A majority of commercial rose varieties bloom repeatedly throughout the year, as compared to most rose species, other woody ornamentals, and fruit crops that bloom once a year. This recurrent flowering feature of the commercial roses resulted from a flowering mutation named everblooming (evb). The mutation is recessive to once blooming and is found in the rose species Rosa chinensis. Although several molecular maps have been developed for rose, little is known about the evb gene, except for its classic genetics. The purpose of this study was to develop a large-insert bacterial artificial chromosome (BAC) library as a starting tool for molecular cloning and analysis of the evb gene by map-based cloning. To construct the large-insert BAC library, nuclear megabase-size DNA was isolated from the recurrent blooming diploid species, Rosa chinensis cv. Old Blush. The DNA was then partially digested with BamHI and separated on agarose gels by multi-phase pulsed-field gel electrophoresis. Size selected fragments estimated between 100 kb and 150 kb in size were cloned into the pECBAC1 BAC vector and the clones having rose DNA inserts were arrayed in 80 384-well microplates individually, with each clone being barcoded. The library contains 30,720 clones, has an average insert size of 108 kb and covers roughly 5.9x genome equivalents, with a >99% probability of isolating a single-copy clone from the library. The library is now available to be screened with the genes cloned from other species that control vernalization and floral development and will be used in mapbased cloning of the evb gene using a Rosa wichuraiana (‘Basye’s Thornless’) x ‘Old Blush’ backcross population.

BACKGROUND:Sugarcane breeding has significantly progressed in the last 30 years, but achieving additional yield gains has been difficult because of the constraints imposed by the complex ploidy of this crop. Sugarcane cultivars are interspecific hybrids between Saccharum officinarum and Saccharum spontaneum. S. officinarum is an octoploid with 2n=80 chromosomes while S. spontaneum has 2n=40 to 128 chromosomes and ploidy varying from 5 to 16. The hybrid genome is composed of 70-80%S. officinaram and 5-20%S. spontaneum chromosomes and a small proportion of recombinants. Sequencing the genome of this complex crop may help identify useful genes, either per se or through comparative genomics using closely related grasses. The construction and sequencing of a bacterial artificial chromosome (BAC) library of an elite commercial variety of sugarcane could help assembly the sugarcane genome.RESULTS:A BAC library designated SS_SBa was constructed with DNA isolated from the commercial sugarcane variety SP80-3280. The library contains 36,864 clones with an average insert size of 125 Kb, 88% of which has inserts larger than 90 Kb. Based on the estimated genome size of 760-930 Mb, the library exhibits 5-6 times coverage the monoploid sugarcane genome. Bidirectional BAC end sequencing (BESs) from a random sample of 192 BAC clones sampled genes and repetitive elements of the sugarcane genome. Forty-five per cent of the total BES nucleotides represents repetitive elements, 83% of which belonging to LTR retrotransposons. Alignment of BESs corresponding to 42 BACs to the genome sequence of the 10 sorghum chromosomes revealed regions of microsynteny, with expansions and contractions of sorghum genome regions relative to the sugarcane BAC clones. In general, the sampled sorghum genome regions presented an average 29% expansion in relation to the sugarcane syntenic BACs.CONCLUSION:The SS_SBa BAC library represents a new resource for sugarcane genome sequencing. An analysis of insert size, genome coverage and orthologous alignment with the sorghum genome revealed that the library presents whole genome coverage. The comparison of syntenic regions of the sorghum genome to 42 SS_SBa BES pairs revealed that the sorghum genome is expanded in relation to the sugarcane genome.

O maracujá-amarelo (Passiflora edulis f. flavicarpa) é uma frutífera de importância econômica no Brasil, sendo apreciado para a produção de suco concentrado e para o consumo in natura, além de ser usado pela indústria farmacêutica na extração da passiflorina. O presente trabalho visou à exploração da biblioteca genômica inserida em BACs (Ped-B-Flav) por meio da técnica de BAC-end sequencing, visando prover os primeiros insights sobre a composição e organização genômica da espécie, além de gerar novos candidatos a marcadores moleculares. Ao todo foram realizadas 9.979 reações de sequenciamento com eficiência média de 89 %, resultando em 8.821 BES de alta qualidade, com tamanho variando entre 100 pb e 1.255 pb, tendo em média 596 pb, totalizando cerca de 5,7 Mpb de informação genômica. Foram identificados, ao todo, 610 potenciais novos marcadores microssatélites. Os motivos de tetranucleotídeos foram os mais abundantes, ou seja, 28,9 % do total, sendo as repetições AATT aquelas observadas com maior frequência, com 131 ocorrências. Foram identificados e classificados 4.394 (19,69 %) elementos repetitivos. Dentre estes elementos, os grupos dos retrotransposons gypsy e copia-like foram os mais abundantes, correspondendo a 10,08 % e 7,93 % das ocorrências, respectivamente. Além disso, foram encontradas 767 (8,7 %) sequências com alta identidade a regiões codificadoras de proteínas. Estas sequências foram classificadas e anotadas de acordo com o vocabulário controlado GeneOntology. Análises de mapeamento genômico comparativo revelaram três regiões microssintênicas com o genoma de Populus trichocarpa, uma com o genoma de Vitis vinifera e uma com o genoma de Arabdopisis thaliana, além de evidenciarem uma série de regiões rearranjadas em relação aos genomas de referência. O presente estudo mostrou que os BES de Passiflora edulis são uma excelente fonte de informações sobre o genoma da espécie, principalmente no que tange à diversidade gênica, identificação de elementos transponíveis e ao potencial para o desenvolvimento de novos marcadores genéticos. Igualmente, foi possível empregar essas sequências na identificação de regiões microssintênicas entre o genoma do maracujá-amarelo e de outras espécies vegetais próximas. / Yellow passion fruit (Passiflora edulis f. flavicarpa) is of considerable economic importance to Brazil. It is used to produce juice concentrate and also marketed for consumption as a fresh fruit. In the pharmaceutical industry, it is used to produce passiflora extract. The aim of this study was to explore the BAC (Bacterial Artificial Chromosome) genomic library (Ped-B-Flav) using BAC-end sequencing (BES) to provide some initial insights into the composition and organization of the species genome, and to generate new candidates for molecular markers. Altogether, 9,979 sequencing reactions were performed, with an average efficiency of 89 %, resulting in 8,821 high-quality BES, of average length ranging from 100 bp to 1255 bp, and consisting of an average 596 bp, totaling some 5.7 Mb of genomic information. In all, we identified 610 potential new microsatellite markers. Tetranucleotide motifs (28.9%) were the most abundant and AATT was the most frequently observed motif, with 131 occurrences. We identified and classified 4,394 (19.69 %) repetitive elements. Retrotransposon gypsy (10.8%) and copia-like (7.93%) elements were the most abundant. Furthermore, we found 767 (8.7 %) sequences very similar to those of protein coding regions. These sequences were classified and annotated according to gene ontology controlled vocabulary. Comparative genomic mapping revealed three regions showing microsynteny with the genome of Populus trichocarpa, one with Vitis vinifera genome and one with the Arabdopisis thaliana genome. In addition it revealed a series of rearranged regions in comparison to the reference genomes. This study showed that Passiflora edulis BES form an excellent source of information on the genome of the species, especially in regard to genetic diversity, identification of transposable elements and potential for the development of new genetic markers. It was also possible, using these sequences, to identify regions showing microsynteny with other plant species.

Centromeres are responsible for sister-chromatid cohesion, kinetochore formation, and accurate transmission of chromosomes. Rice provides an excellent model for organizational and functional studies of centromeres since several of its chromosomes contain limited amounts of satellite and other repetitive sequences in their centromeres. To facilitate molecular characterization of the centromeres, we screened several BIBAC and BAC libraries of japonica and indica rice, using several centromere-specific repeat elements as probes. The positive clones were identified, fingerprinted and integrated into our whole genome physical map databases of the two rice subspecies. BAC/BIBACbased physical maps were constructed for the centromeric regions of the subspecies. To determine whether the genomic organization of the centromeres has changed since the cultivated rice split from its progenitor and to identify the sequences potentially playing an important role in centromere functions, we constructed a large-insert BIBAC library for the wild progenitor of Asian cultivated rice, O. rufipogon. The library contains 24,192 clones, has an average insert size of 163 kb, and covers 5 x haploid genome of wild rice. We screened the wild rice library with two centromere 8-specific overgo probes designed from the sequences flanking centromere 8 of japonica rice. A BIBACbased map was constructed for wild rice centromere 8. Two of the clones, B43P04 and B15E04, were found to span the entire region of the wild rice centromere and thus selected for sequencing the centromere. By sequencing the B43P09 clone, a 95% genomic sequence of the long arm side of wild rice centromere 8 was obtained. Comparative analysis revealed that the centromeric regions of wild rice have a similar gene content to japonica rice, but the centromeric regions of japonica rice have undergone chromosomal rearrangements at both large scale and nucleotide levels. In addition, although the 155-bp satellite repeats showed dramatic changes at the middle region, they are conserved at the 5' and 3' ends of satellite monomers, suggesting that those regions might have important functional roles for centromeres. These results provide not only new insights into genomic organization and evolution, but also a platform for functional analysis of plant centromeres.

O maracujá-amarelo (Passiflora edulis f. flavicarpa) é uma frutífera de importância econômica no Brasil, sendo apreciado para a produção de suco concentrado e para o consumo in natura, além de ser usado pela indústria farmacêutica na extração da passiflorina. O presente trabalho visou à exploração da biblioteca genômica inserida em BACs (Ped-B-Flav) por meio da técnica de BAC-end sequencing, visando prover os primeiros insights sobre a composição e organização genômica da espécie, além de gerar novos candidatos a marcadores moleculares. Ao todo foram realizadas 9.979 reações de sequenciamento com eficiência média de 89 %, resultando em 8.821 BES de alta qualidade, com tamanho variando entre 100 pb e 1.255 pb, tendo em média 596 pb, totalizando cerca de 5,7 Mpb de informação genômica. Foram identificados, ao todo, 610 potenciais novos marcadores microssatélites. Os motivos de tetranucleotídeos foram os mais abundantes, ou seja, 28,9 % do total, sendo as repetições AATT aquelas observadas com maior frequência, com 131 ocorrências. Foram identificados e classificados 4.394 (19,69 %) elementos repetitivos. Dentre estes elementos, os grupos dos retrotransposons gypsy e copia-like foram os mais abundantes, correspondendo a 10,08 % e 7,93 % das ocorrências, respectivamente. Além disso, foram encontradas 767 (8,7 %) sequências com alta identidade a regiões codificadoras de proteínas. Estas sequências foram classificadas e anotadas de acordo com o vocabulário controlado GeneOntology. Análises de mapeamento genômico comparativo revelaram três regiões microssintênicas com o genoma de Populus trichocarpa, uma com o genoma de Vitis vinifera e uma com o genoma de Arabdopisis thaliana, além de evidenciarem uma série de regiões rearranjadas em relação aos genomas de referência. O presente estudo mostrou que os BES de Passiflora edulis são uma excelente fonte de informações sobre o genoma da espécie, principalmente no que tange à diversidade gênica, identificação de elementos transponíveis e ao potencial para o desenvolvimento de novos marcadores genéticos. Igualmente, foi possível empregar essas sequências na identificação de regiões microssintênicas entre o genoma do maracujá-amarelo e de outras espécies vegetais próximas. / Yellow passion fruit (Passiflora edulis f. flavicarpa) is of considerable economic importance to Brazil. It is used to produce juice concentrate and also marketed for consumption as a fresh fruit. In the pharmaceutical industry, it is used to produce passiflora extract. The aim of this study was to explore the BAC (Bacterial Artificial Chromosome) genomic library (Ped-B-Flav) using BAC-end sequencing (BES) to provide some initial insights into the composition and organization of the species genome, and to generate new candidates for molecular markers. Altogether, 9,979 sequencing reactions were performed, with an average efficiency of 89 %, resulting in 8,821 high-quality BES, of average length ranging from 100 bp to 1255 bp, and consisting of an average 596 bp, totaling some 5.7 Mb of genomic information. In all, we identified 610 potential new microsatellite markers. Tetranucleotide motifs (28.9%) were the most abundant and AATT was the most frequently observed motif, with 131 occurrences. We identified and classified 4,394 (19.69 %) repetitive elements. Retrotransposon gypsy (10.8%) and copia-like (7.93%) elements were the most abundant. Furthermore, we found 767 (8.7 %) sequences very similar to those of protein coding regions. These sequences were classified and annotated according to gene ontology controlled vocabulary. Comparative genomic mapping revealed three regions showing microsynteny with the genome of Populus trichocarpa, one with Vitis vinifera genome and one with the Arabdopisis thaliana genome. In addition it revealed a series of rearranged regions in comparison to the reference genomes. This study showed that Passiflora edulis BES form an excellent source of information on the genome of the species, especially in regard to genetic diversity, identification of transposable elements and potential for the development of new genetic markers. It was also possible, using these sequences, to identify regions showing microsynteny with other plant species.

Sporisorium scitamineum é um fungo basidiomiceto causador do carvão da cana-de-açúcar, uma doença com impacto negativo no cultivo da cana-de-açúcar, e com ocorrência em todos os países produtores. A manifestação da doença na cultura da cana depende da formação de uma hifa dicariótica a partir da anastomose de duas hifas haplóides compatíveis com relação ao tipo de reação sexual (mating-type). O controle do cruzamento sexuado (mating) é realizado pela expressão de um conjunto de genes presentes em dois loci, a e b. O locus a codifica um lipopeptídeo com função de feromônio e um receptor de feromônio, responsáveis pelo reconhecimento de células compatíveis e fusão de hifas, enquanto o locus b codifica fatores de transcrição que controlam a expressão de genes responsáveis pela manutenção das hifas dicarióticas durante o processo de infecção e crescimento do fungo dentro da planta. Apesar de desempenharem função essencial no processo de infecção e manutenção da doença em cana-de-açúcar, o conhecimento a respeito da organização genômica ou da função dos demais genes presentes nos loci a e b em S. scitamineum e em outros fungos causadores de carvão é ainda incipiente. Desta forma, o objetivo geral do presente trabalho foi isolar as regiões genômicas relacionadas aos genes de cruzamento em S. scitamineum e analisar comparativamente com regiões similares já descritas e depositadas em bancos de dados públicos. Para o isolamento destas regiões, foi construída uma biblioteca genômica em BAC de uma linhagem haplóide de S. scitamineum, a Ssc39 (+), isolada de uma variedade de cana-de-açúcar com sintomas de alta susceptibilidade. Foram selecionados 11 clones por PCR. Os insertos foram sequenciados e utilizados para confirmação da montagem dos loci no sequenciamento do genoma do fungo. Apesar do fungo S. scitamineum apresentar sistema bipolar de reação sexual assim como o fungo U. hordei, as análises comparativas de ambos os locus indicaram que S. scitamineum apresenta maior similaridade com o fungo S. reilianum principalmente com o alelo a1, no qual apresenta sistema tetrapolar de reação sexual. A anotação e caracterização dos genes do tipo de reação sexual (mating type) possibilitaram a comparação e melhor entendimento sobre esses genes de grande importância na patogenicidade e no ciclo de vida do fungo. / Sporisorium scitamineum is a basidiomycete fungus causing the smut disease in sugarcane, with a negative impact on the cultivation of sugarcane, and occurring in all producing countries. The manifestation of the disease in sugarcane crop depends on the formation of a dikaryotic hyphae originated of the anastomosis of two haploid mating type compatible cells. The control of the sexual crossing (mating) is performed by expression of a set of genes present in two loci, a and b. The locus a encodes a lipopeptide with the function of pheromone and pheromone membrane receptor responsible for cell recognition and compatible hyphal fusion, whereas the locus b encodes transcription factors that control the expression of genes responsible for the maintenance of the dikaryotic hyphal growth in plant. Although they play an essential role in the maintenance of infection and disease in sugarcane process, knowledge about the genomic organization and function of other genes in these two loci of S. scitamineum and other smut fungi is still incipient. Thus, the overall goal of this work was to isolate genomic regions related to the mating type in S. scitamineum and to perform a comparative analyze with similar regions described and deposited in public databases. For the isolation of these regions, we constructed a genomic BAC library of a haploid strain of S. scitamineum, the Ssc39 (+), isolated from a variety of sugarcane with symptoms of high susceptibility. Eleven clones were selected by PCR. The inserts were sequenced and used to confirm the assembly of both loci in the genome sequencing of the fungus. Although S. scitamineum belongs to the class of bipolar system of sexual response as well as the fungus U. hordei , the comparative analysis of both loci indicated that S. scitamineum shows greater similarity to the S. reilianum mainly with A1 allele, which has a tetrapolar system sexual response. The annotation of the genes and characterization mating type genes enabled the comparison and better understanding of the importance of these genes in the life cycle of the fungus.

Sporisorium scitamineum é um fungo basidiomiceto causador do carvão da cana-de-açúcar, uma doença com impacto negativo no cultivo da cana-de-açúcar, e com ocorrência em todos os países produtores. A manifestação da doença na cultura da cana depende da formação de uma hifa dicariótica a partir da anastomose de duas hifas haplóides compatíveis com relação ao tipo de reação sexual (mating-type). O controle do cruzamento sexuado (mating) é realizado pela expressão de um conjunto de genes presentes em dois loci, a e b. O locus a codifica um lipopeptídeo com função de feromônio e um receptor de feromônio, responsáveis pelo reconhecimento de células compatíveis e fusão de hifas, enquanto o locus b codifica fatores de transcrição que controlam a expressão de genes responsáveis pela manutenção das hifas dicarióticas durante o processo de infecção e crescimento do fungo dentro da planta. Apesar de desempenharem função essencial no processo de infecção e manutenção da doença em cana-de-açúcar, o conhecimento a respeito da organização genômica ou da função dos demais genes presentes nos loci a e b em S. scitamineum e em outros fungos causadores de carvão é ainda incipiente. Desta forma, o objetivo geral do presente trabalho foi isolar as regiões genômicas relacionadas aos genes de cruzamento em S. scitamineum e analisar comparativamente com regiões similares já descritas e depositadas em bancos de dados públicos. Para o isolamento destas regiões, foi construída uma biblioteca genômica em BAC de uma linhagem haplóide de S. scitamineum, a Ssc39 (+), isolada de uma variedade de cana-de-açúcar com sintomas de alta susceptibilidade. Foram selecionados 11 clones por PCR. Os insertos foram sequenciados e utilizados para confirmação da montagem dos loci no sequenciamento do genoma do fungo. Apesar do fungo S. scitamineum apresentar sistema bipolar de reação sexual assim como o fungo U. hordei, as análises comparativas de ambos os locus indicaram que S. scitamineum apresenta maior similaridade com o fungo S. reilianum principalmente com o alelo a1, no qual apresenta sistema tetrapolar de reação sexual. A anotação e caracterização dos genes do tipo de reação sexual (mating type) possibilitaram a comparação e melhor entendimento sobre esses genes de grande importância na patogenicidade e no ciclo de vida do fungo. / Sporisorium scitamineum is a basidiomycete fungus causing the smut disease in sugarcane, with a negative impact on the cultivation of sugarcane, and occurring in all producing countries. The manifestation of the disease in sugarcane crop depends on the formation of a dikaryotic hyphae originated of the anastomosis of two haploid mating type compatible cells. The control of the sexual crossing (mating) is performed by expression of a set of genes present in two loci, a and b. The locus a encodes a lipopeptide with the function of pheromone and pheromone membrane receptor responsible for cell recognition and compatible hyphal fusion, whereas the locus b encodes transcription factors that control the expression of genes responsible for the maintenance of the dikaryotic hyphal growth in plant. Although they play an essential role in the maintenance of infection and disease in sugarcane process, knowledge about the genomic organization and function of other genes in these two loci of S. scitamineum and other smut fungi is still incipient. Thus, the overall goal of this work was to isolate genomic regions related to the mating type in S. scitamineum and to perform a comparative analyze with similar regions described and deposited in public databases. For the isolation of these regions, we constructed a genomic BAC library of a haploid strain of S. scitamineum, the Ssc39 (+), isolated from a variety of sugarcane with symptoms of high susceptibility. Eleven clones were selected by PCR. The inserts were sequenced and used to confirm the assembly of both loci in the genome sequencing of the fungus. Although S. scitamineum belongs to the class of bipolar system of sexual response as well as the fungus U. hordei , the comparative analysis of both loci indicated that S. scitamineum shows greater similarity to the S. reilianum mainly with A1 allele, which has a tetrapolar system sexual response. The annotation of the genes and characterization mating type genes enabled the comparison and better understanding of the importance of these genes in the life cycle of the fungus.

Our ability to map and sequence whole genomes is one of the most important developments in biological science. It will provide us with an unprecedented insight into the genetic background of phenotypic traits, such as disease resistance, reproduction and growth and also makes it feasible to study the processes of genome evolution. The main focus of this thesis has been to develop a linkage map of the horse (Equus caballus) genome. A secondary aim was to expand the number of physically mapped genes in the horse, allowing comparative analyses with data from the human genome map. Finally, attempts were made to identify single nucleotide polymorphisms (SNPs) on the horse Y chromosome. The development of a genome map relies on the information generated by both linkage and cytogenetical studies. To integrate genetical and physical assignments in the very early phase of equine genome map construction, 19 polymorphic microsatellite markers were isolated from lambda phage clones which, in parallel, were physically assigned to chromosomes by fluorescent in situ hybridization (FISH). The microsatellites were simultaneously mapped by linkage analysis in a Swedish reference pedigree. A first primary male autosomal linkage map of the domestic horse was constructed by segregation analysis of 140 genetic markers within eight half-sib families with, in total, 263 offspring. One hundred markers were arranged into 25 linkage groups, 22 of which could be assigned physically to 18 different chromosomes. The total map distance contained within linkage groups was 679 cM. The presented map provides an important framework for future genome mapping in the horse. Our contribution to the comparative horse genome map, was the presentation of map data for 12 novel genes using FISH and somatic cell hybrid mapping. AD chromosomal assignments except one were in agreement with human-horse Zoo-FISH data. The exception concerned the CLU gene which was mapped by synteny to ECA2 while human-horse Zoo-FISH data predicted that it would be located on ECA9. The level of SNPs on the horse Y chromosome was also investigated by DNA sequencing and denaturing high performance liquid chromatography (DHPLC) of Y chromosome-specific fragments derived mainly from BAC clone subcloning. The amount of genetic variability was found to be very low, consistent with low male effective population size.

La qualité du fruit est un critère incontournable de sélection chez les Rosacées fruitières, et l’acidité constitue une composante majeure de la qualité organoleptique. Toutefois, les mécanismes physiologiques et moléculaires contrôlant l'acidité des fruits restent mal connus. Chez le pêcher, le caractère non acide du fruit est contrôlé par le locus D. Une descendance F2 de 208 individus issus d'un croisement entre une variété de pêche non acide ‘Ferjalou Jalousia®’ et une variété de nectarine normalement acide, ‘Fantasia’ (JxF) a été analysée pour différents caractères agronomiques dont l’acidité du fruit. Cette descendance a servi à la réalisation d’une carte génétique et ainsi à la localisation du locus D sur le groupe de liaison 5 (GL5). Ce locus co-localise avec des QTL à effet majeur impliqués dans l’acidité titrable, le pH, la teneur en acides organiques ainsi que des QTL à effet plus faible pour la teneur en sucres solubles. De nombreux gènes candidats impliqués dans la synthèse des acides organiques, la dégradation et le stockage vacuolaire avaient été précédemment étudiés. Cependant, aucun gène candidat n’a encore été cartographié dans la région du locus D, excluant ainsi leur rôle direct dans le contrôle de l’acidité du fruit. Ceci s’explique par la complexité des voies métaboliques des acides organiques et par l’implication de transporteurs, de canaux et de pompes à protons qui rendent l'identification du ou des gène(s) associés au locus D plus complexe par une approche gène candidat. Par conséquent, une approche de clonage positionnel a été menée dans la présente étude afin d’identifier le ou les gène(s) intervenant dans le contrôle de l’acidité du fruit chez le pêcher dans le but de comprendre les mécanismes moléculaires et physiologiques sous-jacents. La recherche de marqueurs liés au locus D a été réalisée par BSA-AFLP. Trente quatre marqueurs AFLP ont été cartographiés sur le GL5 et les six marqueurs les plus proches ont été convertis en marqueurs SCAR codominants. Une carte génétique fine de la région contenant le locus D a ensuite été réalisée à partir d’une descendance F2 élargie à 1 718 individus et à l’aide des six marqueurs SCAR et de trois marqueurs microsatellites précédemment cartographiés dans cette région. L’ensemble des données de génotypage et de phénotypage des individus ayant subi un événement de recombinaison dans la région du locus d’intérêt, a permis la localisation précise du locus D dans un intervalle de 0,4 cM. En parallèle, une banque BAC d’une couverture estimée à 15 fois la taille du génome haploïde du pêcher a été réalisée et son criblage a permis d’évaluer le rapport distance physique/distance génétique dans cette région à 250 kb/cM. Après deux étapes de marche sur le chromosome, une carte physique de la région a été construite en intégrant 16 marqueurs issus du séquençage d’extrémités de BAC. Un clone BAC de 98 kb contenant l’allèle D et un autre de 78 kb contenant l’allèle d ont été séquencés. L’annotation des séquences des deux allèles a mis en évidence onze gènes candidats. De nouveaux marqueurs développés à partir des séquences de ces deux BAC ont ensuite permis de préciser la localisation du locus d’intérêt dans un intervalle de 16 kb. Dans cette région deux gènes ont été identifiés : un gène de résistance et un gène codant pour un transporteur. Une approche transcriptionnelle a été initiée en complément du clonage positionnel afin de fournir un premier élément pouvant confirmer l’implication d’un ou plusieurs gène(s) candidat(s) positionnel(s) dans l’acidité du fruit chez le pêcher. / Acidity is an essential component of the organoleptic quality of fleshy fruits. However, the physiological and molecular mechanisms that control fruit acidity remain unclear. In peach low-acidity is determined at the D locus by the dominant allele. A peach progeny of 208 F2 individuals obtained from a cross between ‘Ferjalou Jalousia®’ (a low-acid peach) and ‘Fantasia’ (a normally acid nectarine) varieties (JxF) was analyzed for several agronomical traits. This peach F2 progeny segregating for several mendelian traits, was analyzed for fruit quality traits including fruit acidity and used for the construction of a genetic linkage map. The D locus was mapped to the proximal end of linkage group 5 (LG5) and co-localized with major QTLs involved in the control of fruit pH, titratable acidity and organic acid concentration and minor QTLs for sugar concentration. Several candidate genes involved in organic acids synthesis, degradation or vacuolar storage have previously been studied. However, none of these candidate genes were located on in the region of the D locus, excluding their direct role in the control of fruit acidity by the D locus. The complexity of organic acids metabolic pathways as well as the involvement of transporters and channels and related proton pumps has hampered, so far, the identification of the gene(s) associated to the D locus using a candidate gene approach. Thus, in order to investigate the molecular and physiological bases of fruit acidity in peach, a positional cloning strategy of the D locus was undertaken for the isolation of the gene(s) underlying this trait. Using a BSA-AFLP method, 34 AFLP markers were mapped to the LG5, and the six nearest markers were transformed into codominant SCAR markers. These SCAR markers and three previously mapped SSR markers were used to genotype an F2 segregating progeny extended to 1,718 F2 individuals. A high-resolution map of the D locus was realized after genotyping and phenotyping recombinant individuals. Using these recombinant plants we delimited the D locus to a genetic interval of 0.4 cM. We also constructed a peach BAC library with a covering estimated at 15 x the peach haploid genome. The screening of the BAC library with tightly linked markers indicated that 1 cM corresponds to 250 kb at the vicinity of the D locus and allowed the construction of the physical map in two walks integrating 16 markers obtained from the BACends sequences. Two BAC clones harbouring the D locus were identified and sequenced; one BAC clone of 98 kb containing the D dominant allele and another one of 78 kb containing the d recessive allele. Eleven predicted genes were found in the sequenced region. A new set of markers was developed which allowed the localization of the D locus in a 16 kb interval. In this region, two genes were identified: a resistance gene and a gene encoding for a transporter. A transcriptional approach was initiated in addition to the positional cloning strategy to provide a first element which could confirm the involvement of one or more identified positional candidate gene(s) in the control of peach fruit acidity.