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Myotonic dystrophy type 2 (DM2) is caused by the extreme expansion (from < 30 repeats in normal individuals to ~ 11,000 for the full mutation in certain patients) of the repeating tetranucleotide CCTG•CAGG sequence in the intron of the zinc finger protein 9 (ZNF9) gene. The genetic instabilities of the CCTG•CAGG repeats were investigated to evaluate the molecular mechanisms responsible for these massive expansions. The effects of replication, recombination, repair and transcription on the genetic instabilities have been investigated in COS-7 cells and E. coli model systems. A replication assay was established in COS-7 cells wherein the CCTG•CAGG repeats cloned proximal to the SV40 origin of replication resulted in expansions and deletions in a length and orientation-specific manner, whereas the repeats cloned distal to the same origin were comparatively stable. These results fit with our data obtained from biochemical studies on synthetic oligonucleotides since these biochemical studies revealed that the d(CAGG)26 oligomer had a marked propensity to adopt a hairpin structure as opposed to its complementary d(CCTG)26 that lacked this capacity. Furthermore, a genetic assay in E. coli was used to monitor the intramolecular frequency of recombination. This assay revealed that the tetranucleotide repeats were indeed hot spots for recombination. Moreover, studies conducted in SOS-repair mutants showed that recombination frequencies were much lower in a SOS¯ strain as compared to a SOS+ strain. However, experiments conducted to ascertain the level of induction of the SOS response revealed that the SOS pathway was not stimulated in our studies. These results revealed that although breaks may occur within the repeats, the damage is most likely repaired without induction of the SOS response contrary to previous beliefs. Thus, a complex interplay of replication, recombination, and repair is likely responsible for the expansions observed in DM2.

A trinucleotide repeat (TNR) is a 3 base pair (bp) DNA sequence tandemly repeated in an array. In humans, TNR sequences have been found to be associated with at least 14 severe neurological diseases including Huntington disease, myotonic dystrophy and several of the spinocerebellar ataxias. Such diseases are caused by an expansion of the repeat sequence beyond a threshold length and are characterized by non-Mendelian patterns of inheritance which lead to genetic anticipation. Although the mechanism of the genetic instability in these arrays is not yet fully understood, various models have been suggested based on the in vitro observation that TNR sequences can form secondary structures such as pseudo-hairpins. In order to investigate the mechanisms responsible for instability of TNR sequences, a study was carried out on Escherichia coli cells in which TNR arrays had been integrated into the chromosomal lacZ gene. This genetic assay was used to identify proteins and pathways involved in deletion and/or expansion instability. Deletion instability was clearly dependent on orientation of the TNR sequence relative to the origin of replication. Interestingly, it was found that expansion instability is not dependent on the orientation of the repeat array relative to the origin of replication. The replication fork reversal pathway and the RecFOR mediated gap repair pathway were found to have no statistically significant influence on the instability of TNR arrays. However, the protein UvrD was found to affect the deletion instability of TNR sequences. The roles of key helicase genes were investigated for their effects on instability of chromosomal CTG•CAG repeats. Mutation of the rep gene increased deletion in the CTG leading-strand orientation of the repeat array, and expansion in both orientations - destabilizing the TNR array. RecQ helicase was found to have a significant effect on TNR instability in the orientation in which CAG repeats were present on the leading-strand relative to the origin of replication. Mutation of the recQ gene severely limited the number of expansion events in this orientation, whilst having no effect on deletions. This dependence of expansions on RecQ was lost in a rep mutant strain. In a rep mutant expansions were shown to be partially dependent on the DinG helicase. All together, these results suggest a model of TNR instability in which expansions are due to events occurring at either the leading or lagging strand of an arrested replication fork, facilitated by helicase action. The identity of the helicase implicated is determined by the nature of the arrest.

The tumour suppressor p53-binding protein 1 (53BP1) is phosphorylated following DNA double strand breaks (DSBs); however, little is understood about the upstream signaling pathways that control this phosphorylation. Additionally, it is not known how these processes combine with 53BP1 to control the survival of cells following DNA damage such as that imparted by ionizing radiation (IR), which is the basis of radiotherapy. In this thesis, I have shown that 53BP1 is phosphorylated specifically in S-phase cells, but not relocalized to intranuclear foci, in response to severe oxygen stress. This occurs with only partial dependence on the ATM kinase (Chapter 2). Following IR, I find that both ATM and DNA-PKcs contribute to intranuclear phosphorylated 53BP1 foci, but that this phosphorylation is independent of proximal signaling molecules that control the localization of 53BP1 to initial DSBs (Chapter 3). Furthermore, I show that 53BP1 loss confers sensitivity to IR and this can be further augmented by inhibition of ATM and DNA-PKcs kinases suggesting that there are both 53BP1-dependent and -independent pathways of survival from IR (Chapter 4). These findings may have important implications for molecular pathology and personalized medicine as 53BP1 has recently been found to be activated or lost in subsets of human tumours. I have collaborated to initiate the development of a novel system to interrogate the implications of 53BP1 loss as traditional siRNA approaches in human cancer cells were not feasible (Chapter 5 and Appendix 2). This system can be used in vivo as tumour xenografts to further understand how 53BP1 and the tumour microenvironment interact endogenously and in response to IR. I also present the possibility and proof of concept for the use of 53BP1 as a biomarker in primary human prostate cancer tissue where little is known about 53BP1 biology (Chapter 5).

The tumour suppressor p53-binding protein 1 (53BP1) is phosphorylated following DNA double strand breaks (DSBs); however, little is understood about the upstream signaling pathways that control this phosphorylation. Additionally, it is not known how these processes combine with 53BP1 to control the survival of cells following DNA damage such as that imparted by ionizing radiation (IR), which is the basis of radiotherapy. In this thesis, I have shown that 53BP1 is phosphorylated specifically in S-phase cells, but not relocalized to intranuclear foci, in response to severe oxygen stress. This occurs with only partial dependence on the ATM kinase (Chapter 2). Following IR, I find that both ATM and DNA-PKcs contribute to intranuclear phosphorylated 53BP1 foci, but that this phosphorylation is independent of proximal signaling molecules that control the localization of 53BP1 to initial DSBs (Chapter 3). Furthermore, I show that 53BP1 loss confers sensitivity to IR and this can be further augmented by inhibition of ATM and DNA-PKcs kinases suggesting that there are both 53BP1-dependent and -independent pathways of survival from IR (Chapter 4). These findings may have important implications for molecular pathology and personalized medicine as 53BP1 has recently been found to be activated or lost in subsets of human tumours. I have collaborated to initiate the development of a novel system to interrogate the implications of 53BP1 loss as traditional siRNA approaches in human cancer cells were not feasible (Chapter 5 and Appendix 2). This system can be used in vivo as tumour xenografts to further understand how 53BP1 and the tumour microenvironment interact endogenously and in response to IR. I also present the possibility and proof of concept for the use of 53BP1 as a biomarker in primary human prostate cancer tissue where little is known about 53BP1 biology (Chapter 5).

Le Stress Réplicatif est caractérisé par une accumulation de fourches bloquées et est connu pour être une source majeure d'instabilité génétique dans les cellules humaines. Le Stress Réplicatif et l'instabilité génétique sont des marqueurs précoces de la tumorigenèse. Il est connu que les fourches de réplication bloquées peuvent dégénérer en cassures double brin. En effet, après un stress réplicatif prolongé (24h) induit par l'hydroxyurée (HU), l'endonucléase MUS81-EME1 peut promouvoir l'effondrement des fourches de réplication. Cette endonucléase prévient l'accumulation de régions sous-répliquées en G2 et des défauts de ségrégation chromosomique en mitose. Dans cette étude, en suivant l'apparition de cassures double brin (CDB) par les techniques sensibles d'essai comète neutre et de QIBC (Quantitative Image-Based Cytometry), nous avons pu mettre en évidence que l'effondrement des fourches est un événement qui peut être visualisé rapidement suite au stress réplicatif (dès 2h après HU). Nous avons pu caractériser cet effondrement rapide comme étant un mécanisme indépendant de MUS81, sous unité catalytique du complexe MUS81-EME1. De plus, en réalisant des extinctions de l'expression de gènes par siARN, nous avons identifié deux nucléases, Artémis et XPF, comme étant impliquées dans ce mécanisme d'effondrement rapide des fourches de réplication. Nos résultats suggèrent un rôle de ce mécanisme d'effondrement rapide dans la prévention d'intermédiaires mitotiques et de la transmission de lésions aux cellules filles. Nous avons également identifié l'ADN polymérase alternative, Pol theta comme étant un facteur impliqué dans la prévention de la mort cellulaire induite par ce mécanisme. L'exploration de données de qPCR sur des prélèvements de tissus cancéreux nous a permis d'identifier la surexpression de Pol theta comme étant corrélée à des gènes de la HR. Ceci suggère un potentiel mécanisme d'adaptation pour prévenir de l'accumulation de fourches effondrées dans les cellules cancéreuses. L'ensemble de ces données révèle que les cellules humaines ont acquis au cours de l'évolution la capacité de cliver rapidement des fourches bloquées qui pourrait s'avérer importante pour la stabilité du génome, notamment en contexte de stress réplicatif. / Replicative stress is characterized by an accumulation of stalled replication forks and is known to be a major source of genetic instability in human cells. Replicative stress and genetic instability are early markers of tumorigenesis. It is known that stalled replication forks can degenerate into double strand breaks (DSB), a process called replication fork collapse. Indeed, after an extended replicative stress (24h) induced by hydroxyurea (HU), the endonuclease MUS81-EME1 can promote the collapse of replication forks. This endonuclease prevents accumulation of under replicated regions in G2 and mitotic segregation defects. Here, by monitoring DSB with sensitive neutral comet assay and QIBC (Quantitative Image-Based Cytometry) approaches, we found that replication forks can also collapse rapidly after replicative stress (as early as 2 hours after HU). We characterised this rapid replication fork collapse as a MUS81-independent mechanism. Moreover, by performing siRNA based knock down, we identified two nucleases, Artemis and XPF, involved in rapid replication fork collapse mechanism. Our results point toward a role of this rapid collapse mechanism in preventing mitotic intermediates and lesion transmission to daughter cells. Also, we identified the role of an alternative DNA polymerase Pol theta as a molecular factor involved in preventing this mechanism to induce cell death. Data mining of expression data from tumour samples allowed us to identify Pol theta verexpression as correlated with HR genes, underpinning a potential adaptation mechanism to prevent collapsed fork accumulation in cancer cells. Collectively, these data reveal that human cells have evolved a quick cleavage response to stalled forks that might be important for genome stability notably in cells undergoing replicative stress.

La stabilité du génome est compromise par les déséquilibres du pool de dNTPs qui affectent la vitesse de progression des fourches de réplication. Par exemple, la déficience en cytidine désaminase (CDA) conduit à un excès de dCTP qui induit un ralentissement des fourches de réplication. Les résultats obtenus au cours de ma thèse ont permis de mettre en évidence un nouveau mécanisme par lequel un déséquilibre du pool de nucléotides compromet la complétion de la réplication et la ségrégation correcte des chromosomes. En utilisant des techniques de peignage moléculaire, de microscopie électronique et d’imagerie cellulaire permettant de quantifier le niveau basal de PAR, nous avons montré que la réplication incomplète de l’ADN lorsque la CDA est absente est due à l’inhibition partielle de PARP-1, et n’est pas liée au ralentissement de la vitesse de progression des fourches de réplication. En effet, l’accumulation intracellulaire de dCTP inhibe l’activité de PARP-1 ce qui réduit l’activation de Chk1 et l’efficacité des points de contrôle situés en aval, favorisant ainsi l’accumulation de séquences d’ADN non répliquées en mitose. Celles-ci conduisent alors à la formation de ponts anaphasiques ultrafins (UFBs) entre les chromatides sœurs au niveau de sites difficiles à répliquer tels que les centromères et les sites fragiles. Ces résultats ont des implications directes dans le syndrome de Bloom (BS), une maladie génétique rare combinant prédisposition aux cancers et instabilité génétique. Ce syndrome est la conséquence de la mutation du gène BLM, codant pour une hélicase RecQ du même nom. La déficience en BLM conduit à une chute de l’expression de la CDA résultant en une augmentation des UFBs entièrement due à l’inhibition de PARP-1 par la dCTP, indépendamment de BLM. Ces travaux décrivent ainsi une conséquence pathologique encore inconnue du déséquilibre du pool de nucléotides et révèlent un rôle inattendu de PARP-1 dans la surveillance des séquences d’ADN non répliquées prévenant leur accumulation en mitose et les défauts de ségrégation des chromosomes associés. / Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA) deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, we found that in CDA-deficient cells DNA replication was unsuccessful due to the partial inhibition of basal PARP-1 activity, rather than slower fork speed. Indeed, the intracellular accumulation of dCTP inhibits PARP-1 activity compromising the activation of Chk1 and the downstream checkpoints efficiency, allowing the subsequent accumulation of unreplicated DNA in mitosis. This unreplicated DNA leads to the formation of ultrafine anaphase bridges (UFBs) between sister-chromatids at “difficult-to-replicate” sites such as centromeres and fragile sites. These results have direct implications for Bloom syndrome (BS), a rare genetic disease combining susceptibility to cancer and genomic instability. BS results from mutation of the BLM gene, encoding BLM, a RecQ 3’-5’ DNA helicase, a deficiency of which leads to CDA downregulation. BS cells thus have a CDA defect, resulting in a high frequency of UFBs due entirely to dCTP-dependent PARP-1 inhibition and independent of BLM status. Our results describe previously unknown pathological consequences of the distortion of dNTP pools and reveal an unexpected role for PARP-1 in preventing unreplicated DNA accumulation in mitosis and in preventing chromosome segregation defects.

Incidência de melanoma cutâneo maligno (MM) está aumentando em torno de 2,5 a 4% por ano no mundo. Os principais fatores de risco são história familiar de MM, múltiplos nevos benignos ou atípicos, e fatores adicionais como a imunossupressão, sensibilidade solar e exposição à radiação ultravioleta (UV). A instabilidade genômica é responsável pelo acúmulo de mutações que frequentemente estão envolvidas na transformação maligna. Podemos estudar a instabilidade genômica através de duas formas: microssatélites e RAPD (Random Amplified Polymorphic DNA). Na instabilidade genética o DNA repetitivo pode sofrer alterações. Através da Instabilidade de microssatélites (MSI) e da perda da heterozigosidade (LOH) podemos diferenciar tecidos normais de tumorais. A técnica de RAPD (baseada na PCR) produz fingerprints utilizados para detectar instabilidade genômica, polimorfismos, mutações e translocações quando comparados à fingerprints de amostras normais. No estudo de nove microssatélites encontramos um aumento de MSI (p=0.0132). D9S50 apresentou o maior número de alterações (28,5%) em nevos e MMs. D6S252, D9S52 e D9S180 são candidatos à marcador de prognóstico de MM porque apresentaram alterações (MSI + LOH) apenas em MMs. Na análise de 15 primers de RAPD em 12 amostras de MMs obtivemos 100 % de alteração com relação ao número ou posição das bandas. Os primers OPA-2 e OPA-14 são capazes de detectar alterações genéticas nos MMs. Dos padrões obtidos foram encontradas bandas que estavam ausentes nos tumores e estas foram clonadas e seqüenciadas. Estes procedimentos evidenciaram alterações em 9q33 e 12q15. O RAPD propicia o estudo do genoma humano sem a definição prévia de um lócus. Assim, podemos detectar alterações até então desconhecidas aumentando o conhecimento sobre a genômica tumoral. / The incidence of malignant skin melanoma (MM) increases around 2,5 a 4% each year in the world. The main risk factors are family history of MM, multiple benign or atypical nevi, and additional factors such as immunossuppression, sun sensibility and UV exposure. Genomic instability is responsible for a collection of mutations that are frequently involved in malignant transformation, and it can cause alterations in repetitive DNA sequences. There are two ways of studying genomic instability: microsatellites and RAPD (Random Amplified Polymorphic DNA). Through microsatellite instability (MSI) and loss of heterozygosis (LOH) we can separate normal from tumoral tissues. RAPD technique (which is based on PCR) generates fingerprints used for detection of genomic instability, polymorphisms, mutations and translocations that can be compared with fingerprint generated from normal tissue. Studying nine microsatellite, we found an increased MSI (p=0.0132). D9S50 showed the greater number of alterations (28,5%) in nevi and MM. D6S252, D9S52 e D9S180 are candidates for MM prognostic marker for showing alterations (MSI+LOH) in melanomas only. The analysis of 15 RAPD primers in 12 MM samples showed 100% of alteration related to the number or location of the bands. OPA-2 and OPA-14 primers are capable of detecting genetic alterations in MM. In the patterns obtained, two bands which were absent in tumors were found, and they were cloned and submitted to sequencing. These procedures highlighted alterations in loci 9q33 e 12q15. RAPD makes it possible to study the genome without a previous definition of a locus. So we are able to detect alterations so far unknown, increasing our knowledge on tumor genetics.

La bactérie Deinococcus radiodurans est connue pour sa capacité à résister à un grand nombre de traitements génotoxiques parmi lesquels on peut citer l’exposition aux rayons ionisants, aux ultra-violets, à la mitomycine C, à la dessication et au stress oxydant. Elle est capable lors d’une exposition à des doses extrêmes de rayons γ générant des centaines de cassures de l’ADN de reconstituer un génome intact en seulement 2 à 3 heures via un mécanisme original, l’ESDSA, impliquant une synthèse massive d’ADN pendant la phase de réparation des cassures de l’ADN. En plus de mécanismes efficaces de réparation de l’ADN, elle possède un kit de survie comprenant une compaction importante du nucléoïde, des mécanismes de protection des protéines contre l’oxydation, une réponse originale aux lésions de l’ADN et des protéines spécifiques induites après irradiation. Tous ces facteurs contribuent au maintien de l’intégrité du génome et à la survie de la cellule lors de l’exposition à différents agents génotoxiques. Souvent considéré comme un organisme ayant une stabilité génomique exceptionnelle, cette bactérie possède dans son génome un grand nombre de séquences répétées et des éléments mobiles et est par ailleurs naturellement compétente. Ce sont autant de facteurs pouvant participer à la variabilité génétique de cette espèce. Je me suis donc intéressée lors de ma thèse à deux processus pouvant participer à l’instabilité génétique chez D. radiodurans : la recombinaison entre séquences répétées et la transformation naturelle.L’introduction dans le génome de D. radiodurans de séquences répétées directes de 438 pb séparées par des régions d’ADN d’une longueur allant de 1479 pb à 10 500 pb m’a permis de mettre en évidence le rôle majeur joué par l’appariement simple brin (Single Strand Annealing ou SSA) impliquant la protéine DdrB, spécifique des Deinococcaceae, joue un rôle majeur dans la recombinaison « spontanée » entre les séquences répétées en absence de la recombinase RecA. L’absence de DdrB dans des souches déficientes pour la recombinaison augmente davantage la perte de viabilité observée dans ces souches ce qui suggère que le SSA participe à la prise en charge de fourches de réplication bloquées, source majeure d’instabilité génétique en absence de stress extérieur, si ces fourches ne peuvent être prise en charge par des voies impliquant des protéines de recombinaison. Je me suis également intéressée à la transformation naturelle et aux protéines impliquées dans ce processus chez D. radiodurans. J’ai pu démontrer que la protéine DprA impliquée dans la protection de l’ADN simple brin et le chargement de RecA sur l’ADN simple brin internalisé lors de la transformation de nombreuses espèces comme Streptococcus pneumoniae, Bacillus subtilis ou Helicobacter pylori, est également impliquée dans la transformation chez D. radiodurans. J’ai pu montrer également qu’en plus de jouer un rôle majeur dans la transformation par de l’ADN plasmidique, DdrB est impliquée dans la transformation par de l’ADN génomique si la protéine DprA est absente. / The bacterium Deinococcus radiodurans is known for its ability to withstand a large number of genotoxic treatments, including exposure to ionizing or ultraviolet radiation, mitomycin C, desiccation, and oxidative stress. It is able, upon exposure to extreme doses of γ-radiation generating hundreds of DNA breaks, to reconstitute an intact genome in only 2 to 3 hours via an ESDSA mechanism, involving massive DNA synthesis during DNA double strand break repair. Together with efficient DNA repair mechanisms, D. radiodurans possesses a survival kit comprising significant compaction of its nucleoid, protection mechanisms against protein oxidation, an original response to DNA damage and specific proteins induced after irradiation. All of these contribute to the maintenance of genomic integrity and cell survival upon exposure to various genotoxic agents. In spite of the idea that D. radiodurans is an organism with outstanding genomic stability, this bacterium has in its genome a large number of repeat sequences and mobile elements and is also naturally competent. All these factors contribute to the genetic variability of species. I was interested in two processes that can play a role in genetic variability in D. radiodurans: recombination between repeated sequences and natural transformation.The introduction, into the genome of D. radiodurans, of 438 bp direct repeated sequences separated by DNA regions ranging from 1,479 bp to 10,500 bp in length allowed me to demonstrate the major role of Single Strand Annealing (SSA) involving the DdrB protein specific for Deinococcaceae, in the "spontaneous" recombination between the repeated sequences in the absence of the RecA recombinase. The absence of DdrB in strains deficient for recombination further increased the loss of viability observed in these strains, suggesting that SSA is required for the management of blocked replication forks, a major source of genetic instability in the absence of external stress when these forks cannot be rescued by pathways involving recombination proteins.I was also interested in the natural transformation and proteins involved in this process in D. radiodurans. I demonstrated that DprA protein involved in DNA single strand protection and loading of RecA on single-stranded DNA internalized during transformation of many species such as Streptococcus pneumoniae, Helicobacter pylori, or Bacillus subtilis, is also involved in this process in D. radiodurans. I also showed that, in addition to playing a major role in transformation by plasmid DNA, DdrB is also involved in transformation by genomic DNA of cells devoid of the DprA protein.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The potato (Solanum tuberosum L.), Solanaceae, ranked fourth in quantity food production, exceeded only by wheat, rice and corn. The potato crop is propagated vegetative form, which requires high quality plant for income maintenance in the field. However, culture is susceptible to diseases, mainly viruses so it is necessary to use techniques such as culture of shoot tips for cleaning and subsequent clonal propagation in a system that maintains the quality of plant propagation material such as hydroponics. However, it is known that tissue culture induced genetic variability in micropropagated material. Changes collectively termed somaclonal variation. As a result, strategies are needed to control and monitor this phenomenon in potato cultivars due to the possibility of loss of genetic traits for which have been improved. Given the above, this study aimed to evaluate the in vitro behavior of ramets, evaluate characteristics of the tubers produced hydroponically, evaluate the phenotypic stability of ramets from tissue culture through 26 minimum description of potato compare the phenotypic stability of ramets of each cultivar according to the degree of differentiation of the explants that originated ramets, indicating potential minimum descriptors to monitor the phenotypic stability, to analyze the variability over the subcultures of the growing season and the field through microsatellite markers from potato cultivar Asterix and Atlantic. The results showed that ramets both of Asterix as Atlantic differ on in vitro behavior for the variables. There is variability in characteristics of tuber-seed in the ramets of potato cultivars Atlantic and Asterix, from the cultivation of basic generation zero (G0) in a closed hydroponic system. Ramets of Asterix have a different behavior of ramets Atlantic in relation to minimum descriptors. There is great variability and intraclonal divergence in relation to the phenotypic varieties in minimum descriptors in ramets of Asterix and Atlantic. The degree of differentiation of explants that form these ramets has no effect on the phenotypic stability of Asterix and Atlantic. Growth habit and pigmentation of the main stem descriptors can be useful in monitoring the genetic purity of Asterix. Growth habit, vegetative and Tuber shape have potential use in monitoring the intraclonal variability in Atlantic. The potato cultivars Asterix and Atlantic show high variability in intraclonal microsatellite markers. The pattern of instability in molecular phenotypes is different among potato cultivars Asterix and Atlantic. Ramets regenerated from shoot tip derived and indirect organogenesis are also unstable. The subculture time, alone, seems to have no effect on the incidence of somaclonal variation in potato cultivar Asterix and Atlantic. / A batata (Solanum tuberosum L.) ocupa o quarto lugar em nível mundial em produção, sendo superada apenas pelo trigo, arroz e milho. A cultura da batata é propagada de forma vegetativa e, devido à suscetibilidade a doenças, é imprescindível o emprego da cultura de ápices caulinares para efetuar limpeza clonal e, na sequência, da micropropagação das partes aéreas para a obtenção de mudas livres de patógenos para satisfazer a demanda dos agricultores. Entretanto, a cultura de tecidos pode induzir variabilidade genética no material micropropagado, a qual é denominada variação somaclonal. Face ao exposto, o objetivo geral do presente estudo consistiu na análise da variação somaclonal nas cultivares de batata Asterix e Atlantic, com o intuito de contribuir para uma melhor compreensão e, também, para o desenvolvimento de estratégias direcionadas ao controle e monitoramento desse fenômeno, e, simultaneamente, para a obtenção de variantes de utilidade para programas de melhoramento genético. Com esta finalidade, as diferentes etapas do sistema de produção de batata-semente foram avaliadas por descritores morfológicos, morfoagronômicos e marcadores microssatélites. Os resultados revelaram que os rametes de Asterix e Atlantic apresentam extensiva variabilidade intraclonal, observando-se padrões diferenciados nas duas cultivares em resposta às fontes de variação estudadas. Há variabilidade intraclonal na estabilidade fenotípica in vitro e em características de tubérculo-semente nos rametes das cultivares de batata Atlantic e Asterix, provenientes do cultivo de plantas básicas da geração inicial (G0) em sistema hidropônico. Plantas e tubérculos de primeira geração clonal apresentam grande variabilidade intraclonal e divergência em relação ao padrão fenotípico das cultivares nos descritores mínimos nos rametes de Asterix e Atlantic. O grau de diferenciação dos explantes que originam os rametes não tem efeito na estabilidade fenotípica de Asterix e Atlantic. Os rametes das duas cultivares apresentam elevada variabilidade genética intraclonal em marcadores microssatélites. O padrão de instabilidade nos fenótipos moleculares é diferenciado em Asterix e Atlantic. Rametes regenerados a partir de ápices caulinares e derivados de organogênese indireta são igualmente instáveis em nível molecular. O tempo de subcultivo pode ser usado como ferramenta de controle da variação somaclonal nas cultivares de batata Asterix e Atlantic, pela análise dos fenótipos moleculares.