Les téosintes Zea mays ssp. parviglumis et ssp. mexicana sont les apparentés sauvages les plus proches du maïs cultivé. Elles occupent au Mexique des niches écologiques distinctes bien délimitées par l’altitude. Zea mays ssp. parviglumis pousse dans des environnements chauds et humides situés à moins de 1800m d’altitude, tandis que Zea mays ssp mexicana pousse dans des environnements plus secs et froids (au-dessus de 1800m). Nous nous sommes intéressés aux bases génétiques de l’adaptation locale des téosintes à l’altitude. Dans ce but, 37 populations ont été échantillonnées le long de deux gradients altitudinaux, utilisés comme réplicats biologiques. Deux populations extrêmes de chaque gradient ainsi que deux populations de moyenne altitude de chaque sous-espèce du gradient 1 ont été séquencées à faible profondeur afin de découvrir des polymorphismes dont les fréquences alléliques sont contrastées entre les extrêmes tout en contrôlant pour la différenciation entre les sous-espèces. A partir de méthodes de détection de la sélection intra- et inter-populationnelle, des locus candidats ont été détectés. Une revue bibliographique a permis d’établir des correspondances entre nos candidats et des régions précédemment décrites pour leurs effets phénotypiques sur des caractères adaptatifs. Nous montrons ainsi un rôle important des interactions sol-plante et de la pilosité des feuilles dans l’adaptation à l’altitude. Pour valider certains de nos candidats, 270 polymorphismes ont été génotypés sur les 37 populations afin de réaliser des études de clines de fréquence. Parallèlement, nous avons mis en œuvre un dispositif de caractérisation phénotypique (2 lieux x 2 années) afin de tester l’association entre ces polymorphismes et 18 caractères mesurés en champ. Nous discutons des apports méthodologiques de notre étude aussi bien du point de vue des technologies haut débit que de la détection de la sélection. Notre dispositif devra être pleinement exploité pour valider nos candidats. Les perspectives incluent la poursuite de l’étude sur d’autres types de polymorphismes ainsi qu’un suivi temporel des populations. / Teosintes Zea mays ssp parviglumis (parviglumis) and Zea mays ssp mexicana (mexicana) are the closest wild relatives of cultivated maize. They occupy distinct ecological niches in Mexico, well separated by altitude. Zea mays ssp. parviglumis encountered below 1800m grows in wet and warm conditions, while mexicana grows in drier and colder climates (above 1800m). In order to investigate the genetic bases of local adaptation to altitude, we sampled 37 populations along two altitudinal gradients. We sequenced the two most extreme populations of each gradient as well as two intermediate populations - one from each subspecies along gradient 1. We searched for polymorphisms with contrasted allele frequencies between the extremes while accounting for subspecies differentiation. Using both inter- and intra- population methods we identified several candidate loci. Based on a literature review, we confronted them with regions previously described as involved in phenotypic variation of adaptive traits. Our results highlight the role of plant-soil interactions and leaves hairiness in the adaptation to altitude. To validate further a subset of 270 polymorphisms chosen among our best candidates, we genotyped them on the 37 populations with the aim of performing clinal analyzes of allele frequencies. In parallel, we undertook phenotypic evaluation trials (2 locations x 2 years) to test the association of these polymorphisms with 18 traits measured in the field. We discuss the methodological contributions of our study both from the standpoint of high throughput technologies and detection of selective footprints. Our setting will be fully exploited to validate our candidates. Perspectives include the discovery and assessment of the contribution of other types of polymorphisms and the temporal follow-up of populations.

Objective: Mutations in the parkin (PARK2) gene have been associated with autosomal recessive early-onset Parkinson's disease (EOPD) with various frequencies in different populations. The aim of the study is to describe phenotypic characteristics of Czech EOPD patients, to evaluate the influence of environmental risk factors, and to determine the frequency of parkin allelic variants in patients and healthy controls. Methods: A total of 70 EOPD patients (age at onset ≤ 40 years) and 75 controls were phenotyped and screened for the sequence variants and exon rearrangements in the parkin gene. Results: The main features in the phenotype of the patients' sample were: the absence of cognitive deficit, high occurrence of dystonia, depression, hyperhidrosis, an excellent response to dopaminergic therapy, early onset of dyskinesia and motor fluctuation. Patients with mutations in the parkin gene had significantly lower age at onset. The agricultural occupation and work with chemicals increased the risk of EOPD, however the coffee drinking appeared to be a protective factor. Parkin mutations were identified in five patients (7.1%): the p.R334C point mutation was present in one patient, four patients had exon deletions. The detected mutations were observed in the heterozygous state except one homozygous...

Recently the suspected precursor lesions of ductal adenocarcinoma of the pancreas have been called Pancreatic intraepithelial neoplasia (PanIN) and graded according to the degree of dysplasia. To correlate each grade of PanIN with molecular genetic alterations, we determined the frequency of allelic losses at chromosomal arms 9p (the location of the p16 gene), 17p (p53 gene) and 18q (DPC4/SMAD4 gene) in 81 microdissected PanINs, using a combination of whole genome amplification and microsatellite analysis. In addition, p53 and Dpc4 protein expression was determined by immunohistochemistry. Essentially no allelic losses were identified in the non-dysplastic PanIN-1 lesion. In PanIN-2 with low grade dysplasia the frequency of allelic losses at chromosomal region 9p, 17p and 18q was 20%, 33% and 17%, respectively, which increased to 46%, 77% and 58%, respectively, in PanIN-2 with moderate dysplasia, to 87%, 60% and 88% in PanIN-3 with high grade dysplasia, and to 100%, 91%, and 82% in the invasive carcinomas. The progressive occurrence of allelic losses at all three loci strongly supports the PanIN progression model for pancreatic carcinoma. Nuclear p53 and loss of Dpc4 protein expression was associated only with PanIN-3 and invasive carcinomas, consistent with the model that inactivation of p53 and DPC4 are late events in pancreatic carcinogenesis. Since the aberrant protein expression patterns, were preceded, however by a sharp increase in allelic losses from PanIN-2 with low grade dysplasia to PanIN-2 with moderate dysplasia it is suggested that the increasing grade of dysplasia in the PanIN lesions identify biologically relevant steps towards invasive carcinoma. The discrepancy between alleic loss frequencies and p53 and DPC4 expression also raises the possibility that additional tumor suppressor genes on chromosomes 17p and 18q promote early pancreatic carcinogenesis.

No description available.

No description available.

Genetic variation is a strong risk factor for many human diseases, including diabetes, cancer, cardiovascular disease, depression, autoimmunity and asthma. Most of the disease genes identified so far alter the amino acid sequences of encoded proteins. However, a significant number of genetic variants affecting complex diseases may alter the regulation of gene transcription. The map of the regulatory elements in the human genome is still to a large extent unknown, and it remains a challenge to separate the functional regulatory genetic variations from linked neutral variations. The objective of this thesis was to develop methods for the identification of genetic variation with a potential to affect the transcriptional regulation of human genes, and to analyze potential regulatory polymorphisms in the CD36 glycoprotein, a candidate gene for cardiovascular disease. An in silico tool for the prediction of regulatory polymorphisms in human genes was implemented and is available at www.cisreg.ca/RAVEN. The tool was evaluated using experimentally verified regulatory single nucleotide polymorphisms (SNPs) collected from the scientific literature, and tested in combination with experimental detection of allele specific expression of target genes (allelic imbalance). Regulatory SNPs were shown to be located in evolutionary conserved regions more often than background SNPs, but predicted transcription factor binding sites were unable to enrich for regulatory SNPs unless additional information linking transcription factors with the target genes were available. The in silico tool was applied to the CD36 glycoprotein, a candidate gene for cardiovascular disease. Potential regulatory SNPs in the alternative promoters of this gene were identified and evaluated in vitro and in vivo using a clinical study for coronary artery disease. We observed association to the plasma concentrations of inflammation markers (serum amyloid A protein and C-reactive protein) in myocardial infarction patients, which highlights the need for further analyses of potential regulatory polymorphisms in this gene. Taken together, this thesis describes an in silico approach to identify putative regulatory polymorphisms which can be useful for directing limited laboratory resources to the polymorphisms most likely to have a phenotypic effect.

Made available in DSpace on 2014-07-29T15:30:34Z (GMT). No. of bitstreams: 1 CRISTINA RODRIGUES DE OLIVEIRA.pdf: 2212867 bytes, checksum: a424437b0d0a13929b5f608708fec44a (MD5) Previous issue date: 2007-02-13 / The FcγRIIA, receptor for the Fc portion of IgG, expressed by macrophages, neutrophils, platelets and dendritic cells, bind to the subclasses of IgG with variable affinity, that can be influenced by the polymorphism in the gene that encodes this receptor. The substitution of the amino acid arginine (R) for histidine (H), in the 131 position, defines three allelic patterns, the homozygote H/H, R/R and the heterozygote H/R, conferring to the FcγRIIA H/H131 a greater affinity to the IgG2 and IgG3 subclasses. This can result in different responses to diverse pathogens. Studies show the importance of Fcγ receptors on the macrophage infection by amastigote forms of Leishmania sp, in addition to those for the complement (CR3) and for mannose (MR). Besides that, genetic factors related to the hosts are involved in the immune response to the leishmaniasis, among them, the FcγRs. Until this time, we haven t found studies relating the receptor FcγRIIA to the leishmaniasis in humans. This way, this work consists in analyzing the allelic polymorphism in the gene that encodes the FcRIIA in individuals with American Tegumentary Leishmaniasis (ATL), evaluate if this polymorphism would be a genetic fact of susceptibility or resistance for this disease, if this would be influencing in the development of the different clinical forms of the ATL, as well in the healing process of the lesions. The FcγRIIA H/R131 polymorphism was analyzed in 88 blood samples of individuals with leishmaniasis and in 98 samples of healthy individuals (control group), using PCR, amplifying a segment of the gene that encodes the FcRIIA, followed by allele-specific enzymatic digestion and agarose gel eletrophoresis 3%. These results showed that the genotypic and allelic distribution of the FcγRIIA H/R131 were similar in the patients with leishmaniasis and in the control group, as well in patients with different clinical forms of the ATL. Concerning the resolution time of the disease, among the patients that had their lesions healed within one month of treatment or after this period, there was a predominance of the alleles R and H, respectively, however these differences were not significant. This way, our study suggests that the allelic polymorphism of the FcγRIIA H/R131 possibly is not a genetic factor of host that is associated to the protection or pathogenesis in the American Tegumentary Leishmaniasis. / O FcyRIIA, receptor para região Fc de IgG, expresso por macrófagos, neutrófilos, plaquetas e células dendríticas, liga-se às subclasses de anticorpos IgG com afinidade variável, que pode ser influenciada pelo polimorfismo alélico no gene que codifica este receptor. A troca do aminoácido arginina (R) para histidina (H) na posição 131 determina três padrões alélicos: os homozigotos H/H e R/R e o heterozigoto H/R, conferindo ao FcγRIIA H/H131 maior afinidade para as subclasses IgG2 e IgG3. Isto pode resultar em diferentes respostas frente a patógenos diversos. Alguns estudos demonstram a importância dos receptores Fcγ na infecção de macrófagos por formas amastigotas de Leishmania sp, em adição aos receptores para complemento (CR3) e para manose (MR). Além disso, vários fatores genéticos do indivíduo estão envolvidos na resposta imune à leishmaniose, dentre esses os FcyRs. Até o presente momento, não encontramos estudos relacionando o receptor FcγRIIA com a leishmaniose em humanos. Assim, o presente trabalho teve como objetivo analisar o polimorfismo alélico no gene que codifica o receptor FcyRIIA em indivíduos com Leishmaniose Tegumentar Americana (LTA), avaliar se este polimorfismo seria um fator genético de susceptibilidade ou resistência para esta doença, se estaria influenciando no desenvolvimento das diferentes formas clínicas da LTA, bem como no tempo de cura das lesões. O polimorfismo do FcγRIIA H/R131 foi analisado em 88 amostras sangüíneas de indivíduos com LTA e em 98 amostras de indivíduos saudáveis (grupo controle), através da PCR, amplificando um segmento do gene que codifica o FcγRIIA, seguida de digestão enzimática alelo-específica e eletroforese em gel de agarose 3%. Nossos resultados demonstraram que a distribuição genotípica e alélica do FcγRIIA - H/R131 foi similar nos pacientes com leishmaniose e no grupo controle, bem como nos pacientes com as diferentes formas clínicas da LTA. Em relação ao tempo de resolução da doença, nos pacientes que tiveram suas lesões curadas com até um mês de tratamento ou após esse período, houve um predomínio do alelo R e do H, respectivamente, entretanto essas diferenças não foram significativas. Assim, nosso estudo sugere que o polimorfismo alélico do FcγRIIA H/R131 possivelmente não é um fator genético do hospedeiro que esteja associado com proteção ou patogênese na Leishmaniose Tegumentar Americana.

Abstract Adaptation through polygenic selection is a prominent feature in nature. Still, the genetic backgrounds of polygenic adaptations are often unknown. The challenges of resolving adaptive processes are related to selection being distributed over several loci with often small effect sizes. Also, even a low level of population substructure can obstruct the inference. Further, demographic factors in the history of the species, such as population size changes and range expansions leave a confounding footprint in the background genomic variation. In this thesis, polygenic adaptation was studied with Scots pine (Pinus sylvestris L.), a widespread ecologically and economically important conifer. In this thesis, timing of bud set – an adaptive polygenic trait – was studied at the level of the phenotype in a common garden study, and at the genomic level by examining the sequence and allele frequency variation patterns in bud set timing related loci, with a sampling across a latitudinal transect in Europe. An association study, combining these two levels, was carried out with a new Bayesian multipopulation method. The congruence of allozyme and nucleotide level diversity was estimated, the level of neutral genetic population structure surveyed, and a demographic background model for statistical inference of selective signals redefined. Allozyme variation seemed to correlate well with the nucleotide level variation at the between species level, but within population, at the individual allozyme coding loci, does not describe the underlying level of nucleotide variation well. Indications of recent colonization history affecting the level of differentiation between populations were seen, and the need to control for the background effects of simultaneous range expansion and adaptation shown. Lower phenotypic and additive genetic variation in timing of bud set was found in northern compared to central European populations. Signs of heterogeneity in genetic basis of this trait were also found between these areas, which could indicate different timekeeping mechanisms due to different environmental cues in the two regions. The results in this thesis are of value to the study of adaptation, but also for breeding, conservation and prediction of responses of forest trees to future climate change. / Tiivistelmä Sopeutuminen perustuu usein polygeenisiin ominaisuuksiin. Näiden ominaisuuksien geneettiset taustat ovat silti vielä pitkälti selvittämättä. Sopeutumisominaisuuksien genetiikan selvittämistä vaikeuttaa valinnan vaikutusten jakautuminen usean, usein pienivaikutuksisen lokuksen kesken. Lisäksi vähäinenkin populaatiorakenne hankaloittaa geenien tunnistamista. Myös lajin historiassa tapahtuneet demografiset muutokset, kuten populaatiokoon vaihtelut ja kolonisaatio jättävät jälkensä genomiin. Väitöskirjassani tutkin polygeenistä sopeutumista ekologisesti ja taloudellisesti tärkeän havupuulajin, metsämännyn (Pinus sylvestris L.) avulla. Väitöskirjassani tutkin metsämännyn silmunmuodostuksen ajoitusta sekä fenotyypin että sekvenssimuuntelun tasolla. Ajoitusta mitattiin eri leveysasteilta peräisin olevista eurooppalaisista populaatioista yhteiskasvatuskokeessa, ja sekvenssimuuntelua sekä alleelifrekvenssien jakautumista tutkittiin vastaavasta näytteestä. Geenikartoituskokeessa yhdistettiin nämä kaksi muuntelun tasoa hyödyntäen uutta, usean populaation tutkimiseen soveltuvaa analyysimenetelmää. Lisäksi tutkin allotsyymimuuntelun ja nukleotidimuuntelun keskinäistä tarkkuutta geneettisen diversiteetin kuvaajina, neutraalin populaatiorakenteen tasoa, sekä demografian vaikutusta metsämännyn genomissa. Allotsyymimuuntelun todettiin kuvaavan hyvin lajien välisiä diversiteettieroja. Populaation sisällä yksittäisten allotsyymien heterosygotia ei korreloinut entsyymiä koodaavan geenin muuntelun määrän kanssa. Pohjoisten populaatioiden vähäisemmät keskinäiset erot verrattuna keskieurooppalaisiin antoivat viitteitä siitä, että viimeisimmän jääkauden jälkeiset kolonisaatiotapahtumat voivat edelleen vaikuttaa populaatioiden erilaistumisasteeseen. Assosiaatiotutkimuksessa osoitettiin, kuinka tärkeää yhtäaikaisen sopeutumisen ja kolonisaation huomioiminen on sopeutumisominaisuuksien tutkimisessa. Fenotyyppinen muuntelu silmunmuodostuksen ajoituksessa oli vähäisempää pohjoisissa populaatioissa. Lisäksi löysimme merkkejä geneettisestä heterogeenisuudesta silmunmuodostuksen taustalla pohjoisten ja keskieurooppalaisten metsämäntyjen välillä, mikä voi johtua vuodenajan vaihtelun mittaamiseen käytettävien ympäristösignaalien eriytymisestä näiden alueiden välillä. Väitöskirjassani saadut tulokset hyödyttävät paitsi sopeutumistutkimusta, myös jalostus- ja luonnonsuojelututkimusta sekä ilmastonmuutoksen vaikutusten arviointia.

Tesis por compendio / [ES] Un objetivo fundamental en los programas de conservación es mantener la diversidad genética y la estrategia de gestión más eficiente para lograrlo es aplicar el método de Contribuciones Óptimas. Este método optimiza las contribuciones de los candidatos a reproductores minimizando el parentesco global ponderada, lo que conduce a niveles más altos de diversidad genética, medida como heterocigosis esperada, y a un control efectivo del aumento de consanguinidad. El parámetro fundamental de este método es la matriz de parentesco. Esta matriz se ha obtenido tradicionalmente a partir del pedigrí, pero la disponibilidad actual de genotipos para un gran número de polimorfismos de un solo nucleótido (SNP) nos permite estimarla con una mayor precisión. Sin embargo, se han propuesto muchas medidas de parentesco genómico y se desconoce qué medida es la más apropiada para minimizar la pérdida de diversidad genética. Por lo tanto, el objetivo general de esta tesis fue investigar la eficiencia de diferentes matrices genómicas de parentesco en la gestión de poblaciones en programas de conservación, cuando se aplica el método de Contribuciones Óptimas. Las matrices comparadas fueron aquellas basadas en: i) la proporción de alelos compartidos por dos individuos (CSIM); ii) las desviaciones del número observado de alelos compartidos por dos individuos respecto del número esperado (CL&H); iii) la matriz de relaciones genómicas obtenida a través el método 1 de VanRaden (CVR1); iv) la matriz de relaciones genómicas obtenida a través el método 2 de VanRaden (CVR2); v) la matriz de relaciones genómicas obtenida a través el método de Yang (CYAN); y vi) segmentos idénticos por descendencia (CSEG). Los resultados para una sola generación, usando miles de SNP genotipados en individuos de una población cultivada de rodaballo, mostraron grandes diferencias en la magnitud de los seis coeficientes de parentesco. Las correlaciones entre los diferentes coeficientes variaron mucho, siendo las más bajas aquellas entre CSIM, CL&H o CSEG y CVR2 o CYAN. La gestión que utiliza matrices basadas en la proporción de alelos o segmentos compartidos (CSIM, CL&H y CSEG) retuvieron una mayor diversidad que aquella que utiliza matrices de relaciones genómicas (CVR1, CVR2 y CYAN). Como era de esperar, maximizar la heterocigosis llevó los alelos hacia frecuencias intermedias. Sin embargo, alejar las frecuencias alélicas de las frecuencias iniciales puede ser indeseable, ya que se pueden perder adaptaciones particulares al medio. Se utilizaron simulaciones estocásticas para investigar la eficiencia de CL&H y CVR2 en el manejo de poblaciones no divididas a lo largo de 50 generaciones y ambas matrices se compararon tanto en términos de la diversidad genética como en términos de los cambios asociados en las frecuencias alélicas. EL uso de CL&H en el método de Contribuciones Óptimas llevó a una mayor diversidad genética pero también en un mayor cambio de frecuencias alélicas que el uso de CVR2. Las diferencias entre estrategias fueron menores cuando sólo se usaron SNP con una frecuencia del alelo menos común (MAF) por encima de un umbral particular (MAF > 0.05 y MAF > 0.25) para calcular CL&H y CVR2 así como cuando se aplicó el método de Contribuciones Óptimas en poblaciones con censo más pequeños (se pasó de N = 100 a N = 20). La evaluación de CL&H y CVR2 se extendió a poblaciones subdivididas, también a través de simulaciones por ordenador. En poblaciones subdivididas, la diversidad genética se distribuye en dos componentes: dentro y entre subpoblaciones. Cuando se otorga un mayor peso al componente dentro de subpoblaciones, es posible restringir los niveles de consanguinidad. Bajo este escenario, la utilización de CL&H resultó ser la mejor opción para gestionar este tipo de poblaciones, ya que mantuvo una mayor diversidad global, condujo a una menor consanguinidad y a cambios en las frecuencias similares a los observados cuando se utilizó CVR2. / [CA] Un objectiu fonamental en els programes de conservació és mantindré la diversitat genètica i l'estratègia de gestió més eficient per aconseguir-ho és aplicar el mètode de contribucions òptimes. Aquest mètode optimitza les contribucions dels candidats a reproductors minimitzant el parentiu global ponderat, cosa que condueix a nivells més alts de diversitat genètica, mesurada com a heterozigosi esperada, i a un control efectiu de l'augment de consanguinitat. El paràmetre fonamental d'aquest mètode és la matriu de parentiu. Aquesta matriu s'ha obtingut tradicionalment a partir del pedigrí, però la disponibilitat actual de genotips per a un gran nombre de polimorfismes d'un sol nucleòtid (SNP) ens permet estimar-la amb més precisió. Tot i això, s'han proposat moltes mesures de parentiu genòmic i es desconeix quina mesura és la més apropiada per minimitzar la pèrdua de diversitat genètica. Per tant, l'objectiu general d'aquesta tesi va ser investigar l'eficiència de diferents matrius genòmiques de parentiu en la gestió de poblacions en programes de conservació, quan s'aplica el mètode de Contribucions Òptimes. Les matrius comparades van ser aquelles basades en: i) la proporció d'al·lels compartits per dos individus (CSIM); ii) les desviacions del nombre observat d'al·lels compartits per dos individus respecte del nombre esperat (CL&H); iii) la matriu de relacions genòmiques obtinguda a través del mètode 1 de VanRaden (CVR1); iv) la matriu de relacions genòmiques obtinguda a través del mètode 2 de VanRaden (CVR2); v) la matriu de relacions genòmiques obtinguda a través del mètode de Yang (CYAN); i vi) segments idèntics per descendència (CSEG). Els resultats per a una sola generació, usant milers d'SNP genotipats en individus d'una població cultivada de turbot, van mostrar grans diferències en la magnitud dels sis coeficients de parentiu. Les correlacions entre els coeficients van variar molt, sent les més baixes aquelles entre CSIM, CL&H o CSEG i CVR2 o CYAN. La gestió que utilitza matrius basades en la proporció d'al·lels o segments compartits (CSIM, CL&H i CSEG) van retindré una diversitat més gran que aquella que utilitza matrius de relacions genòmiques (CVR1, CVR2 i CYAN). Com calia esperar, la màxima heterozigosi va portar els al·lels cap a freqüències intermèdies. No obstant això, allunyar les freqüències al·lèliques de les freqüències inicials pot ser indesitjable, ja que es poden perdre adaptacions particulars al medi. Es van fer servir simulacions estocàstiques per investigar l'eficiència de CL&H i CVR2 en el maneig de poblacions no dividides al llarg de 50 generacions i les dues matrius es van comparar tant en termes de la diversitat genètica com en termes dels canvis associats a les freqüències al·lèliques. L'ús de CL&H en el mètode de Contribucions Òptimes va resultar en una major diversitat genètica però també en un canvi més gran de freqüències al·lèliques que l'ús de CVR2. Les diferències entre estratègies van ser menors quan només es van fer servir SNP amb una freqüència de l'al·lel menys comú (MAF) per sobre d'un llindar particular (MAF > 0.05 i MAF > 0.25) per calcular CL&H i CVR2 així com quan es va aplicar el mètode de Contribucions Òptimes en poblacions amb cens més petit (es va passar de N = 100 a N = 20). L'avaluació de CL&H i CVR2 es va estendre a poblacions subdividides, també a través de simulacions per ordinador. En poblacions subdividides, la diversitat genètica es distribueix en dos components: dins i entre subpoblacions. Quan s'atorga un pes més gran al component dins de subpoblacions, és possible restringir els nivells de consanguinitat. Sota aquest escenari, la utilització de CL&H va resultar ser la millor opció per gestionar aquest tipus de poblacions, ja que va mantindré una diversitat global més gran, va conduir a una menor consanguinitat i a canvis en les freqüències similars als observats quan es va utilitzar CVR2. / [EN] A main objective in conservation programs is to maintain genetic diversity, and the most efficient management strategy to achieve it is to apply the Optimal Contributions method. This method optimizes the contributions of breeding candidates by minimizing the global weighted coancestry. This leads to the highest levels of genetic diversity, when measured as expected heterozygosity, and to an effective control of the increase of inbreeding. The fundamental parameter of the method is the coancestry matrix which, traditionally, has been obtained from pedigree data. The current availability of genome-wide information allows us to estimate coancestries with higher precision. However, many different genomic coancestry measures have been proposed and it is unknown which measure is more efficient to minimize the loss of genetic diversity. Thus, the general aim of this thesis was to investigate the efficiency of different genomic coancestry matrices in the management of conserved populations when the Optimal Contributions method is applied to maximize genetic diversity. The matrices compared were those based on: i) the proportion of shared alleles (CSIM); ii) deviations of the observed number of alleles shared by two individuals from the expected number (CL&H); iii) the realized relationship matrix obtained by VanRaden's method 1 (CVR1); iv) the realized relationship matrix obtained by VanRaden's method 2 (CVR2); v) the realized relationship matrix obtained by Yang¿s method (CYAN); and vi) identical by descent segments (CSEG). Results for a single generation using thousands of SNP genotyped in individuals from a farm turbot population, showed large differences in the magnitude of the six coancestry coefficients. Moreover, pairwise correlations were those between coefficients greatly varied (especially for self-coancestry). The lowest correlations between CSIM, CL&H or CSEG and CVR2 or CYAN. Management with matrices based on the proportion of shared alleles or on segments (CSIM, CL&H and CSEG) retained higher variability than those based on realized genomic relationship matrices (CVR1, CVR2 and CYAN). As expected, maximizing heterozygosity pushed alleles toward intermediate frequencies. However, moving allele frequencies away from initial frequencies may be undesirable as particular adaptations to the environment can be lost. Stochastic simulations were used to investigate the efficiency of CL&H and CVR2 in the management of an undivided population across 50 generations and both matrices were compared not only in terms of the genetic diversity maintained but also in terms of the associated changes in allele frequencies across generations. The use of CL&H in the Optimal Contribution method resulted in a higher genetic diversity but also in a higher change of allele frequencies than the use of CVR2. The differences between strategies were reduced when only SNPs with a minimum allele frequency (MAF) above a particular threshold (MAF > 0.05 and MAF > 0.25) were used to compute CL&H and CVR2 as well as when the Optimal Contributions method was applied in populations of smaller sizes (N = 20 vs N = 100). The evaluation of CL&H and CVR2 was extended to subdivided populations, also via computer simulations. When populations are subdivided into different breeding groups, it is possible to give different weights to the within- and between-subpopulation components of genetic diversity. When a higher weight is given to the within-subpopulation component, the levels of inbreeding can be restricted. In this scenario, the use of CL&H was the best option for managing subdivided populations as it maintained more global diversity, led to less inbreeding and to changes in frequencies similar to those observed when using CVR2 when a large weight was given to the within-subpopulation term. / Esta tesis doctoral se realizó en el Instituto de Investigación y Tecnología Agraria y Alimentaria (INIA-CSIC) de Madrid. El trabajo expuesto en el capítulo 2 se realizó en parte en el Instituto Roslin, de la Universidad de Edimburgo durante una estancia predoctoral. Los trabajos expuestos en la tesis han sido financiados con una beca predoctoral FPI (BES-2017-081070) y a través de proyectos del Plan Estatal de I+D+i del Ministerio de Ciencia e Innovación (proyectos CGL2016-75904-C2-2-P y PID2020- 114426GB-C22) y de la Unión Europea (‘European Union‘s Seventh Framework Program, KBBE.2013.1.2-10, under Grant Agreement No. 613611, FISHBOOST project’ y ‘European Commission Horizon 2020, Framework Programme through Grant Agreement No. 727315, MedAID project’). / Morales González, E. (2023). Management of Genetic Diversity in Conservation Programs Using Genomic Coancestry [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/195231 / Compendio

L’auto-incompatibilité (AI) est une barrière reproductive prézygotique qui permet aux pistils d’une fleur de rejeter leur propre pollen. Les systèmes d’AI peuvent prévenir l’autofertilisation et ainsi limiter l’inbreeding. Dans l’AI gamétophytique, le génotype du pollen détermine son propre phénotype d’incompatibilité, et dans ce système, les déterminants mâles et femelles de l’AI sont codés par un locus multigénique et multi-allélique désigné le locus S. Chez les Solanaceae, le déterminant femelle de l’AI est une glycoprotéine stylaire extracellulaire fortement polymorphique possédant une activité ribonucléase et désignée S-RNase. Les S-RNases montrent un patron caractéristique de deux régions hypervariables (HVa et HVb), responsables de leur détermination allélique, et cinq régions hautement conservées (C1 à C5) impliquées dans l’activité catalytique ou la stabilisation structurelle de ces protéines. Dans ce travail, nous avons investigué plusieurs caractéristiques des S-RNases et identifié un nouveau ligand potentiel aux S-RNases chez Solanum chacoense. L’objectif de notre première étude était l’élucidation du rôle de la région C4 des S-RNases. Afin de tester l’hypothèse selon laquelle la région C4 serait impliquée dans le repliement ou la stabilité des S-RNases, nous avons généré un mutant dans lequel les quatre résidus chargés présents en région C4 furent remplacés par des résidus glycine. Cette protéine mutante ne s’accumulant pas à des niveaux détectables, la région C4 semble bien avoir un rôle structurel. Afin de vérifier si C4 est impliquée dans une liaison avec une autre protéine, nous avons généré le mutant R115G, dans lequel un acide aminé chargé fût éliminé afin de réduire les affinités de liaison dans cette région. Ce mutant n’affectant pas le phénotype de rejet pollinique, il est peu probable que la région C4 soit impliquée dans la liaison des S-RNases avec un ligand ou leur pénétration à l’intérieur des tubes polliniques. Enfin, le mutant K113R, dans lequel le seul résidu lysine conservé parmi toutes les S-RNases fût remplacé par un résidu arginine, fût généré afin de vérifier si cette lysine était un site potentiel d’ubiquitination des S-RNases. Toutefois, la dégradation des S-RNases ne fût pas inhibée. Ces résultats indiquent que C4 joue probablement un rôle structurel de stabilisation des S-RNases. Dans une seconde étude, nous avons analysé le rôle de la glycosylation des S-RNases, dont un site, en région C2, est conservé parmi toutes les S-RNases. Afin d’évaluer la possibilité que les sucres conjugués constituent une cible potentielle d’ubiquitination, nous avons généré une S11-RNase dont l‘unique site de glycosylation en C2 fût éliminé. Ce mutant se comporte de manière semblable à une S11-RNase de type sauvage, démontrant que l’absence de glycosylation ne confère pas un phénotype de rejet constitutif du pollen. Afin de déterminer si l’introduction d’un sucre dans la région HVa de la S11-RNase pourrait affecter le rejet pollinique, nous avons généré un second mutant comportant un site additionnel de glycosylation dans la région HVa et une troisième construction qui comporte elle aussi ce nouveau site mais dont le site en région C2 fût éliminé. Le mutant comportant deux sites de glycosylation se comporte de manière semblable à une S11-RNase de type sauvage mais, de manière surprenante, le mutant uniquement glycosylé en région HVa peut aussi rejeter le pollen d’haplotype S13. Nous proposons que la forme non glycosylée de ce mutant constitue un allèle à double spécificité, semblable à un autre allèle à double spécificité préalablement décrit. Il est intéressant de noter que puisque ce phénotype n’est pas observé dans le mutant comportant deux sites de glycosylation, cela suggère que les S-RNases ne sont pas déglycosylées à l’intérieur du pollen. Dans la dernière étude, nous avons réalisé plusieurs expériences d’interactions protéine-protéine afin d’identifier de potentiels interactants polliniques avec les S-RNases. Nous avons démontré que eEF1A, un composant de la machinerie de traduction chez les eucaryotes, peut lier une S11-RNase immobilisée sur résine concanavaline A. Des analyses de type pull-down utilisant la protéine eEF1A de S. chacoense étiquetée avec GST confirment cette interaction. Nous avons aussi montré que la liaison, préalablement constatée, entre eEF1A et l’actine est stimulée en présence de la S11-RNase, bien que cette dernière ne puisse directement lier l’actine. Enfin, nous avons constaté que dans les tubes polliniques incompatibles, l’actine adopte une structure agrégée qui co-localise avec les S-RNases. Ces résultats suggèrent que la liaison entre eEF1A et les S-RNases pourrait constituer un potentiel lien fonctionnel entre les S-RNases et l’altération du cytosquelette d’actine observée lors des réactions d’AI. Par ailleurs, si cette liaison est en mesure de titrer les S-RNases disponibles à l’intérieur du tube pollinique, ce mécanisme pourrait expliquer pourquoi des quantités minimales ou « seuils » de S-RNases sont nécessaires au déclenchement des réactions d’AI. / Self-incompatibility (SI) is a prezygotic reproductive barrier that allows the pistil of a flower to specifically reject their own (self-) pollen. SI systems can help prevent self-fertilization and avoid inbreeding. In gametophytic SI (GSI), the genotype of the pollen determines its breeding behaviour and in this system both female and male specificity determinants of SI are under the control of a multigenic and multiallelic locus called the S-locus. In Solanaceae, the female determinant of SI is a highly polymorphic stylar-expressed extracellular glycoprotein with RNase activity called the S-RNase. S-RNases show a distinct pattern of two hypervariable (HVa and HVb) regions, responsible for their allelic specificity, and five highly conserved regions (C1 to C5) thought to be involved in either the catalytic activity or the structural stabilization of the protein. In this work, we analyzed and characterized several conserved features of the S-RNases and also identified a potential novel S-RNase interactant in Solanum chacoense. The aim of our first study was to investigate the role of the C4 region of S-RNases. To test the hypothesis that the C4 region may be involved in S-RNase folding or stability, we examined a mutant in which the four charged residues in the C4 region were replaced with glycine. This mutant did not accumulate to detectable levels in styles, supporting a structural role for C4. To test the possibility that C4 might be involved in binding another protein, we prepared an R115G mutant, in which a charged amino acid was eliminated to reduce any potential binding to this region. This mutant had no effect on the pollen rejection phenotype of the protein, and thus C4 is likely not involved in either ligand binding or S-RNase entry inside pollen tubes. Finally, a K113R mutant, in which the only conserved lysine residue in all the S-RNases was replaced with arginine, was generated to test if this residue was an S-RNase ubiquitination site. However, S-RNase degradation was not disrupted in this mutant. Taken together, these results indicate that the C4 region likely plays a structural role. In a second study, we analyzed the role of S-RNase glycosylation. All S-RNases share a conserved glycosylation site in the C2 region. To test the possibility that the sugar residues might be a target for ubiquitination, a transgenic S11-RNase lacking its single glycosylation site was examined. This construct behaved similarly to a wild type S11-RNase, demonstrating that the lack of glycosylation does not confer constitutive pollen rejection. To determine if the introduction of an N-linked glycan in the HVa region would affect pollen rejection, a construct containing a second N-glycosylation site inside the HVa region of the S11-RNase and a construct containing only that N-glycosylation site inside the HVa region were prepared. The first construct rejected S11 pollen normally, but surprisingly, plants expressing the construct lacking the C2 glycosylation site rejected both S11 and S13 pollen. We propose that the non-glycosylated form is a dual specific allele, similar to a previously described dual-specific allele that also had amino acid replacements in the HV regions. Interestingly, this phenotype is not observed in the mutant containing two glycosylation sites, which suggests that the sugar residues are not removed during S-RNase entry into the pollen. In the final study, S-RNase-binding assays were performed with pollen extracts to detect potential interacting proteins. We found that concanavalin A-immobilized S11-RNase bound eEF1A, a component of the eukaryotic translational machinery. This interaction was validated by pull-down experiments using a GST-tagged S. chacoense eEF1A. We also found that a previously documented actin binding to eEF1A was markedly increased in the presence of S-RNases, although S-RNases alone do not bind actin. Lastly, we observed that actin in incompatible pollen tubes has an unusual aggregated form which also co-labels with S-RNases. This suggests that binding between S-RNases and eEF1A could provide a potential functional link between the S-RNase and the alteration of the actin cytoskeleton that occurs during the SI reaction. Furthermore, if eEF1A binding to S-RNases acted to titrate the amount of free S-RNase in the pollen tube, this binding may help explain the threshold phenomenon, where a minimum quantity of S-RNase in the style is required to trigger the SI reaction.