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USA soybean germplasm has a narrow genetic base that could be augmented by alleles from the wild species Glycine soja which positively influence agronomic traits. The objective of this study was to identify such alleles for yield and yield component QTL (quantitative trait loci). Two populations of 150 BC2F4 lines were generated from a mating between recurrent parent Glycine max 7499 and donor parent Glycine soja PI 245331 with one line in each population tracing back to the same BC2 plant. Population A was used for the QTL identification analysis and population B was used for the QTL verification test. The population A lines were genotyped at 120 SSR marker loci and one phenotype marker, covering a total map length of 1506 cM in 20 linkage groups with an average interval size of 12.5 cM. There were nine putative QTL significantly (Pandlt;0.0001, LODandgt;3.0) associated with yield and yield component traits across 3 environments. One QTL for seed yield was identified using the combined data; the G. soja allele at satt511 on LG-A1 was associated with increased seed yield (LOD=4.3) with an additive yield effect of 190 235 kg ha-1 depending on the QTL analysis method. The phenotypic variance accounted for by the QTL at satt511 was 12%. This QTL also provided a significant yield increase across environments in the validation population; lines that were homozygous for the G. soja allele at satt511 demonstrated a 6.3% (P=0.037) yield increase over lines that were homozygous for the G. max allele. One seed filling period QTL was identified at satt335 (LOD=4.0) on LG-F with an additive effect of +1 day. This QTL also provided a +1 day additive effect (LOD=3.3) on maturity. These results demonstrate the potential of using exotic germplasm to improve soybean yield.

Soybean (Glycine max) is an important crop in the world in terms of total production and usage. It is also among the least diverse species. The main objectives of the present study were 1) to determine differences between ISSR and RAPD marker systems in detecting genetic variation in soybeans and 2) to identify and characterize accession- diagnostic molecular markers in G. max accessions. Genomic DNAs from 108 G. max accessions from 11 different gene pools were analyzed using several ISSR and RAPD primers. The levels of polymorphic loci detected with the two marker systems were in general moderate and similar.. Overall, 82% of genetic distance values were above 0.40 based on ISSR analysis. However, RAPD data revealed that the accessions from different countries are closely related with 64% genetic distance values below 0.40. The dendrograms constructed with ISSR data revealed that the South Korean accessions formed an out-group while the RAPD analysis showed that accessions from Sweden were separate from the other 10 gene pools. One variety-diagnostic marker generated with ISSR 5 primer was identified in the accession Kao Chien Tao from China. This marker was cloned, and sequenced. Although RAPD and ISSR marker systems detected similar levels of genetic variability, they target different regions of the soybean genome, resulting in different clustering of the 11 gene pools indicating different genetic relatedness among them. This finding demonstrates the usefulness of both marker systems in assessing diversity and relatedness among Glycine max gene pools.

Sporisorium reilianum is a basidiomycetous fungus that causes head smut in sorghum and maize. Infection requires the formation of a dikaryon between spores of compatible mating type and leads to a change from yeast-like to hyphal growth within the host plant. This switch is controlled through mating type loci. Among forty four different sorghum isolates of S. reilianum (SRS), including five different pathotypes, only three compatible pairs were detected, leading to the establishment of haploid cultures with three different “a locus” mating types, as verified by mRNA expression. Amino acid sequence comparison showed that the pheromone gene components of the a mating type loci of sorghum isolates (SRS) are highly similar to those of maize isolates (SRZ). Genetic diversity was measured using amplified fragment length polymorphism (AFLP) to compare isolates collected from the same and different hosts. AFLP analysis showed polymorphism both within and between SRS and SRZ isolates, with isolates from each host tending to show greater similarity. However, the study did not reveal patterns that could be associated with pathotype of sorghum isolates. A recent outbreak of sorghum downy mildew in Texas has led to the discovery of both metalaxyl fungicide resistance and a new pathotype, P6, in the causal organism, Peronosclerospora sorghi. To identify sources of resistance against P6, a total of 336 sorghum lines (245 mini-core lines from ICRISAT, India, 67 elite accessions from KSU, Kansas and 24 accessions from Texas) were used in a greenhouse study. Fifty two mini-core and 20 accessions from Kansas and 13 from Texas were recorded with ≤10% infection and characterized as resistant for further confirmation. Out of the 52 resistant mini-core accessions, 20 were photo-insensitive and showed 0 % infection. Eleven out of 20 from Kansas showed 0% infection. To quantify the genetic diversity among resistant accessions a high throughput ABI Prism 3100 DNA sequencing system was used for DNA fingerprinting with 60 Simple Sequence Repeat (SSR) markers representing all 10 sorghum linkage groups. Analysis of SSR patterns showed high diversity among the resistant sorghum accessions that were collected from different geographical regions and include the five defined races of Sorghum bicolor. Accessions that are not closely related were most likely to represent unique sources of genetic resistance to P. sorghi.

Title from first page of PDF document. Includes bibliographical references (p. 24-26).

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Deciphering patterns of intraspecies population genetic structuring in commercially important shark species is essential for an integrated fisheries management approach to conservation of regional biodiversity. The common smoothhound shark, Mustelus mustelus, is an overexploited, commercially and recreationally important shark species in South Africa. Considering the vulnerable status of the common smoothhound shark and due to very limited available genetic information, this study aimed to develop molecular markers, assess patterns of genetic diversity and population connectivity along the South African coast using multilocus data generated from 12 microsatellite markers and the mitochondrial gene, NADH dehydrogenase subunit 4 (ND4). The cross-species amplification of microsatellites proved useful for genetic diversity and population genetic analysis of the common smoothhound shark. These microsatellites could aid in the molecular characterisation of other endemic and cosmopolitan species and provide valuable tools for the conservation of potentially threatened or exploited shark species. For the microsatellite data, moderate levels of genetic diversity based on the heterozygosity, allelic richness and haplotype diversity were found in a total of 144 individuals sampled across eight study populations. Estimates for pairwise population differentiation, F-statistics, AMOVA and factorial correspondence analysis (FCA) indicated significant genetic structure within and between west- and east coast populations. Additionally, Bayesian clustering analyses detected two putative ancestral gene pools, supporting the presence of a biogeographic barrier at the Cape Agulhas region and therefore genetic discontinuity between the Indian and Atlantic Ocean samples. On the contrary, mitochondrial data indicated that common smoothhound shark is genetically homogenous with substantial interoceanic gene flow. Such conflicting signals found between nuclear and mitochondrial DNA (mitonuclear discordance) can be attributed to a number of factors and could simply be due to the inherent differences in marker properties or an indication of sex biased dispersal. Despite an indication of an expanding common smoothhound shark population based on both marker types, a contemporary genetic bottleneck may have gone undetected as genetic divergence was very low in some of the study populations. Nonetheless, contemporary restriction to gene flow and historical demographics such as range expansion are proposed as the most likely forces explaining genetic structure in present-day common smoothhound sharks in South Africa. For future sustainable exploitation of common smoothhound shark, the possible existence of two genetically differentiated populations and observed asymmetric gene flow along the South African coast should be taken into consideration. It is also recommended that in the future further evaluations of finescale genetic structure and seasonal migration patterns in this commercially important species are conducted in order to allow integration of this knowledge into existing fisheries management practices. / AFRIKAANSE OPSOMMING: Die ontsyfering van patrone van intraspesie populasie genetiese struktuur in kommersieel belangrike haai spesies is noodsaaklik vir 'n geïntegreerde bestuursbenadering tot visserue en bewaring van plaaslike biodiversiteit. Die hondhaai, Mustelus mustelus, is 'n oorbenutte, kommersiële en sporthengelary belangrike haai spesie in Suid-Afrika. Met inagneming van die kwesbare status van die hondhaai en as gevolg van baie beperkte beskikbare genetiese inligting, het hierdie studie gepoog om molekulêre merkers te ontwikkel, asook om die patrone van genetiese diversiteit en populasie struktuur te ondersoek langs die Suid- Afrikaanse kus deur middel van multilokus data gegenereer uit 12 mikrosatelliet merkers en die mitokondriale geen, NADH dehidrogenase subeenheid 4 (ND4). Die kruis-spesie amplifisering van mikrosatelliete was nuttig vir genetiese diversiteit en populasie genetiese analise van die hondhaai. Hierdie mikrosatelliete kan moontlik help met die molekulêre karakterisering in ander inheemse en kosmopolitaanse spesies en kan as waardevolle hulpmiddels dien in die bewaring van potensieel bedreigde en oorbenutte haai spesies. Vir die mikrosatelliet data is matige vlakke van genetiese diversiteit gevind gebaseer op heterosigositeit, alleliese rykheid en haplotipe diversiteit gevind in 'n totaal van 144 individue getoets oor agt studie populasies. Skattings vir paarsgewyse populasie differensiasie, Fstatistieke, AMOVA en faktoriale ooreenstemming analise het betekenisvolle genetiese struktuur aangedui binne en tussen wes- en ooskus populasies. Daarbenewens, het Bayesian groepering analise twee potensiele voorvaderlike geenpoele waargeneem, ter ondersteuning van die teenwoordigheid van 'n biogeografiese versperring by die Cape Agulhas gebied en dus genetiese diskontinuïteit tussen die Indiese en Atlantiese Oseaan monsters. In teenstelling het die mitokondriale data aangedui dat hierdie haai spesie geneties homogeen is met aansienlike interoseaniese geenvloei. Sulke teenstrydige tekens tussen kern en mitokondriale DNS (mitokern onenigheid) kan toegeskryf word aan 'n aantal faktore en kan eenvoudig wees as gevolg van die inherente verskille in merker eienskappe of 'n aanduiding van geslags sydigeverspreiding. Ten spyte van 'n aanduiding van 'n groeiende hondhaai populasie gebaseer op beide merker tipes, kon 'n hedendaagse genetiese bottelnek onopgemerk gegaan het aangesien genetiese divergensie baie laag was in sommige van die studie populasies. Nietemin, hedendaagse restriksie van geenvloei en historiese demografie soos verbreding van reeks voorkoming word voorgestel as die mees waarskynlike dryfkragte wat genetiese struktuur in die hedendaagse hondhaaie in Suid-Afrika verduidelik. Vir toekomstige volhoubare benutting van die spesie, moet die moontlike bestaan van twee geneties verskillende populasies en waargenome asimmetriese geenvloei langs die Suid-Afrikaanse kus in ag geneem word. Vir die toekoms word dit ook aanbeveel dat verdere evaluerings van fyn-skaal genetiese struktuur en seisoenale migrasie patrone in hierdie kommersiël belangrike spesie uitgevoer word om die integrasie van hierdie kennis in die bestaande bestuur van visserye praktyke toe te laat. / National Research Foundation (NRF)

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Vitis vinifera is the woody crop most susceptible to intracellular pathogens. Currently 70 pathogens infect grapevine, of which 63 are of viral origin. Grapevine leafroll-associated virus 3 (GLRaV-3) is the type species of the genus Ampelovirus, family Closteroviridae. It is considered to be the primary causative agent of Grapevine leafroll disease (GLD) globally; however, the etiology of GLD is not completely understood. Here we report on the viral populations present in GLD symptomatic grapevines across the Western Cape province, South Africa. A widespread survey was performed to screen 315 grapevines for 11 grapevine-infecting viruses using RT-PCR. Additionally, GLRaV-3 variant groups were distinguished with high-resolution melt (HRM) curve analysis used in conjunction with real-time RT-PCR. Members of the family Closteroviridae were detected with the highest frequency, particularly GLRaV-3 that was detected in 87% of tested plants. Nextgeneration sequencing (NGS) is capable of detecting known and novel viruses without prior knowledge of viral sequences and when used in a metagenomic approach is able to detected viral populations within diseased vines. A total of 17 grapevine samples were subjected to NGS using either an Illumina MiSeq or HiSeq 2500 instrument to determine the virome within GLD vines. Collectively, more than 190 million reads were generated through NGS. Read datasets were trimmed and filtered for quality and subjected to both read-mapping and de novo assembly. Contigs assembled de novo were analyzed with BLAST (Basic Local Alignment Search Tool) against the NCBI (National Centre for Biotechnology Information) database and it was determined that GLRaV-3 was the best-represented virus, comprising 97.5% of the assembled contigs. Grapevine virus F (GVF) was detected for the first time in South African vineyards through de novo assemblies and the complete genome sequence validated through direct Sanger sequencing. The complete genome of GVF isolate V5 spans 7 539 nucleotides and shares 89.11% nucleotide identity to existing GVF genomes. The data generated through this study will assist in further understanding the etiology of GLD, support the current hypothesis of GLRaV-3 as the primary contributor to GLD, aid in understanding virus associations in diseased vines and potentially develop systems in which to control disease spread and symptom severity. / AFRIKAANSE OPSOMMING: Vitis vinifera is die houtagtige oes wat die mees vatbaarste is vir intrasellulêre patogene. Tans word wingerde deur 70 patogene geïnfekteer, waarvan 63 van virale oorsprong is. Grapevine leafrollassociated virus 3 (GLRaV-3) is die tipe spesie van die genus Ampelovirus, familie Closteroviridae. Dit word globaal beskou as die primêre oorsaak van Wingerd krulblaar-siekte (GLD), alhoewel die etiologie van GLD nie heeltemal begryp word nie. In hierdie verslag word die virale populasies teenwoordig in GLD simptomatiese wingerde oor die Wes-Kaap provinsie in Suid-Afrika gerapporteer. ‘n Wydverspreide opname was uitgevoer om 315 wingerde met 11 wingerdinfekterende virusse te ondersoek, deur gebruik te maak van tru-transkripsie polimerase ketting reaksie (PKR). Verder is variantgroepe van GLRaV-3 onderskei met hoë-resolusie smeltingskurweanalise, tesame met die gebruik van in-tyd tru-transkripsie PKR. Die hoogste frekwensie was van die lede van die familie Closteroviridae, veral GLRaV-3 wat in 87% van die ondersoekte plante gevind is. Nuwe-generasie volgorderbepaling (NGS) beskik oor die vermoë om bekende en nuwe virusse te herken in virale populasies in geaffekteerde wingerde sonder vorige kennis van virale volgorderbepalings en wanneer dit in ‘n metagenomiese benadering gebruik word kan die virale bevolkings binne siek wingerde ontdek. ‘n Totaal van 17 wingerd-steekproewe was blootgestel aan NGS deur die gebruik van of ‘n Illumina MiSeq of ‘n HiSeq 2500 instrument om die virome te bepaal van GLD wingerde. In totaal is meer 190 miljoen lesings gegenereer deur NGS. Hierdie data lesings was verwerk en gefilter vir kwaliteit om onderwerp te word vir beide kartering en de novo samestellings. Contigs verkry deur de novo samestellings was geanaliseer met BLAST (Basic Local Alignment Search Tool) teenoor die NCBI (National Centre for Biotechnology Information) databasis en dit was vasgestel dat GLRaV-3 was die mees-verteenwoordigende virus, bestaande uit 97.5% van die saamgestelde contigs. Grapevine virus F (GVF) was vir die eerste keer in Suid- Afrikaanse wingerde waargeneem deur de novo samestellings en die volledige genoom volgordger is geverifieer deur middel van direkte Sanger volgorderbepaling. Die volledige genoom van GVF isoleer V5 spanwydte van 7539 nukleotiedes en deel 89.11% nukleotied identiteite van bestaande GVF genome. Die gegenereerde data van hierdie studie sal bykomende begrip van die etiologie van GLD bystaan, die huidige hipotese van GLRaV-3 as die primêre bydraer tot GLD ondersteun, verhoogde begrip van virus-assosiasies in wingerdsiektes verseker en potensiële sisteme ontwikkel om siektes en simptome te beheer.

Lactobacilos são um grupo de bactérias relacionadas com cárie dental. Há falta de estudos sobre a biologia populacional dos lactobacilos na cárie dental. O objetivo do estudo foi avaliar o efeito das modificações ambientais na composição, diversidade genética dos lactobacilos e Identificar a filogenia dos Lactobacillus paracasei isolados do biofilme. Lactobacilos foram isolados em um modelo de formação de biofilme in situ antes e depois de 28 dias de exposição à solução de sacarose 20%. As colônias foram randomicamente selecionadas do meio Rogosa Ágar e subcultivadas (n=222, 31 antes e 191 após período de exposição à sacarose). Os isolados foram identificados usando o seqüenciamento parcial dos genes pheS ou rpoA. As espécies de lactobacilos predominantes encontradas foram L. paracasei, L. fermentum e L. rhamnosus. A diferença na composição e na diversidade genética de lactobacilos associada às modificações ambientais no biofilme foi analisada através de PCR utilizando palíndromes repetitivos extragênicos (REP-PCR). Após a fase com sacarose, um maior número de lactobacilos pode ser encontrado no biofilme (p=0,001). A prevalência de L.fermentum foi similar na fase sem sacarose (6/11 indivíduos colonizados) e na fase com sacarose (8/11 indivíduos colonizados) (p=0,721). A prevalência de L. rhamnosus e L. paracasei aumentou no biofilme de 2/11 para 8/11 indivíduos (p= 0,028) e de 2/11 para 7/11 indivíduos colonizados (p=0,012) após exposição a sacarose, respectivamente. A prevalência de L. gasseri foi baixa e em ambas as fases (p=1,00). As espécies de Lactobacilos apresentaram maior diversidade na fase com sacarose (2 a 3 espécies por indivíduo) do que na fase sem sacarose (0 a 2 espécies por indivíduo) (p=0,045). Na maioria dos casos, diferentes genótipos estavam presentes na fase sem sacarose em comparação à fase com sacarose (p=0,01). Aqueles lactobacilos identificados como L. paracasei foram também submetidos à tipificação através do seqüenciamento de múltiplos loci (MLST). No MLST, foi obtida a sequência parcial de 7 genes de referência: fusA, ileS, lepA, leuS, pyrG, recA, e recG. Sete indivíduos apresentavam L. paracasei (n=75) e 14 seqüências de tipificação (ST) foram encontradas. Verificou-se que indivíduos não relacionados podem apresentar uma mesma ST e que múltiplas STs estão presentes por indivíduo. Três indivíduos apresentavam STs previamente isoladas de produtos alimentícios lácteos. Resultados conflitantes na comparação entre REP-PCR e MLST foram observados na genotipagem de L. paracasei. Diferentes números de padrões foram obtidos para os L. paracasei de acordo com o método molecular utilizado (14 com MLST versus 25 com REP-PCR). Para algumas cepas, REP-PCR foi mais discriminatório do que MLST. Em outros casos, cepas pertencentes a diferentes STs foram agrupadas pelo REP-PCR, mostrando que o MLST foi mais discriminatório do que o REP-PCR. Em poucos casos, MLST e REP-PCR apresentaram o mesmo poder discriminatório. Em geral, REP-PCR mostrou um maior poder discriminatório em comparação ao MLST, mas pouca concordância foi apresentada entre os dois métodos. Assim, MLST pode contribuir na identificação da diversidade genética obtida pelo REP-PCR em alguns casos. Os dados obtidos permitiram concluir que após exposição a sacarose observa-se (a) aumento no número de lactobacilos e de superfícies colonizadas; e (b) aumento na diversidade genética e de espécies. Além disso, observou-se que (c) alguns lactobacilos orais podem apresentar origem exógena, e que (d) a combinação dos métodos MLST e REP-PCR aumenta o poder discriminatório quanto à diversidade genética de L. paracasei. / Lactobacilli are a group of bactéria related to dental caries. There is a lack of studies on the population biology of this organisms in dental caries. The aim of the study was to evaluate the effect of the environmental changes in the composition, genetic diversity and identify the filogeny of Lactobacillus paracasei isolated from biofilm. Lactobacilli were isolated from a biofilm model, formed in situ prior to and during a 28-day period of exposure to 20% sucrose solution. The lactobacillus colonies were randomly selected from Rogosa Agar medium and subcultured (n=222, 31 prior to and 191 following a sucrose exposure period). The isolates were identified using pheS or rpoA gene sequence analysis. The predominant lactobacilli were L. paracasei, L. fermentum and L. rhamnosus. The difference in composition and genetic diversity of lactobacilli related to environmental changes in biofilm was analysed by repetitive extragenic palindromic PCR (REP-PCR). After the sucrose phase, a higher number of lactobacilli could be found in dental biofilm (p=0.001). The prevalence of L. fermentum was similar in the non-sucrose (6/11 subjects) compared to the sucrose phase (8/11 subjects) (p=0.721). Prevalence of L. rhamnosus and L. paracasei increased in biofilm from 2/11 to 8/11 subjects (p= 0.028) and from 2/11 to 7/11 subjects (p=0.012) after sucrose exposure, respectively. L. gasseri prevalence was low in both phases (p=1.00). Lactobacilli exhibited greater species diversity (2 or 3 species per subject) in the sucrose phase, than those isolated from the non-sucrose phase (0-2 species per subject) (p=0.045). In most of the cases different genotypes were present in the non-sucrose phase in comparison to the sucrose phase. Those lactobacilli identified as L. paracasei were subjected to multilocus sequencing typing (MLST). In MLST, partial sequences of seven housekeeping genes fusA, ileS, lepA, leuS, pyrG, recA, and recG was obtained. Seven subjects harboured L. paracasei (n=75) and these represented 14 sequence types (ST). Comparison of the STs showed that unrelated subjects may harbour the same ST and that individuals harbour multiple STs. Three subjects harboured STs previously isolated from dairy products. There were mixed results among REP-PCR patterns compared with the MLST in the L. paracasei genotyping. Different numbers of patterns were obtained for L. paracasei according to the molecular technique used (14 MLST versus 25 REP-PCR patterns). For some strains, REP-PCR was more discriminatory than MLST. In other cases, strains belonging to different ST were grouped together by REP-PCR, showing that MLST was more discriminatory than REP-PCR. In few cases MLST and REP-PCR presented the same discriminatory power. REP-PCR showed a greater discriminatory power in comparison to MLST, but little agreement was observed between this two methods. Therefore, MLST could enhance the genetic diversity obtained by REP-PCR. The present data support that after sucrose exposure there is (a) an increase in the number of lactobacilli and colonized surfaces; and (b) increase in lactobacilli species and genetic diversity. Also it was found that (c) some oral lactobacilli may be of exogenous origin; and (d) the combination of MLST and REP-PCR increased the discriminatory power in genotyping L. paracasei.

Made available in DSpace on 2015-05-14T16:53:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-12-18Bitstream added on 2015-05-14T16:59:00Z : No. of bitstreams: 1 000822497.pdf: 618015 bytes, checksum: 10e49b7cf4610742761f400fa33c5d06 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A arara-azul-grande (Anodorhynchus hyacinthinus), maior psitacídeo do mundo, é uma espécie considerada em vulnerabilidade de extinção (IUCN) principalmente devido à perda de habitat e ao tráfico ilegal. Para estabelecer estratégias que visem a conservação da espécie considerar a composição genética de suas populações é muito importante. Comumente para o estudo da diversidade genética e estrutura genética populacional são utilizados marcadores moleculares que podem ser prospectados no genoma, dentre os quais destacam-se os locos microssatélites. Atualmente, entretanto, não há locos microssatélites polimórficos espécie-específícos identificados em A. hyacinthinus. Esta espécie pode ser encontrada em três regiões possivelmente isoladas, o que evidencia a necessidade de medidas de manejo integradas para a preservação do pool gênico da espécie. Assim, os objetivos principais desse trabalho foram: (i) desenvolver primers para locos microssatélites em A. hyacinthinus, e (ii) analisar a diversidade e a estrutura genética populacional da espécie. Como resultado seis locos espécie-específicos foram prospectados (AnH6, AnH10, AnH17, AnH33, AnH23 e AnH34), dos quais cinco mostraram-se polimórficos em A. hyacinthinus. Além disso, os seis pares de primers mostraram potencial aplicabilidade para estudos populacionais em outras sete espécies de psitacídeos neotropicais. Nas análises populacionais, A. hyacinthinus apresentou um baixo índice de variabilidade genética em comparação à reportada para outros psítacídeos. Esse resultado indica que a baixa variabilidade encontrada não deve ter relação com a utilização restrita de locos heterólogos em trabalhos anteriores, pois foram utilizados tanto locos espécie-específicos quanto locos heterólogos nas análises do presente estudo, sugerindo que esta é uma característica intrínseca da espécie. Além disso, considerando-se a análise de estruturação ... / Hyacinth macaw (Anodorhynchus hyacinthinus), the largest parrot in the world, is considered vulnerable to extinction (IUCN) mainly due to habitat loss and illegal trade. To establish strategies for the conservation of the species must be regarded the genetic makeup of their populations. Commonly for the study of genetic diversity and population genetic structure are used molecular markers, highlighting specially the microsatellite loci, which in turn need to be isolated for population studies. However, currently there is no polymorphic microsatellite loci identified in A. hyacinthinus. Additionally, the species is distributed in three apparently isolated areas, thus requiring integrated management in order to preserve the gene pool of the species. Thus, the main objectives of this study were: (i) the development of primers for microsatellite loci in Anodorhyncus hyacinthinus, and (ii) the analysis of the diversity and population genetic structure of all areas of occurrence of the species. Six loci species-specific prospected were obtained (AnH6, AnH10, AnH17, AnH23, AnH33 and AnH34) and five of these primers proved to be polymorphic. In addition, these six pairs of primers showed potential applicability for population studies after they were tested in seven Neotropical parrot species. Regarding the population analyzes using both specific and heterologous loci, A. hyacinthinus presented a low level of genetic variability compared to other parrots, thereby indicating that the low variability found should not be related to the use of only heterologous loci in previous studies, suggesting that this is an intrinsic characteristic of this species. Moreover, considering in the analysis of genetic structure by Bayesian clustering of microsatellite data it could not be found genetic structure between subpopulations of four regions [North Pantanal (NP), South Pantanal (SP), Para (PA) and northeast (NE)]. In contrast, Rst indices indicated ...

Lactobacilos são um grupo de bactérias relacionadas com cárie dental. Há falta de estudos sobre a biologia populacional dos lactobacilos na cárie dental. O objetivo do estudo foi avaliar o efeito das modificações ambientais na composição, diversidade genética dos lactobacilos e Identificar a filogenia dos Lactobacillus paracasei isolados do biofilme. Lactobacilos foram isolados em um modelo de formação de biofilme in situ antes e depois de 28 dias de exposição à solução de sacarose 20%. As colônias foram randomicamente selecionadas do meio Rogosa Ágar e subcultivadas (n=222, 31 antes e 191 após período de exposição à sacarose). Os isolados foram identificados usando o seqüenciamento parcial dos genes pheS ou rpoA. As espécies de lactobacilos predominantes encontradas foram L. paracasei, L. fermentum e L. rhamnosus. A diferença na composição e na diversidade genética de lactobacilos associada às modificações ambientais no biofilme foi analisada através de PCR utilizando palíndromes repetitivos extragênicos (REP-PCR). Após a fase com sacarose, um maior número de lactobacilos pode ser encontrado no biofilme (p=0,001). A prevalência de L.fermentum foi similar na fase sem sacarose (6/11 indivíduos colonizados) e na fase com sacarose (8/11 indivíduos colonizados) (p=0,721). A prevalência de L. rhamnosus e L. paracasei aumentou no biofilme de 2/11 para 8/11 indivíduos (p= 0,028) e de 2/11 para 7/11 indivíduos colonizados (p=0,012) após exposição a sacarose, respectivamente. A prevalência de L. gasseri foi baixa e em ambas as fases (p=1,00). As espécies de Lactobacilos apresentaram maior diversidade na fase com sacarose (2 a 3 espécies por indivíduo) do que na fase sem sacarose (0 a 2 espécies por indivíduo) (p=0,045). Na maioria dos casos, diferentes genótipos estavam presentes na fase sem sacarose em comparação à fase com sacarose (p=0,01). Aqueles lactobacilos identificados como L. paracasei foram também submetidos à tipificação através do seqüenciamento de múltiplos loci (MLST). No MLST, foi obtida a sequência parcial de 7 genes de referência: fusA, ileS, lepA, leuS, pyrG, recA, e recG. Sete indivíduos apresentavam L. paracasei (n=75) e 14 seqüências de tipificação (ST) foram encontradas. Verificou-se que indivíduos não relacionados podem apresentar uma mesma ST e que múltiplas STs estão presentes por indivíduo. Três indivíduos apresentavam STs previamente isoladas de produtos alimentícios lácteos. Resultados conflitantes na comparação entre REP-PCR e MLST foram observados na genotipagem de L. paracasei. Diferentes números de padrões foram obtidos para os L. paracasei de acordo com o método molecular utilizado (14 com MLST versus 25 com REP-PCR). Para algumas cepas, REP-PCR foi mais discriminatório do que MLST. Em outros casos, cepas pertencentes a diferentes STs foram agrupadas pelo REP-PCR, mostrando que o MLST foi mais discriminatório do que o REP-PCR. Em poucos casos, MLST e REP-PCR apresentaram o mesmo poder discriminatório. Em geral, REP-PCR mostrou um maior poder discriminatório em comparação ao MLST, mas pouca concordância foi apresentada entre os dois métodos. Assim, MLST pode contribuir na identificação da diversidade genética obtida pelo REP-PCR em alguns casos. Os dados obtidos permitiram concluir que após exposição a sacarose observa-se (a) aumento no número de lactobacilos e de superfícies colonizadas; e (b) aumento na diversidade genética e de espécies. Além disso, observou-se que (c) alguns lactobacilos orais podem apresentar origem exógena, e que (d) a combinação dos métodos MLST e REP-PCR aumenta o poder discriminatório quanto à diversidade genética de L. paracasei. / Lactobacilli are a group of bactéria related to dental caries. There is a lack of studies on the population biology of this organisms in dental caries. The aim of the study was to evaluate the effect of the environmental changes in the composition, genetic diversity and identify the filogeny of Lactobacillus paracasei isolated from biofilm. Lactobacilli were isolated from a biofilm model, formed in situ prior to and during a 28-day period of exposure to 20% sucrose solution. The lactobacillus colonies were randomly selected from Rogosa Agar medium and subcultured (n=222, 31 prior to and 191 following a sucrose exposure period). The isolates were identified using pheS or rpoA gene sequence analysis. The predominant lactobacilli were L. paracasei, L. fermentum and L. rhamnosus. The difference in composition and genetic diversity of lactobacilli related to environmental changes in biofilm was analysed by repetitive extragenic palindromic PCR (REP-PCR). After the sucrose phase, a higher number of lactobacilli could be found in dental biofilm (p=0.001). The prevalence of L. fermentum was similar in the non-sucrose (6/11 subjects) compared to the sucrose phase (8/11 subjects) (p=0.721). Prevalence of L. rhamnosus and L. paracasei increased in biofilm from 2/11 to 8/11 subjects (p= 0.028) and from 2/11 to 7/11 subjects (p=0.012) after sucrose exposure, respectively. L. gasseri prevalence was low in both phases (p=1.00). Lactobacilli exhibited greater species diversity (2 or 3 species per subject) in the sucrose phase, than those isolated from the non-sucrose phase (0-2 species per subject) (p=0.045). In most of the cases different genotypes were present in the non-sucrose phase in comparison to the sucrose phase. Those lactobacilli identified as L. paracasei were subjected to multilocus sequencing typing (MLST). In MLST, partial sequences of seven housekeeping genes fusA, ileS, lepA, leuS, pyrG, recA, and recG was obtained. Seven subjects harboured L. paracasei (n=75) and these represented 14 sequence types (ST). Comparison of the STs showed that unrelated subjects may harbour the same ST and that individuals harbour multiple STs. Three subjects harboured STs previously isolated from dairy products. There were mixed results among REP-PCR patterns compared with the MLST in the L. paracasei genotyping. Different numbers of patterns were obtained for L. paracasei according to the molecular technique used (14 MLST versus 25 REP-PCR patterns). For some strains, REP-PCR was more discriminatory than MLST. In other cases, strains belonging to different ST were grouped together by REP-PCR, showing that MLST was more discriminatory than REP-PCR. In few cases MLST and REP-PCR presented the same discriminatory power. REP-PCR showed a greater discriminatory power in comparison to MLST, but little agreement was observed between this two methods. Therefore, MLST could enhance the genetic diversity obtained by REP-PCR. The present data support that after sucrose exposure there is (a) an increase in the number of lactobacilli and colonized surfaces; and (b) increase in lactobacilli species and genetic diversity. Also it was found that (c) some oral lactobacilli may be of exogenous origin; and (d) the combination of MLST and REP-PCR increased the discriminatory power in genotyping L. paracasei.