticas foram realizadas para cada gene de forma isolada e concatenada. Construiu-se uma árvore filogenética, por meio de inferência Bayesiana, utilizando os genes concatenados. Analisou-se, também, a diversidade genética intraespecífica dos isolados, por meio da construção de uma rede de haplótipos presentes na coleção. As demais caracterizações foram realizadas com 10 isolados selecionados com base na caracterização molecular. A caracterização cultural foi realizada nos meios BDA e SNA e foram avaliados esporulação, taxa de crescimento micelial e análise da coloração, morfologia e topografia das colônias. A caracterização morfológica também foi realizada nos mesmos meios de cultura, por meio da mensuração de 50 conídios por isolado, além da observação de acérvulos, setas e apressórios. A caracterização patogênica foi realizada utilizando-se a variedade BMX Potência inoculada por meio de palitos de dente colonizados com o fungo e por inoculações de suas sementes pelo método da restrição hídrica seguido de semeadura em solo autoclavado. Em ambos os métodos foram observados incidência e severidade de sintomas. A análise molecular dos isolados de Colletotrichum evidenciou ocorrência apenas da espécie C. truncatum associada à cultura da soja na coleção de isolados estudada. No entanto a analise intraespecífica mostrou alto grau de variabilidade, com a presença de 27 haplótipos na coleção, muitos deles idênticos geneticamente a isolados de C. truncatum de outros locais do mundo. Foi encontrada grande variabilidade cultural, morfológica e patogênica entre os isolados. A caracterização patogênica mostrou, nos dois métodos empregados, alta variação na agressividade dos isolados, sendo que os isolados CMES 1075 e CMES 1080 se destacaram dos demais por causarem as maiores lesões na haste da soja e por afetarem mais o desenvolvimento das sementes inoculadas. Não foi possível correlacionar a variabilidade intraespecífica observada nos isolados com sua respectiva origem geográfica. / Diseases are among the main factors that limit high yields in soybean crops. Anthracnose is a major disease of the culture, caused, according to the literature, by Colletotrichum truncatum. In Brazil, there are no detailed studies on the real etiology of the disease. Identification and distribution of Colletotrichum species in different soybean producing regions of Brazil is critical to understand the epidemiology and develop effective control of these diseases. The polyphasic approach was used to characterize 51 isolates from different soybean producing regions in Brazil, using molecular, cultural, morphological and pathogenic analyses. Genomic DNA extraction was performed and polymerase chain reaction (PCR) of GAPDH, HIS3 genes and the ITS region. Phylogenetic analyses were carried for each gene isolated and in the concatenated form. A phylogenetic tree was constructed according to the Bayesian inference, using concatenated genes. We also analyzed the intraspecific genetic diversity of the isolates by building a network of haplotypes in the collection. The remaining characterizations were performed with 10 strains selected based on the molecular characterization. The cultural characterization was performed in PDA and SNA media and sporulation, mycelial growth rate, colonies color, morphology and topography were evaluated. The morphological characterization was also performed in the same culture media by measuring 50 conidia per isolate in addition to observation of acervuli, setae and appressoria. The pathogenic characterization was performed using the variety BMX Potencia inoculated with toothpicks colonized with the fungus as well as by seed inoculations using the water restriction technique followed by sowing in sterilized soil. The evaluation of the pathogenicity was based on disease incidence and severity. The molecular analysis of Colletotrichum isolates showed only occurrence of C. truncatum species associated with soybean in the collection studied. However, the intraspecific analysis showed a high variability degree, with the presence of 27 haplotypes in the collection, many of them genetically identical to strains of C. truncatum elsewhere in the world. There was high cultural, morphological and pathogenic variability among the isolates. Pathogenic characterization showed, in both methods, high variation in aggressiveness of isolates. Isolates CMES 1075 and CMES 1080 were more aggressive due to their ability to cause bigger lesions on soybean stem and affect more severely the development of inoculated seeds. It was not possible to correlate intraspecific variability observed in the isolates with their respective geographical origin.

Amaranth (Amaranthus caudatus L.) is an important pseudocereal in the Andes. The seed has excellent nutritional value (high in protein, essential amino acids, and minerals) and ample capacity for growth in diverse, harsh Andean subsistence-production conditions such as water deficiency, salt stress, and soil mineral nutrient deficiency. The objective of this study was to characterize and quantify the genetic diversity among a series of 178 mostly Peruvian amaranth genotypes using 96 biallelic single-nucleotide polymorphism (SNP) markers. A total of 96 co-dominant, biallelic markers were developed using KASPar™ assays on a 96.96 Fluidigm EP1 array system. The 178 amaranth genotypes included white-opaque and white-translucent (vitreous) putative A. caudatus seed types, along with black-seeded A. hybridus and brown-seeded types, all isolated from among 48 accessions of the CICA-UNSAAC collection. Variation among and within samples and accessions was compared for empirically determined clusters (northern, north-central, south-central, and southern Peruvian Andes). Variation was highest within samples for all groups, but only in white-seeded amaranth was the p-value significant (17.43). The greatest variation among samples was found in the vitreous-seeded group (99.35). The highest average observed heterozygosity within-groups (Ho) was 0.19 in the brown-seeded group, and expected within-group heterozygosity (He) was highest in the vitreous-seeded group (0.359). Cluster analysis (UPGMA), PCA and PCO results partitioned the amaranth accessions into six discrete clusters. Clusters did not manifest obvious structure among accessions, which indicates that genetic diversity has been conserved across a broad region of the Peruvian Andes. The diversity characterization pointed to a center of origin and domestication of A. caudatus in the Ayacucho-Cusco region of southern Peru.

A epidemia de HIV-1 está mudando em São Paulo, com uma crescente preocupação no número de jovens de homens que fazem sexo com homens (HSH). A epidemia é dominada pelos subtipos B, F1, porém tem aumentado a prevalência do subtipo C. O objetivo do estudo foi identificar e caracterizar as redes de transmissão em São Paulo, comparando entre os subtipos epidêmicos, fatores de risco e exposição à terapia antirretroviral. Nós compilamos dados clínicos, epidemiológicos e as sequencias virais dos indivíduos acompanhados no Instituto Adolfo Lutz de Jan 2004 a fev de 2015. Após o controle de qualidade, 2.260 sequencias parciais do gene pol foram subtipadas previamente como B, C e F1 e então incluídas no estudo. 2.107 sequencias únicas controle foram selecionadas pelo blast contra as nossas sequencias no banco de dados de Los Alamos e banco de dados de Portugal. Todas as sequencias foram investigadas para a identificação de mutação de resistência (TDRM). Árvores filogenéticas foram construídas em RaxML para cada subtipo. Nós usamos Cluster Picker para analise da dinâmica de transmissão de acordo com os parâmetros de distância genética e bootstrap. Análises estatísticas foram utilizadas para identificar possíveis correlações dos clusters. 414 (18,3%) da nossa população foi incluída em cluster (2-12 indivíduos). A taxa de cluster não diferiu entre B e F1, no entanto as sequencias do subtipo C se agruparam duas vezes mais (p<0.001). Mais clusters foram identificados entre a população HSH quando comparada a de heterossexuais independente do subtipo (p<0.001). A taxa de TDRM foi maior em cluster que fora de cluster nos subtipos B e F1 (p<0.001 e p=0.009), respectivamente. Apesar do alto número de clusters no subtipo C verificamos baixa prevalência de TDRM. Os indivíduos que estavam em clusters eram 5 anos mais jovens que os de fora de cluster nos subtipos B e C (p<0.001 e 0.02, respectivamente). Nossos resultados indicam que, independente do subtipo, a epidemia de HIV-1 em São Paulo é auto-sustentada pelos pacientes HSH virgens de tratamento. O subtipo C apresentou maior proporção de clusters. No entanto, mais análises são necessárias para clarificar se isto implica em alta taxa de transmissão do subtipo em São Paulo. / The HIV-1 epidemic in Sao Paulo is changing, with a worrying increase in the number of new infections in young men and in the Men who have Sex with Men (MSM). The epidemic is dominated by subtypes B and F1, but theres been a recent increase in the prevalence of subtype C. The aim of this study was to identify and characterize HIV-1 transmission networks in Sao Paulo, comparing across subtype epidemics, risk factors, and HAART exposure therapy. We gathered epidemiological, clinical and viral sequence data from HIV-1 infected individuals followed in Adolfo Lutz Institute/SP-BR from Jan 2004 through Feb 2015. After quality control, 2,260 sequences of the partial pol gene were subtyped as previously reported as B, C and F1 were included on this study. 2,107 unique background control sequences were selected by blasting our sequences against Los Alamos database and the Portuguese HIV-1 database. The dataset was screened for Drug Resistance Mutations to identify transmitted drug resistance mutations (TDRM). Maximum-Likelihood phylogenetic trees were built in RaxML for each subtype separately. We used Cluster Picker to analyze transmission dynamics according the thresholds: genetic distance (0.06) and bootstrap over 90%. Statistical analyzes were performed to identify possible correlates of clustering. Categorical variables were compared using Chi-square or Fishers exact test and quantitative variables were analyzed using Mann-Whitney test (SPSS). 414 (18,3%) of our population were included in clusters (range: 2-12 individuals). The rate of clustering did not differ between subtypes B and F, however more subtype C sequences (40%) significantly clustered together (p<0.001). Furthermore, more clusters were significantly found in the MSM group when compared to heterosexuals for all subtypes, (p<0.001). Also, drug naïve patients were more likely to be in clusters when compared to treated patients in all subtypes (p <0.001). TDRM were more prevalent in clustering than in non-clustering in subtypes B (p <0.001) and F1 (p =0.009), and more related to MSM group. Despite the higher number of clusters, subtype C presented a lower prevalence of TDRM, although without significant difference between cluster and non-cluster. Clustering individuals were also 5 years younger than non-clustering individuals for subtypes B and C (p<0.001 and 0.02, respectively). Our results indicate that, regardless of subtype, the HIV-1 epidemic in Sao Paulo is self-sustained by treatment-naive MSM patients, who transmit the infection before starting treatment. Subtype C presents a higher proportion of patients that cluster together. However, further analyses are necessary to clarify whether this implies a higher transmission rate of this subtype in Sao Paulo.

Concern regarding the effect of energy development on greater sage-grouse (Centrocercus urophasianus) is increasing as the search for fossil fuel intensifies. Sage-grouse may be especially sensitive to energy development because they require large, diverse areas of sagebrush (Artemisia spp.) habitat to complete their life cycle. Additionally, the network of pipelines, roads, and wells required by energy development may fragment sagebrush habitat isolating populations and contributing to genetic drift, inbreeding, local extinction, or rapid divergence. Seep Ridge, located in northeastern Utah, is one area where sage-grouse habitat and energy development plans overlap. Approved leases call for the construction of an additional 4,000 natural gas wells in an area currently occupied by a small sage-grouse population. This research was completed to 1) collect baseline data on the survival, reproductive success and habitat use of the Seep Ridge sage-grouse population, 2) examine sage-grouse habitat use patterns in relation to development, and 3) describe sage-grouse mitochondrial genetic diversity in 3 northeastern Utah populations relative to other parts of the species range. I captured and monitored 16 sage-grouse from the Seep Ridge population in 2007 and 2008. Adult mortality rate of the Seep Ridge population was high (65.2%) and recruitment was low (7.1%) compared to other sage-grouse populations in Utah. Additionally, the monitored sage-grouse used habitats located farther from wells more frequently than habitat located near wells, relative to well spacing. Current habitats occupied by this population do not meet recommended guidelines. No unusual haplotype compositions were observed in the genetic survey of the northeastern Utah sage-grouse populations. However, differences in haplotype composition between the Anthro Mountain and Strawberry Valley populations and other northeastern grouse populations indicate there may be a barrier to gene flow in the area. I also documented that the Seep Ridge population is connected to another population inhabiting Ute Tribal land. This observation suggests that the populations inhabiting Ute Tribal land may constitute a source population to recolonize Seep Ridge during the post-energy development periods. I recommend that mitigation measures incorporate restricting development in breeding habitat, maintaining connections between populations, and actions to reduce adult mortality on the summer range. I also recommend that biologists continue collecting genetic samples from northeastern Utah sage-grouse populations to understand population structure, divergent evolution, and inform decisions concerning translocation

<p>Natural stress and disturbances are important factors affecting the structure and function of ecosystems. However the magnitude of stress has escalated due to anthropogenic activities. Environmental monitoring and toxicity assessments try to protect ecosystems from unwanted human alterations. The aim of this Doctoral thesis was to increase the understanding of the complex effects that environmental stress has on individuals and invertebrate populations. The low saline environment in the Baltic Sea is perceived as stressful for most organisms living there. In Paper I, it was found that Baltic blue mussels living in the less saline northern Baltic Proper (~5 psu) had lower basal metabolism and were more susceptible to toxic exposure than the mussels in the south (~7 psu). There was no genetic differentiation between the mussels from the northern and southern areas while there were genetic differences between mussels from sites within the respective areas (Paper III), indicating that there is not a simple relationship between the health of the mussels and genetic diversity in the microsatellite loci studied. In Paper IV it was found that the heat tolerance of the intertidal dogwhelk <i>Nucella lapillus</i> is oxygen dependent. Increased oxygen levels resulted in higher survival rate. Protein expression profiles also became more similar to those of the controls, compared to the whelks exposed to high temperature and normal oxygen levels. In Paper V and VI it was found that exposure to a single toxicant for more than one generation decreased the genetic diversity in exposed copepod populations even though abundances remained unaltered. In Paper VI, exposure to naturally contaminated sediments, which contained of a mixture of toxicants, did not decrease genetic diversity. However the genetic divergence (<i>F</i><i>ST</i>) within the treatments was very high, probably due to small effective population sizes in the replicates. Likewise in Paper III, the very low blue mussel abundance in the north together with the stressful environment suggests a small effective population in the northern Baltic Proper. In conclusion, my studies show that, measuring effects on several levels, including both functional and structural endpoints will both increase the sensitivity of the tests and increase their ecological relevance. </p>

Natural stress and disturbances are important factors affecting the structure and function of ecosystems. However the magnitude of stress has escalated due to anthropogenic activities. Environmental monitoring and toxicity assessments try to protect ecosystems from unwanted human alterations. The aim of this Doctoral thesis was to increase the understanding of the complex effects that environmental stress has on individuals and invertebrate populations. The low saline environment in the Baltic Sea is perceived as stressful for most organisms living there. In Paper I, it was found that Baltic blue mussels living in the less saline northern Baltic Proper (~5 psu) had lower basal metabolism and were more susceptible to toxic exposure than the mussels in the south (~7 psu). There was no genetic differentiation between the mussels from the northern and southern areas while there were genetic differences between mussels from sites within the respective areas (Paper III), indicating that there is not a simple relationship between the health of the mussels and genetic diversity in the microsatellite loci studied. In Paper IV it was found that the heat tolerance of the intertidal dogwhelk Nucella lapillus is oxygen dependent. Increased oxygen levels resulted in higher survival rate. Protein expression profiles also became more similar to those of the controls, compared to the whelks exposed to high temperature and normal oxygen levels. In Paper V and VI it was found that exposure to a single toxicant for more than one generation decreased the genetic diversity in exposed copepod populations even though abundances remained unaltered. In Paper VI, exposure to naturally contaminated sediments, which contained of a mixture of toxicants, did not decrease genetic diversity. However the genetic divergence (FST) within the treatments was very high, probably due to small effective population sizes in the replicates. Likewise in Paper III, the very low blue mussel abundance in the north together with the stressful environment suggests a small effective population in the northern Baltic Proper. In conclusion, my studies show that, measuring effects on several levels, including both functional and structural endpoints will both increase the sensitivity of the tests and increase their ecological relevance.

Climate change may lead to declines in lake trout (Salvelinus namaycush) populations and change the structure of the ecosystem in which they live. The lake trout is a keystone species in ecosystems of northern temperate lakes and these declines may subsequently reduce the genetic diversity found in these salmonids. Populations that contain greater genetic variation may have an increased capacity to adapt to changes in the ecosystem. Therefore, an understanding of the genetic diversity found in lake trout populations is required for their effective conservation and management. As a result, this study aimed to examine the genetic diversity and phylogeography of lake trout populations in north central Canada.<p>The genetic diversity of lake trout from 19 lakes in Saskatchewan was examined using partial regions of the ND2 and ND5 mtDNA genes. A total of 607 tissue samples were analyzed using PCR-based single stranded conformation polymorphism (SSCP) and DNA sequencing. Although the ND5 gene fragment had minimal intraspecific variation, eleven sequence types were detected in the ND2 gene. Each sequence type differed in relative frequency between and among the lake trout populations sampled. <p>One particular southern lake trout population, Crean Lake, had markedly different genetic composition in comparison to other lakes in the region. In the 1950s and 1960s, Crean Lake was stocked with lake trout from neighbouring Wassegam Lake in an attempt to increase population numbers. The sequence types of Crean Lake trout and their relative frequencies were dramatically different to those in Wassegam Lake. This suggests that the stocked fish may have been unsuccessful in their establishment/reproduction within Crean Lake. Lake trout from this lake also contained the highest frequency (44%) of rare ND2 sequence type B. Sequence type B was only detected in one other lake (La Ronge), at a very low frequency. <p>The mutational changes in the eleven ND2 mitochondrial DNA sequence types represented three different amino acid sequence types. Substitutions of Threonine and Isoleucine occurred, resulting in two polar amino acids with much different hydropathy indexes. This may affect the tertiary structure of the protein, possibly indicating functional differences. Functionally different proteins may be exhibiting characteristics that allow lake trout to flourish in their environment. <p>The fragments of both the ND2 and ND5 genes proved to be valuable for phylogenetic analyses within the Salmonidae. The genetic markers established in the present study provide the basis for future work on population genetics of lake trout. It would be advantageous to broaden the area of study in order to compare the genetic diversity found within the study area to other regions of Canada. This would determine whether the genetic diversity detected in this study is significantly greater than in other populations at a national scale. Management strategies should ultimately attempt to conserve the genetic diversity found within the lake trout populations of north central Saskatchewan.

Invasive species are an increasing threat to biological diversity as well as a leading cause of recent species’ extinctions. Invasives spread quickly and efficiently, and the U.S spends millions of dollars annually in the control and eradication of these species. More information is necessary in order to predict which species may become invasive. Rubus (Rosaceae) was chosen for study because this genus includes various ploidy levels, reproductive modes, and species that are invasive as well as native. Three Rubus species were chosen to represent apomictic and tetraploid invasives (Rubus armeniacus), a sexual and diploid native species (R. occidentalis), and a sexual and diploid invasive species (R. phoenicolasius). Specimens were collected across the U.S. and two different genetic fingerprinting techniques were used; Amplified Fragment Length Polymorphism (AFLP) and Randomly Amplified Fingerprints (RAF). Using three AFLP primers and two RAF primers, genetic similarity was determined and phylograms were constructed. Through statistical analysis and phylogram data it was determined that there might be slightly more genetic diversity in native R. occidentalis than in invasive R. phoenicolasius. Genetic diversity between apomictic and tetraploid Rubus armeniacus and the two sexual and diploid Rubus species were so similar that no distinction could be made, although the mean pairwise distances and mean number of alleles were significantly different. It was also found that geographic distance and genetic similarity do not appear to be related in these three Rubus species. During the course of this study it was also observed that the AFLP technique produced more alleles than the RAF technique, although this difference was not significant.

Climate change may lead to declines in lake trout (Salvelinus namaycush) populations and change the structure of the ecosystem in which they live. The lake trout is a keystone species in ecosystems of northern temperate lakes and these declines may subsequently reduce the genetic diversity found in these salmonids. Populations that contain greater genetic variation may have an increased capacity to adapt to changes in the ecosystem. Therefore, an understanding of the genetic diversity found in lake trout populations is required for their effective conservation and management. As a result, this study aimed to examine the genetic diversity and phylogeography of lake trout populations in north central Canada.<p>The genetic diversity of lake trout from 19 lakes in Saskatchewan was examined using partial regions of the ND2 and ND5 mtDNA genes. A total of 607 tissue samples were analyzed using PCR-based single stranded conformation polymorphism (SSCP) and DNA sequencing. Although the ND5 gene fragment had minimal intraspecific variation, eleven sequence types were detected in the ND2 gene. Each sequence type differed in relative frequency between and among the lake trout populations sampled. <p>One particular southern lake trout population, Crean Lake, had markedly different genetic composition in comparison to other lakes in the region. In the 1950s and 1960s, Crean Lake was stocked with lake trout from neighbouring Wassegam Lake in an attempt to increase population numbers. The sequence types of Crean Lake trout and their relative frequencies were dramatically different to those in Wassegam Lake. This suggests that the stocked fish may have been unsuccessful in their establishment/reproduction within Crean Lake. Lake trout from this lake also contained the highest frequency (44%) of rare ND2 sequence type B. Sequence type B was only detected in one other lake (La Ronge), at a very low frequency. <p>The mutational changes in the eleven ND2 mitochondrial DNA sequence types represented three different amino acid sequence types. Substitutions of Threonine and Isoleucine occurred, resulting in two polar amino acids with much different hydropathy indexes. This may affect the tertiary structure of the protein, possibly indicating functional differences. Functionally different proteins may be exhibiting characteristics that allow lake trout to flourish in their environment. <p>The fragments of both the ND2 and ND5 genes proved to be valuable for phylogenetic analyses within the Salmonidae. The genetic markers established in the present study provide the basis for future work on population genetics of lake trout. It would be advantageous to broaden the area of study in order to compare the genetic diversity found within the study area to other regions of Canada. This would determine whether the genetic diversity detected in this study is significantly greater than in other populations at a national scale. Management strategies should ultimately attempt to conserve the genetic diversity found within the lake trout populations of north central Saskatchewan.

One hundred fifty-five peach (Prunus persica) cultivars, from Asia, Brazil, Europe, and the USA, were examined using eleven Simple Sequence Repeats (SSRs) to study the genetic relationships among low chill as compared to high chill peach germplasm. Data was analyzed by NTSYSpc to form a similarity matrix using Nei and Li’s Dice similarity coefficient. This similarity matrix was then subjected to a cluster analysis and a dendrogram was constructed using the UPGMA (Unweighted Pair-Group Method, Arithmetic Mean) method. A wide range of diversity was detected, from 0.33 coefficient of similarity amongst the Thai peaches to 0.97 between two Brazilian peaches. The most distant clusters were the low chill peaches from Thailand and Taiwan and the local cultivars (both fruit and ornamental types) from China. Among the improved germplasm, there were distinct clusters for the Chinese/Japanese cultivars, three clusters for the Brazilian cultivars and one for the cultivars from the USA and Europe. The Brazilian materials clustered according to breeding programs in São Paulo and Pelotas reflecting the different sets of local cultivars used in the breeding efforts. The largest group investigated was the European/USA peaches. This group subdivided into three distinct clusters, with a general clustering of the low chill germplasm. The low chill accessions from Asia were genetically distant from the improved low chill peaches from the USA or Brazil. The low chill peaches from the Americas were more closely related to the high chill peaches developed in the USA and China/Japan due to the introgression of this germplasm into a low chill background.