Estrutura genética de populações de Amphobotrys ricini, agente causal do mofo cinzento da mamoneira

The gray mold, causal organism Amphobotrys ricini, is one of the major diseases of castor bean. Difficulties in managing plant disease arises form the limited understanding of the genetic structure of A. ricini, their complexity and variability make it difficult to control. Genetic structure can be used to infer the relative impact of different forces that influence the evolution of pathogen populations, that allow to predict the potencial for pathogen populations to envolve in agricultural ecosystems. Growers protect their crop by applying fungicides, but there aren t fungicides to provide significant control of gray mold of castor bean. The objectives of this work were use RAPD to determine the genetic structure of A. ricini subpopulations in Paraíba and assay the sensitivity of A. ricini isolates to azoxystrobin and carbendazim. To determine the genetic structure of A. ricini subpopulations in Paraíba, 23 isolates were colleted from two different geographic location (subpopulation). These isolates were analysed by RAPD using 22 random decamer primers, purchased from OPERON, produced a total of 80 markers polimorphics. The resulting matrixes were analysed using PopGene version 1.32. Sensitivity to azoxystrobin and carbendazim of 30 isolates, colleted form Paraíba and Alagoas, was estimated based on spore germination and colony growth inhibition. The stock solutions were added toV8 medium after sterilization to produce final concentrations of 0, 0.01, 0.1, 1, 10, and 100 µg/ml of carbendazim and 0, 0.001, 0.01, 0.1, 1, and 10 µg/ml of azoxystrobin. All statistical analyses were performed using SAS to estimate the dose that inhibited fungal growth by 50% (ED50 values). The genetic diversity within subpopulations (Hs=0,271) accounted for 92% of the total genetic diversity (Ht=0,293), while genetic diversity between subpopulations (Gst = 0,075) represented only 7,5%. The estimated number of migrants per generation (NM ) was 6,15. Nei s average gene identity across 80 RAPD loci was 0,9468. Individual ED50 values, for the 30 isolates screened for their sensitivity to azoxystrobin, ranged From a maximum of 0,168 µg/ml to a minimum of 0,0036 µg/ml. The ED50 values for carbendazim varied within the range of 0,026 to 0,316 µg/ml

Genética e biologia reprodutiva de vriesea minarum (Bromeliaceae): em busca de estratégias de conservação no quadrilátero ferrífero, minas gerais

Vriesea minarum is a rupiculous bromeliad species, with naturally fragmented populations, restricted to the Iron Quadrangle, Minas Gerais, Brazil. It is a threatened species, which is suffering from habitat loss due to the growth of cities and mining activities. The knowledge of genetic variability in plant populations is one of the main branches of conservation genetics, linking genetic data to conservation strategies while the knowledge about plant reproductive biology can aid in understanding key aspects of their life story, as well as in the comprehension of their distribution and survival strategies. Thus, the study of diversity, richness, and genetic structure, as well as the reproductive biology of populations of V. minarum can contribute to the development of conservation actions. Chapter 1 presents the transferability of 14 microsatellite loci for V. minarum. Among the results of this chapter, we highlight the successful transferability of 10 microsatellite loci described for other species of Bromeliaceae, all of which are polymorphic. In Chapter 2, we present the genetic analyses of 12 populations of V. minarum that are distributed throughout the Iron Quadrangle. We used the 10 microsatellite loci tested in Chapter 1. The results show a low population structuring (Fst = 0.088), but with different values of genetic richness (mean = 2.566) and gene diversity (mean = 0.635) for all populations; and a high inbreeding coefficient (Gis = 0.376). These may be the result of pollinators action and/or efficient seed dispersal, thus allowing a high connectivity among populations of naturally fragmented outcrops. The reproductive biology and floral morphology of a population of V. minarum, located in the Parque Estadual da Serra do Rola-Moça, are studied in Chapter 3. This reserve is the only public environmental protection area where the species occurs. As a result of field experiments and observations, we found that the species has its flowering period from January to March, with flowers that last for two days and that it has a mixed pollination syndrome. It is primarily alogamous, but also has the capacity to be self-ferilized. It is expected that data obtained in chapters 1, 2 and 3 serve as basis for other studies with species from the ferruginous rocky fields, since until now, to our knowledge, there are no other survey of endemic species from the Iron Quadrangle, seeking to merge the genetic knowledge, with the data of the reproductive biology, with the ultimate aim of biodiversity conservation. Considering the great habitat loss for the species by mining, it becomes crucial to analyze the creation of new protected areas for its conservation

Avaliação da atividade antimicrobiana das defensinas recombinantes de ervilha (drr230a) e café (cd1) produzidas em Pichia pastoris

The inefficiency of chemical pesticides to control phytopathogenic fungi in agriculture and the frequent incidence of human diseases caused by bacteria which are resistant to antibiotics lead to the search for alternative antimicrobial compounds. In this context, plant defensins are a promising tool for the control of both plant and human pathogenic agents. Plant defensins are cationic peptides of about 50 amino acid residues, rich in cysteine and whose tridimensional structure is considerably conserved among different plant species. These antimicrobial molecules represent an important innate component from plant defense response against pathogens and are expressed in various plant tissues, such as leaves, tubers, flowers, pods and seeds. The present work aimed at the evaluation of the antimicrobial activity of two plant defensins against different phytopathogenic fungi and pathogenic bacteria to humans. The defensin Drr230a, whose gene was isolated from pea (Pisum sativum), and the defensin CD1,whose gene was identified within coffee (Coffea arabica) transcriptome, were subcloned in yeast expression vector and expressed in Pichia pastoris. The gene cd1 was subcloned as two different recombinant forms: CD1tC, containing a six-histidine sequence (6xHis) at the peptide C-terminal region and CD1tN, containing 6xHis coding sequence at the N-terminal region. In the case of the defensin Drr230a, the 6xHis coding sequence was inserted only at the N-terminal region. Assays of the antimicrobial activity of the purified recombinant proteins rDrr230a and rCD1 against Phakopsora pachyrhizi, causal agent of soybean Asian rust, were performed to analyze the in vitro spore germination inhibition and disease severity caused by the fungus in planta. Both recombinant defensins were able to inhibit P. pachyrhizi uredospore germination, with no difference between the antimicrobial action of either CD1tC or CD1tN. Moreover, rDrr230a and rCD1 drastically reduced severity of soybean Asian rust, as demonstrated by in planta assays. In spite of the fact that rCD1 was not able to inhibit proliferation of the human pathogenic bacteria Staplylococcus aureus and Klebsiella pneumoniae, rCD1 was able to inhibit growth of the phytopathogenic fungus Fusarium tucumaniae, that causes soybean sudden death syndrome. The obtained results show that these plant defensins are useful candidates to be used in plant genetic engineering programs to control agriculture impacting fungal diseases.

Uso sustentável da Copernicia prunifera (Miller) H. E Moore no semiárido potiguar: valorização de saberes e conservação dos recursos genéticos

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Diversificação evolutiva de gobídeos no litoral brasileiro: padrões citogenéticos e ecomorfológicos

The fishes of the order Perciformes are characterized as an important model for understanding the genetic structure of marine populations, because besides they present examples of conservation chromosomal, also they present the karyotype diversification for some groups. Gobiidae family is the most specious in the marine environment. Among its representatives, many species are part of a cryptic fauna little noticed and studied, a wide distribution with behavioral and reproductive characteristics, that make them conducive to the action of biogeographical barriers. Morphologically this family presents reduced body structures through simplification and regressions. Despite their importance in evolutionary inferences, cytogenetics data are incipient facing their species diversity, especially with western Atlantic species. In order to estimate the evolutionary diversity in Gobiidae, it were developed cytogenetic analysis and the standards body, through geometric morphometrics in five species on the Brazilian coast, Coryphopterus glaucofraenum, Bathygobius mystacium, B. soporator, Ctenogobius smaragdus e C. Boleosoma. The data show significant karyotype and morphological diversity among the species. The pericentric inversions and mergers play an important role in chromosomal evolution of this family, causing karyotypic structural and numerical differences in all species. Karyotypic and morphological comparisons among geographic samples of B. soporator from the coast of Maranhão, Rio Grande do Norte and Bahia showed cytogenetics patterns commons, but different morphological patterns. A sample from the Atol das Rocas revealed conspicuous morphological and karyotypic differentiation of another continental populations, confirming the presence of a new island species. The approaches done reveal diversification consistent with characteristics of a group of low vagile and largely able to environmental selection due from peculiar ecological requirements

Estrutura genética de Chromis multilineata (Guichenot, 1853) (Perciformes Pomacentridae) na costa NE do Brasil e ilhas oceânicas: evidências de um passado evolutivo instável.

The fauna of Brazilian reef fishes comprises approximately 320 species distributed along the coast of the mainland and islands ocean. Little is known about the levels of connectivity between their populations, but has been given the interest in the relations between the offshore and the islands of the Brazil, in a biogeographical perspective. The oceanic islands Brazilian hosting a considerable number of endemic species, which are locally abundant, and divide a substantial portion of its reef fish fauna with the Western Atlantic. Among the richest families of reef fish in species are Pomacentridae. This study analyzed through analysis of sequences of the mitochondrial DNA control region (D-loop), the standards-breeding population of C. Multilineata in different areas of the NE coast of Brazil, involving both oceanic islands (Fernando de Noronha Archipelago and of St. Peter and St. Paul) and continental shelf (RN and BA). To this aim, partial sequences were used in the region HVR1 of mtDNA (312pb). The population structure and parameters for the estimates of genetic variability, molecular variance (AMOVA), estimation of the index for fixing (FST) and number of migrants were determined. The phylogenetic relationships between the populations were estimated using neighbor-joining (NJ) method. A group of Bayesian analysis was used to verify population structure, according to haplotype frequency of each individual. The genetic variability of populations was extremely high. The populations sampled show moderate genetic structure, with a higher degree of genetic divergence being observed for the sample of the Archipelago of St. Peter and St. Paul. At smaller geographical scale, the sample of Rio Grande do Norte and the Archipelago of Fernando de Noronha do not have genetic differentiation. Three moderately differentiated population groups were identified: a population group (I), formed by the Rio Grande do Norte (I') and the archipelago of Fernando de Noronha (I''), and two other different groups formed by the island population of the archipelago of Saint Peter and St. Paul (II) and Bahia (III). The genetic patterns found suggest that the species has suffered a relatively recent radiation favoring the absence of shared haplotypes. C. multilineata seems to constitute a relatively homogenous population along the West Atlantic coast, with evidence of a moderate population genetic structure in relation to the Archipelago of St. Peter and St. Paul. These data supports the importance of the dispersal larvae by marine current and the interpopulation similarity this species.

caracterização in situ e estrutura genética de populações de Gossypium mustelinum

Gossypium mustelinum Miers ex Watt is the only cotton species native from Brazil. It is endemic of the semi-arid region from North-east of the country, where it occur near from resilient water sources. The threats to the in situ conservation of the populations are caused by human interference in its habitat, mainly by excessive cattle graze and deforestation. Establish efficient strategies of in situ conservation depend on the accomplishment of a diagnosis of how the specie is found in its natural environment, and the knowledge about the genetic structure of the populations. The objectives of this work were i) to determine the in situ conditions of two populations present in rivers from basin of Rio Paraguaçu at the Bahia State, ii) to evaluate the structure and genetic variability presented in both populations, iii) to establish in situ and ex situ conservation strategies. It were realized collection in november 2007, when was realized in situ characterization of G. mustelinum. SSR markers were used for analyze 218 genotypes deriving from two populations of the G. mustelinum, localized at Tocó river and the Capivara river. The allelic frequencies, the heterozigosity and the F statics were estimated. All the plants were classified as wild and natives, and there was no evidence of the use the plants or its parts. The populations showed different conservation conditions in situ. Few plantlets were found in sites with excessive cattle feed, an indication that the damages in young plants should be high enough to compromise the renovation of the populations. On the other hand, populations were well preserved when the anthropic damages was low or inexistent. The 14 SSR primer pairs amplified 17 loci with a medium number of 5 alleles per locus (a total of 85 alleles). The high level of endogamy estimated (FIS=0,808) and the low observed heterozygosity (H0=0,093) were indicatives that the populations reproduce mainly by selfing, geitonogamy and crosses between related individuals. The genetic diversity was high (HE=0,482) and the differentiation between the populations was very high (FST=0,328). At least two sites from both populations of G. mustelinum must be preserved to achieve suitable in situ conservation. Actions that preserve the gallery forest and keep the cattle away should implemented, and could be as simple as erecting a fence. It is not possible anticipated if the in situ preservation will be possible. Therefore collections and ex situ preservation of representative specimens are essential to conserve the genetic diversity of native G. mustelinum

Desenvolvimento de marcadores microssatélites e estrutura genética espacial da Copernicia prunifera (Miller) H. E. Moore (Arecaceae)

The present study aimed to develop microsatellite markers (SSR) for Copernicia prunifera; and characterize the demographic pattern and the spatial genetic structure (SGS) in different development stages of C. prunifera in a natural population of Rio Grande do Norte (RN) by using ISSR molecular markers. 17 SSR primers pairs were developed, which were tested by using DNA from samples of different populations. The demographic and genetic spatial structure was assessed in a plot with an area of 0.55 ha, where all individuals were georeferenced. The molecular analyses with the use of microsatellite markers pointed out that all built primers pairs, when submitted to PCR, had amplification. They showed sizes of base pairs ranging between 113 and 250 bp. The demographic analyses showed a clustered standard of spatial distribution in the first distance classes, random between 40 and 50 m and segregated in higher distances. Eight ISSR primers were used, thereby producing a total of 102 loci, with 100 of them being polymorphic. Among the three stages, the young showed the highest Nei’s genetic diversity index (He = 0.37); whilst the lowest index was found in the reproductive adults (He = 0.34). The AMOVA results showed a greater genetic differentiation within the development stages (98.61%) in comparison to the interval among the stages (1.39%). The total population (n = 161) showed a positive and significant relationship of kinship in the first distance class (12.3 m). The young showed a significant kinship up to 10.5 m and negative in the fifth distance class (37.6 m). The non-reproductive adults had a positive relationship of kinship in the first distance class (11.0 m) and random distribution of genotypes in the remaining classes. The reproductive adults showed genotypes spatially distributed in a random way. The values for the genetic bottleneck tests proved that the number of loci with excess observed heterozygosity was greater than expected. The SGS results reflect the restricted dispersion of the species, and the bottleneck tests reflect the reduction genotypes provoked by the anthropization of natural environments of C. prunifera.

Plant diversity influencing structure and functioning of Caatinga vegetation

O estudo dos efeitos que a diversidade de espécies pode causar nos processos ecossistêmicos tem crescido vertiginosamente nas últimas duas décadas. Diversos trabalhos experimentais realizados no mundo todo têm demonstrado que uma maior diversidade de plantas contribui para o aumento da produtividade de ecossistemas terrestres. Além disso, esse efeito pode influenciar processos em diversos níveis tróficos, contribuindo assim para a estabilidade dos processos ecossistêmicos a longo prazo. Paralelamente com os estudos do efeito da diversidade, muita atenção tem sido dada para desvendar o papel das características funcionais das espécies no funcionamento dos ecossistemas. Isto porque as características funcionais das espécies têm se mostrado importantes "peças" no entendimento dos efeitos que espécies individuais podem exercer nos ecossistemas e suas respostas ao ambiente. Nesta tese de doutorado eu explorei algumas lacunas de conhecimento dentro dessa área em crescente desenvolvimento conhecida na literatura ecológica como "biodiversidade e funcionamento dos ecossistemas". No primeiro capítulo, eu busquei evidências para mecanismos que podem explicar a relação positiva entre diversidade e funcionamento com foco em cinco mecanismos relacionados às interações entre plantas, tendo como parâmetro de funcionamento a produtividade primária. No segundo capítulo, eu utilizei técnicas para a estimativa de padrões de diversidade em escalas biogeográficas e bases de dados de satélites com longa duração para desvendar se a biodiversidade em escalas macroecológicas promove a estabilidade da produtividade dos ambientes terrestres no semiárido brasileiro. Por fim, o objetivo do terceiro capítulo foi entender como a perda da cobertura vegetal originária do uso da terra por comunidades tradicionais no semiárido brasileiro influenciaria os processos de interações entre plantas e o papel das características funcionais das espécies nessas interações. Acredito que a contribuição individual de cada capítulo preenche lacunas de conhecimento importantes dessa área da Ecologia que ainda se encontra em expansão.