Thesis (MSc)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Haliotis midae, is the only commercially important species of the six abalone species found in South African coastal waters and has become a lucrative commercial commodity. Wild stocks of H. midae are, however, no longer commercially sustainable due to a combination of environmental factors and poaching. The solution to the crisis is artificial production systems in the form of abalone farms. An abalone enhancement programme was initiated in South Africa in 2006, funded by industry and government. This programme focuses on the elucidation of the abalone genome and genetic factors contributing to increased productivity, thereby aiding the commercial production of abalone. The aims of this study, the first of its kind concerning H. midae, were to develop AFLPbased markers (specifically fluorescent AFLP analysis); to monitor the segregation of these markers in a single full-sib family and to use the markers and additional microsatellite markers to generate the first preliminary linkage map for H. midae. Genomic DNA of sufficient quality and purity for fluorescent AFLP analysis was obtained from 3.5-month-old H. midae juveniles. Preliminary linkage maps were constructed using AFLP and microsatellite markers segregating in an F1 family following a pseudo-testcross mapping strategy. Twelve AFLP primer combinations, producing 573 segregating peaks, and 10 microsatellite markers were genotyped in the parents and 108 progeny of the mapping family. Of the 573 segregating AFLP peaks genotyped, 241 segregated in a 1:1 ratio and 332 in a 3:1 ratio. Of these AFLP markers, 90 segregated according to the expected 1:1 Mendelian ratio and 164 segregated according to the expected 3:1 Mendelian ratio at the P = 0.05 level and were used for linkage analysis. Of the 10 microsatellite markers genotyped, nine were informative for linkage mapping analysis. Preliminary male and female genetic linkage maps were developed using markers segregating in the female or male parent. A total of 12 and 10 linkage groups were detected for the female and male maps respectively. The female map covered 1473.5cM and consisted of 56 markers, and the male map covered 738.9cM consisting of 30 markers. Markers with segregation distortion were observed as previously reported in other abalone species and potential homology between one of the linkage groups of the male map and two of the linkage groups of the female map were identified using the 3:1 segregating AFLP markers. In conclusion, the genetic linkage map presented here, despite the fact that it has relatively low genome coverage and low marker density, forms an ideal starting point for more detailed study of the H. midae genome and will provide a scaffold for basic and applied studies in abalone. A high-density linkage map of H. midae should in future be developed with additional co-dominant molecular markers, such as microsatellites, to improve the transferability of the linkage map between different laboratories and among populations. A high-density linkage map will facilitate the mapping of QTL of commercially important traits (i.e. growth) and future MAS breeding programmes. / AFRIKAANSE OPSOMMING: Perlemoenspesie, Haliotis midae, is die enigste spesie van kommersiële belang van die ses wat in die kuswater van Suid-Afrika aangetref word en het ‘n winsgewende handelskommoditeit in Suid-Afrika geword. Die ontginning van natuurlike H. midae populasies is egter, as gevolg van ‘n kombinasie van omgewingsfaktore en stropery nie meer kommersieel volhoubaar nie. Die perlemoenkrisis kan die hoof gebied word deur kunsmatige produksiesisteme op perlemoenplase tot stand te bring. ‘n Perlemoen verbeteringsprogram is in 2006 in Suid-Afrika geïnisieer en word deur die industrie en regering befonds. Die program focus op die ontrafeling van die perlemoen genoom en die genetiese faktore wat bydrae tot verhoogde produksie. Sodanige inligting kan gebruik word om kommersiële perlemoenproduksie te bevorder. Die doel van hierdie studie, die eerste met H. midae, is om AFLP-gebaseerde merkers (spesifiek fluoresserende AFLP analise) te ontwikkel; die segregasie van hierdie merkers te monitor in ‘n enkel volledige verwante familie en die merkers en addisionele mikrosatelliet merkers te gebruik om die eerste voorlopige koppelingskaart vir H. midae te genereer. Genomiese DNS van genoegsame kwaliteit en suiwerheid vir fluoresserende AFLP analise is ge-ekstraeer uit 3.5-maand-oue H. midae individue. Voorlopige koppelingskaart is gekonstrueer deur van segregerende AFLP en mikrosatelliet merkers in ‘n F1 familie gebruik te maak deur ‘n pseudo-kruistoets karteringstrategie te volg. Twaalf AFLP inleier kombinasies, wat 573 segregerende fragmente geproduseer het, en 10 mikrosatelliet merkers is gegenotipeer in die ouers en 108 individue van die nageslag van die karteringsfamilie. Van die 573 segregerende AFLP merkers wat gegenotipeer is, het 241 in ‘n 1:1 verhouding en 332 in ‘n 3:1 verhouding gesegregeer. Van hierdie AFLP merkers, het 90 volgens die verwagte 1:1 Mendeliese verhouding en 164 volgens die 3:1 Mendeliese verhouding by die P = 0.05 gesegregeer vlak en is vir die koppelingsanalise gebruik. Van die 10 mikrosatelliet merkers gegenotipeer, was 9 informatief vir koppeling karteringsanalise. Voorlopige manlike en vroulike genetiese koppelingskaarte is ontwikkel met gebruik te maak van merkers wat in die manlike of vroulike ouer segregeer het. ‘n Totaal van 12 en 10 koppelingsgroepe is onderskeidelik in die vroulike en manlike karate gegenereer. Die vroulike kaart dek 1473.5cM and bestaan uit 56 merkers, terwyl die manlike kaart 738.9cM beslaan het met 30 merkers. Merkers wat segregasie distorsie toon is waargeneem soos voorheen in ander perlemoenspesies gerapporteer. Potensiële ooreenstemming tussen een van die koppelingsgroepe van die manlike kaart en twee van die koppelingsgroepe van die vroulike kaart is aangetoon deur van die 3:1 segregerende AFLP merkers gebruik te maak. Die genetiese koppelingskaarte verskaf wel ‘n relatiewe lae genoomdekking en ‘n lae merkerdigtheid, maar is ‘n ideale vertrekpunt vir meer gedetailleerde studie van die H. midae genoom en dien as ‘n raamwerk vir toekomstige basiese en toegepaste studies in perlemoennavorsing. ‘n Hoëdigtheid koppelingskaart van H. midae moet in die toekoms ontwikkel word met gebruik van bykomstige ko-dominante molekulêre merkers, soos mikrosatelliete. Dit sal die oordraagbaarheid van die koppelingskaart tussen verskillende laboratoria asook tussen populasies verbeter. ‘n Hoëdigtheid koppelingskaart sal die kartering van kwantitatiewe kenmerk loki (KKL) vir kommersieel belangrike kenmerke (onder andere groeikrag) en toekomstige merker bemiddelde seleksie (MBS) teelprogramme moontlik maak.

Thesis (MSc)--University of Stellenbosch, 2008. / ENGLISH ABSTRACT: The five leading abalone producers in South Africa have initiated a genetic enhancement program for Haliotis midae in a collaborative effort to improve economically valuable traits. Several independent objective-specific studies were initiated, including the establishment of a Performance Recording Scheme (PRS), utilised in this study, and necessary to monitor the ongoing performance of individuals as the move from mass-selection to marker assisted selection (MAS) is implemented. The primary objective of this study was parentage assignment of F1 offspring mass-selected for size at approximately one year and allocated to either a “faster” or a “slower” growth group. Nine microsatellite markers were used to genotype juveniles and potential parents, with assignment completed using CERVUS 2.0. Average growth results for Abagold and HIK were comparable for both growth groups. Slight environmental effects, although not statistically significant, were evident as growth advantages for juveniles within the faster growth group at two of the five locations and for juveniles within the slower growth group at one of the five rearing locations. Despite measures to standardise environmental influences, variables are difficult to control within the reality of a production environment; and potential genotype x environment interactions may require further investigation and factoring into future breeding programs. The additional costs associated with MAS often make the technology prohibitive to most aquaculture operations, despite the significant genetic gains to be realised from its implementation. Cost-optimising routine processes such as DNA extractions may be one approach to reduce these additional costs. Chelex®100 appears to be a suitable alternative to the CTAB method – being quick and cost-effective to perform. Applying this method in combination with the high throughput of a robotic platform warrants further evaluation. For the microsatellite development, 50% of positive recombinant clones contained inserts. Sequencing of these clones produced 16% perfect repeats and 47% imperfect repeats for which 52 primer sets were designed and tested. In total, 31 polymorphic microsatellite loci of different motifs and composition were developed. Sixty-one percent of sequenced clones were deemed redundant and pre-screening for both uniqueness and the presence of microsatellites would reduce unnecessary sequencing thus improving the efficiency of the FIASCO method and reducing costs. Nine loci were selected for parentage assignments. Null alleles were present for all the selected markers; however, frequencies were below the critical level of 5%. Parentage yielded 91% and 90% successful assignment for Abagold and HIK respectively; however, observations indicate that a measure of relatedness may exist between breeders. Recommendations with regards to future family breeding include, for both Abagold and HIK, retaining selected breeders based on their respective contributions to the F1 progeny while reassessing the potential of remaining breeding stock under more controlled breeding conditions. No obvious trends were observed for growth with most individuals producing both faster and slower growing offspring. Juveniles will be reassessed at two years to determine whether the size advantage or disadvantages were maintained and to ascertain whether growth advantages/disadvantages may be gender specific. / AFRIKAANSE OPSOMMING: Die vyf mees toonaangewende perlemoen produseerders in Suid Afrika het „n genetiese verbeteringsprogram vir Haliotis midae geinisieer in „n gesamentlike poging om ekonomiese belangrike eienskappe te verbeter. Verskeie onafhanklike fokus-spesifieke studies is geinisieer, insluitend die totstandkoming van „n groeiprestasie aantekenstelsel, soos gebruik in hierdie studie, en wat noodsaaklik is om die aaneenlopende prestasie van individue te moniteer soos daar beweeg word van massa seleksie tot merker bemiddelde seleksie. Die primêre fokus van hierdie studie was die ouerskapsbepaling van F1 nageslag wat massa geselekteer is op ouderdom 1 jaar vir grootte en as of “vinniger” of “stadiger” groeiers geklassifiseer is. Nege mikrosatelliet merkers is gebruik om jong perlemoen individue en moontlike ouers te genotipeer, met die ouerskapstoekenning bereken deur CERVUS 2.0. Groei resultate vir Abagold en HIK was vergelykbaar vir beide groei groepe op drie van die lokaliteite. Geringe omgewingseffekte, alhoewel nie statisties betekenisvol nie, was sigbaar as „n groei voordeel vir jong individue op twee van die vyf lokaliteite. Ongeag maatstawe om omgewingsinvloede te standardiseer, is varieerbares moeilik om te beheer in die produksie omgewing en genotipe x omgewings interaksies mag verdere navorsing vereis en behoort in ag geneem te word in toekomstige telingsprogramme. Die onkoste wat met merker bemiddelde seleksie geassosieer word, maak die tegniek soms onaantreklik vir die meeste akwakultuur operasies; nie teen staande die genetiese voordele wat die gebruik daarvan veroorsaak. Die koste-optimiseering van roetine prosesse, soos byvoorbeeld, DNA ekstraksies, is dalk een aanslag om die addisionele koste te verminder. Chelex®100 blyk „n geskikte alternatief tot die CTAB metode te wees – die tegniek is vinnig en koste-effektief om uit te voer. Die gebruik van hierdie metode in kombinasie met die hoë deurvloei van ‟n robotiese sisteem behoort verder ondersoek te word. Vir die mikrosatelliet ontwikkeling het slegs 50% van die positiewe rekombinante klone invoegings bevat. Nukleotiedvolgorde bepaling van hierdie klone het 16% perfekte herhalings en 47% onderbroke herhalings bevat waaruit 52 inleierstelle ontwikkel en getoets is. In totaal is 31 polimorfiese mikrosatelliet loki van verskillende motiewe en samestelling ontwikkel. Een-en-sestig persent van die volgorde bepaalde klone is oortollig geag en vooraf sifting vir beide uniekheid en die teenwoordigheid van mikrosatelliete sal onnodige volgorde bepaling verhoed, die effektiwiteit van die FIASCO tegniek verhoog sowel as addisionele koste verminder. Nege loki is geselekteer vir ouerskapsbepaling. Nul allele was teenwoordig vir al die geselekteerde merkers, maar die frekwensies was egter laer as die 5% kritieke waarde. Ouerskap is 91% en 90% suksesvol bepaal vir Abagold en HIK onderskeidelik. Waarnemings dui egter daarop dat daar verwantskappe mag wees tussen van die broeidiere. Voorstelle in terme van toekomstige familie teling sluit is, vir beide Abagold en HIK, om geselekteerde broei diere te behou gebaseer op hulle onderskeie bydraes tot die F1 nageslag asook die herevaluaring van die potensiaal van die oorblywende broei diere onder meer beheerde teling toestande. Geen voor-die-handliggende tendense is waargeneem vir groei nie met die meeste individue wat beide vinniger en stadiger groeiende nageslag geproduseer het. Jong individue moet geherevalueer word op tweejarige ouderdom om te bepaal of die groei voordeel of nadele behou is en om te bepaal om groei voordele/nadele geslagspesifiek is.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Haliotis midae (locally also known as perlemoen) is the largest of five endemic species found along the coast of South Africa. It is the only species with commercial value contributing to the exploitation of these animals. Due to declines of natural stocks, farming practices were established during the early 1990s in order to supply the international demand. To facilitate efficient breeding methods and ensure the sustainability of these commercial populations, genetic management, which can be accomplished with the use of molecular markers such as single nucleotide polymorphisms (SNPs), is necessary. Single nucleotide polymorphisms have become the markers of choice in various applications in aquaculture genetics due to their abundance in genomes, reduction in developmental costs and increased throughput of genotyping assays. Identification of SNPs in non-model species such as H. midae can be achieved by in silico approaches. In silico methods are suitable for de novo SNP identification and are both cost- and time-efficient. It is based on the analysis of multiple alignments where mismatches may be reported as candidate SNPs. Various medium-throughput genotyping methods are available to confirm putative SNPs, but the ideal method depends on factors such as cost, accuracy and multiplexing capacity. Although SNP markers can have various applications within the aquaculture environment the focus for this current study was saturating the linkage map of H. midae with additional markers. This would assist in the identification of quantitative trait loci associated with economically important traits, which in turn could ultimately be employed for marker-assisted selection and improved molecular breeding programs. In order to identify in silico SNPs, sequenced transcriptome data from a previous study was used and subjected to a series of criteria: minor allele frequency 10%, minimum coverage 80, 60 bp flanking regions. Selected loci were genotyped using a 192-plex assay with the Illumina GoldenGate genotyping assay with the VeraCode technology on the BeadXpress platform, in individuals from six mapping families. A conversion rate of 69.35% and global success rate of 76.34% was achieved. Polymorphic loci were subjected to linkage analysis using JoinMap® v.4.1 to create sex-average and sex-specific maps and to saturate the current linkage map for H. midae. Along with previously developed markers, 54% of the newly developed SNPs could be successfully incorporated into the linkage map of H. midae. A total of 18 linkage groups were observed with an average marker spacing of 6.9 cM and genome coverage of 79.1%. Bioinformatic analyses and setting stringent criteria to identify SNPs from sequenced transcriptomic data proved to be an efficient way for SNP discovery in the current study. Genotyping of the identified loci with the GoldenGate genotyping assay demonstrated a high success rate; providing a genotyping assay adequate for species with little genomic information. The linkage map created in this study illustrated the utility of SNP markers in conjunction with microsatellite markers for linkage map construction and the adequate marker spacing obtained provides a step closer to quantitative trait loci mapping in this species. / AFRIKAANSE OPSOMMING: Haliotis midae (plaaslik ook bekend as perlemoen) is die grootste van vyf inheemse spesies wat langs die kus van Suid-Afrika aangetref word. Dit is die enigste spesie van kommersiële waarde wat bydraend is tot die uitbuiting van hierdie diere. As gevolg van die afname in hierdie natuurlike hulpbron het boerdery praktyke gedurende die vroeë 1990's ontstaan om in die internasionale aanvraag te voorsien. Ten einde doeltreffende teelmetodes te beoefen en die volhoubaarheid van hierdie kommersiële populasies te verseker is genetiese bestuur, wat bewerkstellig kan word deur die gebruik van molekulêre merkers soos enkel nukleotied polimorfismes (ENPs), baie belangrik. Enkel nukleotied polimorfismes is gewilde merkers in verskeie toepassings in akwakultuur genetika as gevolg van hul oorvloed in genome, verlaagde ontwikkelingskoste en verhoogde deurset van ENP-genotiperingstoetse. Identifisering van ENPs in nie-model spesies soos H. midae kan uitgevoer word deur in siliko benaderings te gebruik wat geskik is vir de novo ENP identifisering en ook tyd- en koste-effektief is. Dit word gebaseer op die analise van veelvuldige inlynstellings waar nukleotiedes wat nie ooreenstem nie as kandidaat ENPs gerapporteer kan word. Om kandidaat ENPs te bevestig, kan verskeie medium-deurset genotiperingsmetodes uitgevoer word, maar die ideale metode word bepaal deur faktore soos koste, akkuraatheid en multipleks kapasiteit. Alhoewel ENP merkers in verskeie toepassing binne die akwakultuur omgewing gebruik kan word was die fokus van die huidige studie om die koppelingskaart van H. midae te versadig. Dit sal bydrae tot die identifisering van kwantitatiewe eienskap lokusse wat gekoppel kan word aan ekonomies belangrike eienskappe wat dan op die beurt weer vir merkerbemiddelde seleksie gebruik kan word en uiteindelik ten opsigte van die verbetering van molekulêre teelprogramme aangewend kan word. Ten einde in siliko ENPs te identifiseer is transkriptoomdata van 'n vorige studie gebruik en onderwerp aan 'n reeks kriteria: geringste alleelfrekwensie 10%, minimum dekking 80, 60 bp gebiede weerskante van polimorfisme. Geïdentifiseerde lokus-genotipering is met behulp van 'n 192-pleks toets uitgevoer met die Illumina GoldenGate genotiperingstoets met die VeraCode tegnologie op die BeadXpress-platform, in individue afkomsitg vanaf ses karteringsfamilies. 'n Omskakelingskoers van 69.35% en 'n algehele sukseskoers van 76.34% is bereik. Polimorfiese lokusse is onderwerp aan koppelings-analise met behulp van JoinMap® v.4.1 om geslags-gemiddelde en geslags-spesifieke kaarte te skep asook om die kaart wat beskikbaar is vir H. midae te versadig. Saam met voorheen ontwikkelde merkers is 54% van die nuut ontwikkelde ENPs suksesvol opgeneem in die kaart van H. midae. 'n Totaal van 18 koppelingsgroepe is verkry met 'n gemiddelde merker-spasiëring van 6.9 cM en 'n genoomdekking van 79.1%. Die gebruik van bioinformatiese analises en streng kriteria om ENPs vanaf transkriptoomdata te identifiseer blyk doeltreffend te wees in hierdie studie. Genotipering van die geïdentifiseerde lokusse met die GoldenGate genotiperingstoets dui op 'n hoë suksessyfer en verskaf 'n voldoende genotiperingstoets aan spesies met min genomiese inligting. Die koppelingskaart in hierdie studie het geïllustreer dat die ENP merkers suksesvol saam met mikrosatelliet merkers gebruik kan word vir koppelingskaart konstruksie en dat die voldoende merker-spasiëring verkry 'n stap nader aan kwantitatiewe eienskap lokus kartering in hierdie spesie bied.

O estresse térmico causa prejuízos para a atividade leiteira. É possível selecionar animais para tolerância ao calor, uma vez que existe variação genética de características produtivas quando avaliadas em diferentes ambientes climáticos. Para uma correta avaliação genética em função de diferentes ambientes, a utilização de modelos que se ajustem aos dados é fundamental. Ao mesmo tempo, ferramentas genômicas podem auxiliar nos processos de avaliação e seleção genética para tolerância ao calor. Nesse contexto, foram desenvolvidos dois estudos. No primeiro, objetivou-se estudar as funções de variância de características de produção e qualidade do leite em relação a um índice de temperatura e umidade (THI), ajustadas por polinômios de Legendre de ordens dois a sete, avaliar qual função melhor se ajusta aos dados e compreender como os componentes de variância e os coeficientes de herdabilidade se comportam em função do THI. Para isso, foram utilizadas 74.470 informações de produção de leite (PROD, kg/dia), escore de células somáticas (ECS), porcentagens de gordura (GOR), proteína (PROT), lactose (LACT), caseína (CAS) e perfil de ácidos graxos (saturados - SAT; insaturados - INSAT; monoinsaturados - MONO; poli-insaturados - POLI; ácido palmítico - C16:0; ácido esteárico - C18:0; e ácido oléico - C18:1) de 5.224 vacas da raça Holandesa avaliadas por meio de modelos de regressão aleatória em 167 valores distintos do THI. À exceção de POLI, houve variação em todos os componentes de variância ao longo do THI para todas as características. As estimativas de herdabilidade variaram de 0,07 a 0,43. Para PROD, GOR, LACT, ECS, SAT e C16:0, as estimativas de herdabilidade diminuíram com o aumento do THI e para CAS, MONO, INSAT e C18:1, aumentaram. Para PROT e C18:0 as estimativas de herdabilidade foram maiores em valores intermediários do THI e menores nos extremos. No segundo estudo, objetivou-se estimar o efeito de marcadores SNP (polimorfismos de nucleotídeo único) sobre as características PROD, CAS, ECS, SAT e INSAT e estimar o valor genético genômico (GEBV) considerando dois valores de THI, um representando o conforto térmico e outro o estresse térmico. Os valores genéticos dos animais para os dois ambientes foram usados como fenótipos nos estudos de associação genômica (GWAS). Foram utilizados genótipos de 1.157 vacas para 60.671 marcadores SNP. Para as análises genômicas, foram utilizadas somente vacas com fenótipo e genótipo. Foi utilizado o método GBLUP (melhor predição linear não-viesada genômica) sob abordagem bicaracterística para realizar o GWAS. Com objetivo de comparar os ambientes, foram considerados os 55 SNP de maior variância aditiva explicada para cada situação e 10% dos animais com maiores valores genéticos genômicos. Em geral, os SNP apresentaram poucas diferenças em suas variâncias aditivas explicadas quando comparados em ambiente de conforto e estresse térmico. Com relação aos valores genéticos genômicos, houve reclassificação dos animais para PROD (correlações 0,90 e 0,90), CAS (correlações 0,88 e 0,86), SAT (correlações 0,88 e 0,87) e INSAT (correlações 0,97 e 0,97). Para ECS, apenas um animal foi reclassificado entre os ambientes (correlações 1,00). O primeiro estudo permitiu avaliar os componentes de variância ao longo de todo o gradiente ambiental. Por sua vez, o segundo estudo permitiu avaliar a variância individual dos marcadores moleculares, complementando o estudo genético das características. Mesmo que pequenas, foi possível verificar diferenças genéticas entre os animais entre os ambientes considerados. / Heat stress causes damage to dairy farming activities. It is possible to select animals for heat tolerance, because there is genetic variation of productive traits when evaluated in different climatic environments. For a correct genetic evaluation according to different environments, the use of models that fit to the data is fundamental. At the same time, genomic tools can aid in the evaluation and genetic selection processes for heat tolerance. In this context, two studies were developed. In the first, the aim was to study the variance functions of milk production and quality traits in relation to a temperature and humidity index (THI), adjusted by Legendre polynomials of orders two to seven, to evaluate which function best fits the data and understand how the variance components and heritability coefficients behave as a function of THI. For this, records of milk yield (MY), somatic cell score (SCS), percentage of fat (FP), protein (PP), lactose (LP), casein (CP) and acid fatty acids (saturated - SAT, unsaturated - UNSAT, monounsaturated - MONO, polyunsaturated - POLY, palmitic acid - C16:0, stearic acid - C18:0; and oleic acid - C18:1) from 5,224 Holstein cows and evaluated using random regression models in 167 different levels of THI were used. With the exception of POLY, there was variation in all variance components along the THI for all traits. Estimates of heritability ranged from 0.07 to 0.43. For MY, FP, LP, SCS, SAT and C16:0, estimates of heritability decreased with increasing THI and for CP, MONO, UNSAT and C18:1 increased. For PP and C18:0, heritability estimates were higher in intermediate THI values and lower at the extremes. In the second study, the objective was to estimate the effect of SNP markers on traits MY, CP, SCS, SAT and UNSAT and to predict the genomic breeding values (GEBV) using these effects, considering two levels of THI: a thermal comfort and an heat stress. The breeding values of the animals for the two environments were used as phenotypes for the genomic wide association studies (GWAS). Genotypes of 1,157 cows to 60,671 SNP markers were used. The GBLUP method (genomic best linear unbiased prediction) was used under a bitrait approach to perform GWAS. With the aim of comparing the environments, the 55 SNP with the greater variance explained for each situation and the 10% animals with the greater GEBV were used. Differences were observed in the variance explained by SNP between the comfort and heat stress environment. There was a re-rankink of animals considering the GEBV for MY (correlations 0.90 and 0.90), CP (correlations 0.88 and 0.86), SAT (correlations 0.88 and 0.87) and UNSAT (correlations 0.97 and 0.97). For SCS, only one animal was reclassified between the environments (correlations 1.00). The first study allowed to evaluate the components of variance along the entire environmental gradient. In turn, the second study allowed to evaluate the individual variance of the molecular markers, complementing the genetic study of the traits. Although small, it was possible to verify genetic differences among the animals among the considered environments.

abstract: Water quality in surface water is frequently degraded by fecal contamination from human and animal sources, imposing negative implications for recreational water use and public safety. For this reason it is critical to identify the source of fecal contamination in bodies of water in order to take proper corrective actions for controlling fecal pollution. Bacteroides genetic markers have been widely used to differentiate human from other sources of fecal bacteria in water. The results of this study indicate that many assays currently used to detect human-specific Bacteroides produce false positive results in the presence of freshwater fish. To further characterize Bacteroides from fish and human, the fecal samples were cultured, speciated, and identified. As a result, forty six new Bacteroides 16S rRNA gene sequences have been deposited to the NCBI database. These sequences, along with selected animal fecal sample Bacteroides, were aligned against human B. volgatus, B. fragilis, and B. dorei to identify multi-segmented variable regions within the 16S rRNA gene sequence. The collected sequences were truncated and used to construct a cladogram, showing a clear separation between human B. dorei and Bacteroides from other sources. A proposed strategy for source tracking was field tested by collecting water samples from central AZ source water and three different recreational ponds. PCR using HF134 and HF183 primer sets were performed and sequences for positive reactions were then aligned against human Bacteroides to identify the source of contamination. For the samples testing positive using the HF183 primer set (8/13), fecal contamination was determined to be from human sources. To confirm the results, PCR products were sequenced and aligned against the four variable regions and incorporated within the truncated cladogram. As expected, the sequences from water samples with human fecal contamination grouped within the human clade. As an outcome of this study, a tool box strategy for Bacteroides source identification relying on PCR amplification, variable region analysis, human-specific Bacteroides PCR assays, and subsequent truncated cladogram grouping analysis has been developed. The proposed strategy offers a new method for microbial source tracking and provides step-wise methodology essential for identifying sources of fecal pollution. / Dissertation/Thesis / Ph.D. Civil and Environmental Engineering 2012

Orientador: Antonio de Goes / Coorientador: Hélida Mara Magalhães / Banca: Rita de Cassia Panizzi / Banca: Luciana Rossini Pinto / Banca: Viviani Vieira Marques / Banca: Rubens Pazza / Resumo: O Brasil é o terceiro maior produtor mundial de frutas, com mais de 40 milhões de toneladas anuais, e uma área plantada em torno de três milhões de hectares. Entretanto, esse setor ressente-se de problemas complexos, de natureza diversa, como os de ordem fitossanitária, dentre eles, destaca-se a antracnose, causada por fungos do gênero Colletotrichum. As perdas resultantes desta doença dão-se em pré e pós-colheita, podendo chegar a 90% de prejuízo dependendo das condições ambientais e do manejo adotado. Nas frutíferas tropicais o Colletotrichum gloeosporioides é descrito com maior frequência, como agente causal da antracnose, porem estudos recentes demonstram que nem sempre apenas essa espécie é a responsável pela doença. De um modo geral, os patógenos descritos em associação com o complexo de sintomas de antracnose em frutas são classificados Colletotrichum gloeosporioides e Colletotrichum acutatum. Estudos recentes demonstram que nem sempre os critérios adotados para a classificação da espécie do patógeno associado aos sintomas são os adequados. Com isso, para maior precisão e clareza é fundamental o estudo completo desses patógenos, envolvendo analises simultâneas das caracteristicas morfoculturais e moleculares / Abstract: Brazil ranks third among the largest fruit producers in the world with more than 40 million tons per year and a planted area of about three million hectares. However, this sector has complex problems of different nature, such as plant diseases and among them, anthracnose. This disease is caused by fungi of the Colletotrichum genus. Losses resulting from this disease happen pre- and post-harvest, reaching 90% of damage depending on environmental conditions and management adopted. Colletotrichum gloeosporioides is described most often as the causal agent of anthracnose in tropical fruits; however, recent studies have shown that this is not always the case. In general, the pathogens described in connection with the complex symptoms of anthracnose in fruits are classified either as Colletotrichum gloeosporioides or Colletotrichum acutatum. But, recent studies have shown that the criteria adopted to classify the pathogen species associated with the symptoms are not always the most appropriate. Thus, simultaneous analysis of morphocultural and molecular characteristics are necessary to classify these pathogen species more accurately / Doutor

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-07-20T21:11:57Z No. of bitstreams: 1 Kathleen Ribeiro SouzaAvaliação de parâmetros moleculares para....pdf: 2252523 bytes, checksum: 982a659125f51498b2c5188068c9718f (MD5) / Made available in DSpace on 2012-07-20T21:11:57Z (GMT). No. of bitstreams: 1 Kathleen Ribeiro SouzaAvaliação de parâmetros moleculares para....pdf: 2252523 bytes, checksum: 982a659125f51498b2c5188068c9718f (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / Apesar do combate recorrente ao mosquito vetor da dengue, o Aedes aegypti, mais de 80% dos estratos da cidade de Salvador-BA apresentam condição de alerta ou risco de surto de dengue. Visto que as abordagens tradicionais para controle do mosquito vetor da dengue não têm produzido os efeitos esperados, o presente estudo avaliou parâmetros moleculares para vigilância entomológica do A. aegypti utilizando ferramentas de geotecnologia e de genética de populações como forma de apoiar o trabalho de campo e ações integradas das instâncias responsáveis pelo controle da dengue. O desenho do estudo apresentou um componente transversal, descrevendo dados sobre a genética de população de larvas de A. aegypti coletadas em Salvador e amostras controle coletadas no ano de 2009 em Jacobina e Vitória da Conquista, além da cepa Rockfeller, e um longitudinal, sobre amostras de quatro áreas (Plataforma, Itapagipe, Tancredo Neves e Itapuã) durante quatro ciclos do LIRAa Salvador entre 2007 a 2009. O DNA de cada larva foi isolado pelo método DNAzol® e genotipado por 5 marcadores SSR através da técnica de PCR e eletroforese capilar. A distribuição espacial dos criadouros foi realizada utilizando-se ortofotos pelo programa Arcview v. 9.3. Para a análise da diferenciação populacional e teste de hipótese foram utilizados os programas GenePop, GenAlEx e Spade, e para inferência populacional utilizamos o programa structure. Os marcadores encontraram-se, em geral, em equilíbrio de H-W e comportaram-se como independentes. Quando utilizamos a estatística Φpt e RST foi possível discriminar significantemente (p<0,05) populações geneticamente diferenciadas de A. aegypti a nível de município, áreas do município de Salvador e estratos pertencentes a estas áreas. O programa structure indicou K igual a 2 populações como ideal para representar os dados, considerando a população de Salvador uma miscigenação de populações de A. aegypti de outras regiões do estado. Os resultados do estudo longitudinal mostraram uma diferenciação entre os ciclos de 2008.3 e 2009.4. As medidas de Ne variaram consideravelmente por área e ciclo evidenciando o efeito de gargalo de garrafa em diferentes períodos em cada área, apesar de não haver correlação com o IIP. A partir dos resultados obtidos, concluímos que o controle vetorial produz alterações sobre a estrutura populacional do A. aegypti, mas que não são efetivas. O uso do georreferenciamento e de informações genéticas do vetor poderiam contribuir para a definição das áreas de abrangência das populações do A. aegypti e para a tomada de decisões a respeito do manejo do tratamento. / Despite the recurring attempts to combat the mosquito vector of dengue, Aedes aegypti, more than 80% of the strata of the city of Salvador-BA show alert condition or risk of an outbreak of dengue. Since traditional approaches to control the mosquito vector of dengue have not produced the expected results, this study evaluated molecular parameters for entomological surveillance of A. aegypti using tools of geotechnology and population genetics as a way of supporting the fieldwork and integrating the actions of the authorities responsible for integrated dengue control. The design of the study had a transverse component, describing data on the population genetics of larvae of A. aegypti collected in Salvador and control samples collected in 2009 in Jacobina and Vitoria da Conquista, in addition to the Rockefeller strain, and a longitudinal samples from four areas (Plataforma, Itapagipe, Tancredo Neves and Itapuã) for four cycles LIRAa of Salvador between 2007 to 2009. The DNA of each larva was isolated by the DNAzol ® method and genotyped with five STR (short tandem repeat) markers by PCR and capillary electrophoresis. The spatial distribution of breeding sites was performed using ortophotos by the program ArcView v.9.3. For the analysis of population differentiation and hypothesis testing the programs GenePop, GenAlEx Spade were used, and for population inference the program structure was used. The markers were usually found to be in H-W equilibrium and behaved as independent. When we use the statistics Φpt and RST it was possible to discriminate (p <0.05) genetically differentiated populations of A. aegypti at the municipal level, between areas of Salvador and strata within these areas. The program structure indicated K equal to 2 populations as ideal to represent the data, considering the population of Salvador is a mixture of populations of A. aegypti in other regions of the state. The results of the longitudinal study showed a difference between the cycles of 2008.3 and 2009.4 Ne measures varied considerably by area and cycle showing the effect of bottleneck in different periods in each area, although no correlation with the IIP. Given these results we conclude that the vector control produces changes on the population structure of A. aegypti, but are not effective. Tthe use of georeferencing and vector’s genetic information could help define the catchment areas of populations of A. aegypti and for making decisions about the management of treatment.

Submitted by Fabieli Loise Braga Feitosa (bifeitosa@hotmail.com) on 2018-09-28T15:30:30Z No. of bitstreams: 1 Tese_Fabieli_Feitosa.pdf: 5506988 bytes, checksum: 8a7ab208cd6c4f7e30cb5680429f2990 (MD5) / Rejected by Neli Silvia Pereira null (nelisps@fcav.unesp.br), reason: Solicitamos que realize correções na submissão seguindo as orientações abaixo: PORTARIA Nº 206, DE 4 DE SETEMBRO DE 2018 Dispõe sobre obrigatoriedade de citação da CAPES O PRESIDENTE DA COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR, no uso das atribuições que lhe foram conferidas pelo art. 26 do (a) Estatuto, aprovado (a) pelo Decreto nº 8977, de 30/01/2017, e CONSIDERANDO o indicado nos Editais da CAPES, nos Termos de Compromisso de Bolsista, nos regulamentos de bolsas no exterior e de bolsas no país, no Manual de AUXPE, e no termo de adesão ao Portal de Periódicos; CONSIDERANDO o constante dos autos do processo nº 23038.013648/2018-51, resolve: Art. 1º Os trabalhos produzidos ou publicados, em qualquer mídia, que decorram de atividades financiadas, integral ou parcialmente, pela CAPES, deverão, obrigatoriamente, fazer referência ao apoio recebido. Art. 2º Para fins de identificação da fonte de financiamento fica autorizada a utilização do código 001 para todos os financiamentos recebidos. Art. 3º Deverão ser usadas as seguintes expressões, no idioma do trabalho: "O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Código de Financiamento 001 Agradecemos a compreensão on 2018-09-28T16:51:12Z (GMT) / Submitted by Fabieli Loise Braga Feitosa (bifeitosa@hotmail.com) on 2018-09-28T16:56:13Z No. of bitstreams: 1 Tese_Fabieli_Feitosa.pdf: 5506988 bytes, checksum: 8a7ab208cd6c4f7e30cb5680429f2990 (MD5) / Rejected by Neli Silvia Pereira null (nelisps@fcav.unesp.br), reason: Solicitamos que realize correções na submissão seguindo as orientações abaixo: PORTARIA Nº 206, DE 4 DE SETEMBRO DE 2018 Dispõe sobre obrigatoriedade de citação da CAPES O PRESIDENTE DA COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR, no uso das atribuições que lhe foram conferidas pelo art. 26 do (a) Estatuto, aprovado (a) pelo Decreto nº 8977, de 30/01/2017, e CONSIDERANDO o indicado nos Editais da CAPES, nos Termos de Compromisso de Bolsista, nos regulamentos de bolsas no exterior e de bolsas no país, no Manual de AUXPE, e no termo de adesão ao Portal de Periódicos; CONSIDERANDO o constante dos autos do processo nº 23038.013648/2018-51, resolve: Art. 1º Os trabalhos produzidos ou publicados, em qualquer mídia, que decorram de atividades financiadas, integral ou parcialmente, pela CAPES, deverão, obrigatoriamente, fazer referência ao apoio recebido. Art. 2º Para fins de identificação da fonte de financiamento fica autorizada a utilização do código 001 para todos os financiamentos recebidos. Art. 3º Deverão ser usadas as seguintes expressões, no idioma do trabalho: "O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Código de Financiamento 001 "This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001" Agradecemos a compreensão on 2018-09-28T17:56:09Z (GMT) / Submitted by Fabieli Loise Braga Feitosa (bifeitosa@hotmail.com) on 2018-09-28T18:41:40Z No. of bitstreams: 1 Tese_Fabieli_Feitosa_2.pdf: 5507005 bytes, checksum: 3602bdd9aaa7e2f1dcb29be2e822d8ff (MD5) / Approved for entry into archive by Neli Silvia Pereira null (nelisps@fcav.unesp.br) on 2018-10-01T17:11:41Z (GMT) No. of bitstreams: 1 feitosa_flb_dr_jabo.pdf: 5507005 bytes, checksum: 3602bdd9aaa7e2f1dcb29be2e822d8ff (MD5) / Made available in DSpace on 2018-10-01T17:11:41Z (GMT). No. of bitstreams: 1 feitosa_flb_dr_jabo.pdf: 5507005 bytes, checksum: 3602bdd9aaa7e2f1dcb29be2e822d8ff (MD5) Previous issue date: 2018-07-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo deste estudo foram realizar associação genômica ampla (GWAS) e predição genômica utilizando haplótipos como marcadores genéticos para o perfil de ácidos graxos da gordura intramuscular do músculo Longissimus dorsi em bovinos Nelore. Foram utilizados dados de 963 machos da raça Nelore terminados em confinamento, provenientes de fazendas que integram três programas de melhoramento genético. O grupo de contemporâneo foi formado por animais nascidos na mesma fazenda e safra, e do mesmo grupo de manejo ao sobreano. Para a determinação do perfil de ácidos graxos foi utilizado para a extração dos lipídeos o método Folch e para a metilação o método Kramer. Para a genotipagem foi utilizado um painel com mais de 777.000 SNPs do BovineHD BeadChip (High-Density Bovine BeadChip) da Illumina. O controle de qualidade foi feito considerando MAF < 0,05 e Call rate < 90%. Após o CQ, 469.981 SNPs permaneceram nas análises. A imputação dos “missing” e o “phasing” do genótipo foram realizados utilizando o software FImpute. A definição dos blocos de haplótipos foi realizada baseada no desequilíbrio de ligação utilizando o software HaploView. No capítulo 2 as análises estatísticas incorporando as informações dos haplótipos foram realizadas utilizando o software ASREML implementando o método REML. O modelo incluiu efeitos fixos do grupo contemporâneo (62 níveis), haplótipo (regressão linear no número de cópias) e idade ao abate como covariável linear. Os valores de p foram ajustados pelo método de FDR. Os haplótipos associados significativamente foram selecionados ao nível de 10% de significância. Para a prospecção dos genes foi utilizado o banco de dados do NCBI na versão UMD3.1 do genoma bovino, Ensembl Genome Browser, DAVID. Foram associados significativamente (p <0,05), 4, 3, 5, 1, 6, 2 e 7 haplótipos com AGS, AGMI, AGPI, AGPI/AGS, n3, n6 e n6/n3, respectivamente. Estes haplótipos significativos abrigam genes envolvidos no metabolismo lipídico, fatores de alongamento e síntese de ácidos graxos de cadeia longa, receptores de hormônios reprodutivos, transporte e uso de ácidos graxos e colesterol, biossíntese e hidrólise de fosfolipídios e constituintes de membrana, constituintes de membranas celulares, metabolismo energético e síntese de proteína quinase. Assim, a identificação desses haplótipos associados pode contribuir para estudos adicionais para validar essas regiões e prospectar genes candidatos que seriam úteis para programas de melhoramento genético para melhorar a qualidade da carne de bovino Nelore. No capítulo 3, o modelo utilizado para estimar componentes de variância, parâmetros genéticos e predizer os valores genômicos incluiu efeitos fixos de grupo contemporâneo e idade ao abate como uma covariável linear, e o efeito genético aleatório aditivo. A acurácia utilizando haplótipos variou de 0,07 a 0,31 e a utilizando SNPs variou de 0,06 a 0,33. O coeficiente de regressão usando os haplótipos variou de 0,07 a 0,74 e utilizando SNPs variou de 0,08 a 1,45. Apesar da precisão de predição não ter sido alta para ambas as abordagens, podemos considerar a utilização da abordagem SNP na seleção genômica, pois é um método computacional mais eficiente. / The aim of this study were genome-wide association (GWAS) and genomic prediction using haplotypes approach for fatty acid profile of intramuscular fat of longissimus dorsi muscle in Nellore cattle. The investigated dataset contained records from 963 Nellore bulls, finished in feedlot (90 days) and slaughter with about two years old. Meat samples of Longissimus dorsi muscle, between the 12th and 13th ribs of the left half-carcasses, were taken to measure the fatty acids (FAs). FAs were quantified by gas chromatography (GC-2010 Plus - Shimadzu AOC 20i autoinjector) using SP-2560 capillary column (100 m x 0.25 mm diameter with 0.02 mm thickness, Supelco, Bellefonte, PA). The animals were genotyped using the high-density SNP panel (BovineHD BeadChip assay 777k, Illumina Inc., San Diego, CA). Those SNP markers with minor allele frequency less than 0.05, call rate less than 90%, monomorphic, located on sex chromosomes, and those with unknown position were removed from the analysis. After genomic quality control, 469,981 SNPs and 892 animals were available for the analyses. Missing genotypes were imputed and genotypes were phased to haplotypes using FImpute software. Haplotype blocks were defined based on linkage disequilibrium using HaploView software. In chapter 2, statistical analyzes incorporating the haplotype information were performed using ASREML software implementing the REML method. The model included fixed effects of the contemporary group (62 levels), haplotype (linear regression in number of copies) and age at slaughter as a linear covariate. The p values were adjusted by FDR method. Haplotypes significantly associated were selected at 10% significance level. For the prospection of the genes, the NCBI database was used in the UMD3.1 version of the bovine genome, the Ensembl Genome Browser and DAVID. There were significantly (p <0.05), 4, 3, 5, 1, 6, 2 and 7 haplotypes associated with SFA, MUFA, PUFA, PUFA/SFA ratio, n3, n6 and n6/n3 ratio, respectively. These significant haplotypes harboring genes involved in lipid metabolism elongation factors and long-chain fatty acid synthesis, reproductive hormone receptors, transport and use of fatty acids and cholesterol biosynthesis and hydrolysis of phospholipids and constituents membrane, cell membrane constituent , energetic metabolism and protein kinase synthesis. Thus, the identification of these associated haplotypes may contribute to further studies to validate these regions and prospect candidate genes that would be useful for breeding programs to improve the quality of Nellore beef. In chapter 3, the model used to estimate variance components, genetic parameters and predict the genomic values included fixed effects of contemporary group and age at slaughter as a linear covariate, and the additive random genetic effect. The accuracy using haplotype approach ranged from 0.07 to 0.31 and using SNP approach ranged from 0.06 to 0.33. The regression coefficient using haplotype approach ranged from 0.07 to 0.74 and using SNP approach ranged from 0.08 to 1.45. Despite prediction accuracy was not high for both approach we can consider use SNP approach in genomic selection because is more efficient computational. / FAPESP: 2015/25304-8/Bolsa de doutorado no país / FAPESP: 2016/24085-3/Bolsa de estágio e pesquisa no exterior / CAPES: 001

Made available in DSpace on 2015-12-10T14:23:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-04-24. Added 1 bitstream(s) on 2015-12-10T14:30:07Z : No. of bitstreams: 1 000852258.pdf: 1391953 bytes, checksum: 0d7e06938cd3251b6670b1cae39a85d1 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Transcrição é o processo catalizado por um complexo enzimático, RNA polimerase (RNAP), responsável pela síntese de RNA mensageiro a partir de uma sequência de DNA. Diferentes estudos experimentais foram realizados para investigar esse processo, como técnicas bioquímicas, de pinça ótica ou magnética, microscopia de força atômica e fluorescência de molécula única. Com os estudos bioquímicos, por exemplo, sabe-se que várias RNAPs podem transcrever uma sequência simultaneamente. O número de diferentes moléculas depende da necessidade da célula, número de RNAP livres, regeneração do promotor e fatores de transcrição. Um dos sistemas mais investigados para o estudo desse processo é a síntese dos genes ribossomais em Escherichia coli. Os genes ribossomais são fundamentais na fisiologia dos organismos, são expressos abundantemente e existem evidências da aceleração da transcrição devido ao comportamento colaborativo entre as RNAPs. Neste trabalho, propomos simular a transcrição múltipla dos genes ribossomais em E. coli com o modelo estocástico e dependente de sequências desenvolvido em nosso laboratório. As reações químicas foram simuladas utilizando o algoritmo de Gillespie. Essa metodologia apresenta uma boa relação entre custo computacional e realismo biológico e inclui alguns parâmetros não utilizados em estudos teóricos prévios. O modelo considera o alongamento em back e forward tracking, identificando os sítios de pausas e colisões entre RNAPs, determinando o tempo de permanência e predizendo a ocorrência de transcrição abortiva ou a aceleração da transcrição devido ao fenômeno colaborativo entre múltiplas RNAPs. A sequência do operon ribossomal rrnB da E. coli foi simulada variando o número de RNAP (R), a força de interação entre RNAPs (F) e a concentração de nucleosídeo trifosfato ([NTP]). Nossos resultados mostraram-se promissores quando utilizamos uma... / The process that produces messenger RNAs from DNA sequences is called transcription, and these reactions are catalyzed by the RNA polimerase enzyme. Many different experimental techniques have been applied to investigate this process including biochemical techniques, optical and magnetic tweezers, atomic force microscopy and single molecule florescence. These biochemical process studies showed that many RNAP molecules operate simultaneously on a single DNA strand. The number of different molecules depends on cellular demands, concentration of free RNAPs, promoter strength and the presence of transcription factors. Escherichia coli ribosomal genes are a popular experimental model to investigate the transcription process. These genes are essential to cell physiology, and they are strongly expressed. There are evidences that some cellular mechanisms collaborate to accelerate their transcription. In this work we investigate the RNAP collaborative transcription in E. coli ribosomal genes using a stochastic and sequence dependent model proposed by our group. The chemical reactions were simulated using a model based on the Gillespie algorithm. This methodology is a good compromise between computational cost and biological realism and includes some ingredients that were missing in previous theoretical studies. The model considers back and forward tracking elongation and it identifies pauses by determining the dwell time on specific sites. The model also predicts abortive transcription and transcription acceleration due to collaborative RNAP interaction. The E. coli rrnB ribosomal operon sequence was simulated by varying (i) the number of RNAP (R) on the DNA strand, (ii) the interaction force between two colliding RNAPs (F) and (iii) the concentration of nucleoside triphosphate ([NTP]). Our results are promising for F =15 pN, R = 50 and [NTP] ... / CNPq: 2013/06683-2

Made available in DSpace on 2016-03-07T19:21:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-08-28. Added 1 bitstream(s) on 2016-03-07T19:25:26Z : No. of bitstreams: 1 000858045.pdf: 980954 bytes, checksum: 6bcdb403af4689c3a8fd68979044e591 (MD5) / Huanglongbing (HLB) doença dos citros emergiu como a principal ameaça à produção de citros no mundo, associado com a bactéria (Candidatus Liberibacter asiaticus - Las) limitada ao floema e ao vetor (Diaphorina citri). No Brasil, o HLB foi relatado pela primeira vez em 2004 e rapidamente se espalhou para todas as regiões geográficas do Estado de São Paulo (SPS), Paraná (PR) e Minas Gerais (MG). No entanto, informações sobre a diversidade genética desta bactéria são pouco conhecidas. Aqui nós testamos a hipótese H0: ambas as regiões geográficas diferentes e espécies de citros não moldam a diversidade genética de populações de Las do Brasil. Para comprovar a hipótese, DNA de 199 amostras de plantas cítricas com sintomas de HLB foram amplificados com 9 conjuntos de iniciadores microssatélites, sendo os iniciadores diretos marcados com corantes fluorescentes. Níveis moderadamente baixos de diversidade genética (HNei = 0,11 - 0,26) foram observados em todas as populações desta bactéria. Com uso da estatística F de Wright (FST), nenhuma diferença estatística foi observada entre populações de Las de todas as sete subdivisões das regiões geográficas do SPS e de MG (FST <0,095). Mas, valores significativos de diferenciação entre populações (FST = 0,118 - 0,191) foram obtidos para populações de Las do PR comparado com as do SPS e MG. Também, valores ainda mais altos e significativos (FST = 0,275 - 0,445) foram observados comparando populações de Las em laranjeiras doce do SPS, MG e PR com as populações de diferentes espécies de citros cultivados na região Sudeste do SPS. Por meio de métodos de agrupamento realizados por estatística Bayesiana e de coordenada principal pode-se agrupar todos os isolados estudados em três populações geneticamente distintas: 1. Um grupo contempla todas as estirpes das regiões do SPS mais MG, 2. Um segundo contendo somente as estirpes de Las do PR, 3. E... / Citrus huanglongbing (HLB) disease emerged as a primary threat for citrus production worldwide, associated with the phloem and vectored (Diaphorina citri) limited - bacterium Candidatus Liberibacter asiaticus (Las). In Brazil, HLB was first reported in 2004 and quickly spread for all geographic regions of the States of Sao Paulo (SP), Parana (PR) and Minas Gerais (MG). However, information about genetic diversity of this bacterium is poorly known. Here we tested the hypothesis H0: different geographic regions and citrus species have no influence on genetic diversity of Las populations in Brazil. To test this hypothesis, total DNA from 199 samples from citrus plants with symptoms of HLB was amplified by 9 sets of forwardfluorescent microsatellites primers. Moderately low levels of genetic diversity (HNei = 0,11 to 0,26) were observed through all populations. By Wright's F-statistics (FST), no statistic difference was observed among Las populations from all the seven previously subdivided geographic regions of SP and MG (FST <0,095). But significant FST values (0,118 to 0,191) were obtained for Las population from PR compared to SP and MG. On the other hand, highest and significant values for FST index (0,275 to 0,445) were observed comparing Las populations from sweet orange trees from SP, MG, and PR and the populations from different citrus species grown at the Southeast region of SP. By Bayesian statistics and principal coordinate analysis all isolates studied were clustered in three genetically different populations: 1. a group that includes all strains from SP regions and MG, 2. A second composed only by Las strains from PR, and 3. a third where strains from different citrus species were grouped. In conclusion, after 10 years of the HLB first report in Brazil, that genetically homogeneous populations of Las infecting sweet orange are present at different geographic regions sampled, with the exception of the populations ...