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.

O Estado do Cearà vem se destacando na Ãrea de produÃÃo de flores e plantas ornamentais nos Ãltimos anos e tem apresentado crescimento em vÃrios aspectos, influenciando, dessa maneira, no desenvolvimento da economia cearense. No entanto, apesar da grande importÃncia econÃmica das rosas no Cearà como tambÃm em outras regiÃes do Brasil, hà carÃncia de pesquisas sobre essas flores no paÃs. As informaÃÃes sÃo escassas quanto, por exemplo, Ãs exigÃncias nutricionais de roseiras nas condiÃÃes de produÃÃo no Brasil, bem como sobre as possÃveis associaÃÃes benÃficas estabelecidas com microrganismos edÃficos. Objetiva-se neste trabalho avaliar o efeito da colonizaÃÃo micorrÃzica arbuscular sobre o desenvolvimento de minirosa em um Neossolo QuartzarÃnico do municÃpio de EusÃbio no Estado do CearÃ. O experimento foi conduzido, inicialmente, em estufa e teve continuidade em condiÃÃes de campo apÃs o transplantio. O trabalho foi realizado na empresa Exotic Paisagismo, no municÃpio de EusÃbio-Ce. O solo utilizado foi um Neossolo QuartzarÃnico o qual foi coletado para anÃlises quÃmicas e microbiolÃgicas. As estacas de minirosa, variedade branca, foram plantadas em bandejas contendo solo estÃril ou natural onde foram mantidas por um perÃodo de 30 dias para o enraizamento. Estas estacas tambÃm receberam Ãgua de lagoa estÃril ou natural durante o perÃodo de formaÃÃo de raÃzes. A fase de enraizamento ocorreu em estufa. ApÃs este perÃodo, as mudas foram transplantadas para vasos plÃsticos contendo somente solo natural. Estas plantas, apÃs transplantio, passaram a receber Ãgua da lagoa apenas natural e dois nÃveis de fÃsforo. O experimento foi destrutivo, com duas Ãpocas de coleta, aos 60 (coleta 1) e 90 (coleta 2) DAT, onde foram retiradas trÃs repetiÃÃes de cada tratamento para a realizaÃÃo de anÃlises. O experimento obedeceu a um delineamento experimental inteiramente casualizado, em esquema fatorial 2 ( dois nÃveis de fÃsforo) x 2 (enraizamento em solo estÃril ou natural) x 2 (irrigaÃÃo com Ãgua da lagoa natural ou Ãgua da lagoa estÃril), com 3 repetiÃÃes. Os parÃmetros a serem avaliados foram: massa da matÃria seca da parte aÃrea MSPA, altura da planta, diÃmetro do caule, nÃmero de rosas, determinaÃÃo de P da parte aÃrea, colonizaÃÃo micorrÃzica arbuscular, densidade de esporos e diversidade de FMA no solo e respiraÃÃo basal do solo. Na coleta 2, nas plantas que receberam o nÃvel subÃtimo de P (P2), a colonizaÃÃo precoce aumentou de forma significativa a produÃÃo de MSPA de plantas enraizadas em solo natural em relaÃÃo Ãs plantas enraizadas em solo estÃril. Os FMA podem ter sido estimulados pelo menor suprimento de fÃsforo. Na coleta 2, o aumento da altura das plantas, influenciado pelas condiÃÃes de enraizamento, pode ter sido promovido pelo enraizamento em solo natural, ou seja, pela presenÃa de FMA durante o perÃodo de formaÃÃo de raÃzes. Na coleta 1, as plantas que receberam os tratamentos T5 e T6 foram as Ãnicas parcelas a nÃo apresentarem rosas aos 60 DAT, sendo que, ambos os tratamentos foram compostos pelo fator enraizamento em solo estÃril, ou seja, na ausÃncia de FMA. Na coleta 2, as plantas que receberam os tratamentos T3 e T4 e que, portanto, foram enraizadas em solo natural (prÃ-colonizadas) apresentaram o maior nÃmero de rosas no momento da coleta e, subseqÃente, contagem das rosas. Em relaÃÃo a colonizaÃÃo micorrÃzica, tanto na coleta 1 quanto na coleta 2 o fÃsforo foi, estatÃsticamente, o Ãnico fator a influenciar nos resultados. O enraizamento em solo natural promoveu um maior desenvolvimento da minirosa no solo com menos P disponÃvel. As estacas de minirosas quando enraizadas em solo apresentaram um menor nÃmero de estacas perdidas, maior sobrevivÃncia ao transplantio e melhor crescimento e vigor do que as plantas cultivadas, comercialmente, em pà de coco e casca de arroz carbonizada. / The state of Cearà has been highlighted in the production of flowers and ornamental plants in recent years and has been growing in many ways influencing in the developing the economy of CearÃ. However, despite the great economic importance of roses in Cearà as well as in other regions of Brazil, there is a lack of research on these flowers in the country. Information is scarce as, for example, the nutritional requirements of roses under the conditions of production in Brazil, as well as the possible beneficial associations established with edaphic microorganisms. The objective of this work was to evaluate the effect of arbuscular mycorrhizal colonization on the development of in a Neosoil Quartzarenic in the EusÃbio county in Cearà state. The experiment was initially conducted under greenhouse conditions and was continued in field conditions after transplanting. The study was conducted at Exotic Landscaping company in the Eusebio county. The soil collected was used for chemical and microbiological analysis. The cuttings rose miniature, white variety, were planted in trays containing sterile soil or natural soil, where they were kept for a period of 30 days for rooting. These cuttings also received sterile water or natural water during the formation of roots. The rooting phase was conducted in greenhouse conditions. After this period, the seedlings were transplanted to plastic pots containing only natural soil. These plants after transplanting began to receive only natural lake water and two different levels of phosphorus. The experiment was destructive, with two samplings periods, to 60 and 90 days after transplanting, and three replicates for each treatment. A factorial completely randomized design 2 (two phosphorus levels) x 2 (roots in sterile soil or natural) x 2 (irrigation with natural pond water or sterile pond water) with four replicates was adopted. The parameters evaluated were: shoot dry matter, plant height, diameter of stalk, number of roses, determination of P in the plant, arbuscular mycorrhizal colonization, spore density and diversity of AMF species and basal soil respiration. To 90 days after transplanting, the plants that received suboptimal levels of P (P2), the early colonization significantly increased the production of shoot dry matter of plants rooted in natural soil when compared to plants rooted in sterile soil. The FMA may have been stimulated by the low supply of phosphorus. To 90 days after transplanting, increased plant height, influenced by the rooting conditions, may have been promoted by the roots in natural soil, or by the presence of AMF during the formation of roots. To 60 days after transplanting, the plants that received the treatments T5 and T6 were the only plots that there were no roses at the this period both treatments were made up by a factor of rooting in sterile soil, eg. in the absence of AMF. To 90 days after transplanting period, the plants that received the treatments T3 and T4 and therefore were rooted in natural soil (pre-colonized) had the largest number of roses at the time of sampling and subsequent counting of roses. Related to mycorrhizal colonization, in both periods, the P factor was, statistically, the only factor to influence the results. Rooting in natural soil promoted the further development of the rose miniature in soil with less P available. The cuttings of rose miniature when rooted in soil had less lost cuttings, increased survival to transplanting and better growth and vigor when compared to plants grown commercially in powder coconut and rice shell.

Les roses à fleurs doubles attirent sélectionneurs et scientifiques depuis de nombreux siècles. L’analyse des taux de ségrégation et cartes génétiques indique que le passage de la fleur simple à la fleur double est dû à une seule mutation dominante située sur le chromosome 3. Cette mutation conduit à une conversion homéotique d’une partie des étamines en pétales, soulignant la possibilité que cette mutation impacte certains gènes du modèle ABC. Il y a quelques années, notre équipe a démontré que l’augmentation du nombre de pétales chez le rosier était corrélée à une restriction de l’expression de RcAGAMOUS (RcAG) vers le centre du méristème floral. Cependant, RcAG étant porté par le chromosome 5, il ne peut être le déterminant génétique de la fleur double. Il a donc été supposé que la mutation en cause se trouvait dans un gène intervenant en amont de RcAG.Récemment, nous avons séquencé, assemblé et publié le génome de Rosa chinensis cv ‘Old Blush’ un ancêtre des rosiers modernes qui produit déjà des fleurs doubles. L’assemblage, de très bonne qualité, nous a aidé à reconstruire la séquence des deux haplotypes de l'intervalle contenant la mutation liée à la fleur double. Nous avons identifié, parmi les 631 gènes de cet intervalle, un gène APETALA2-LIKE (RcAP2L) comme candidat plus que prometteur. En effet, il a été découvert que ce gène existait sous la forme de deux allèles, l’un d’entre eux contenant un grand élément transposable, donnant lieu à un allèle tronqué résistant à l’inhibition par miR172, appelé RcAP2LΔ172. Sachant que la surexpression d’un variant résistant au miR172 entraîne souvent la formation de pétales supplémentaires chez A. thaliana, j’ai démontré que la présence de ce variant corrèle avec le phénotype « fleur double » chez les rosiers d’origine chinoise. Enfin, alors qu’AP2 est capable d’inhiber l’expression d’AG en se liant directement à ses séquences régulatrices chez A. thaliana, j’ai confirmé la capacité des protéines codées par les deux allèles de RcAP2L à lier les séquences régulatrices de RcAG, in vitro. À partir de ces résultats, je propose donc un modèle pouvant expliquer la formation de fleurs doubles chez les rosiers chinois et peut-être d’autres Rosaceae, dans lesquelles la protéine RcAP2LΔ172 peut s’accumuler du fait de sa résistance au miR172 et restreindre davantage l’expression de RcAG au centre du méristème floral. Ainsi, la frontière entre les domaines A et C se trouve elle aussi déplacée vers le centre du méristème, ce qui induit la conversion des étamines en pétales. / Roses exhibiting double flowers have intrigued both breeders and scientists for decades. Based on segregation ratios and genetic maps, it is known that the switch from simple to double flower is due a single dominant locus on chromosome 3. When present in its mutated form, this locus leads to a homeotic conversion of stamens into petals, suggesting a mechanism involving the ABC genes. A few years ago, our team demonstrated that the increase in petal number correlates with a restriction of RCAGAMOUS (RcAG) expression domain towards the center of the floral meristem. However, as RcAG is located on chromosome 5, the causative mutation was assumed to act as a regulator of this gene. Recently, we sequenced, assembled and published the double-flowered Rosa chinensis cv ‘Old Blush’ genome sequence with a high-quality assembly that helped us to reconstruct the sequence of the two haplotypes of the interval containing the double flower mutation. Among the 631 genes from this interval, we identified here an APETALA2-LIKE (RcAP2L) gene as a strong candidate. Indeed, this gene was found to exist as two alleles, with one containing a large transposable element resulting in a truncated, miR172-resistant, variant named RcAP2LΔ172. Knowing that the overexpression of a miR172-resistant variant of AP2 leads to the formation of extra petals (and sometimes stamens) in Arabidopsis, we investigated the presence of this variant in simple and double flower varieties. The presence of RcAP2LΔ172 was found to correlate with the double flower phenotype in Chinese roses and was not observed in any of the simple-flowered roses studied. Finally, as AP2 is able to inhibit AG expression by directly binding to its regulatory sequences in A. thaliana, I confirmed that both RcAP2L proteins are also able to recognize RcAG regulatory sequences in vitro. A working model is thus proposed for double flower formation in rose, that could be valid for other Rosaceae, whereby RcAP2LΔ172 protein may accumulate due to its resistance to miR172 and consequently may repress more RcAG towards the center of the floral meristem, leading to the sliding of the A/C border and thus the conversion of stamens into petals.

BEZERRA, M. E. J. Estudo da colonização micorrízica arbuscular no desenvolvimento de minirosa em um Neossolo Quartzarênico do município de Eusébio - CE. 2011. 46 f. Dissertação (Mestrado em Agronomia/Solos e Nutrição de Plantas) - Centro de Ciências Agrárias, Universidade Federal do Ceará, Fortaleza, 2011. / Submitted by Francisco Lacerda (lacerda@ufc.br) on 2014-09-16T22:37:38Z No. of bitstreams: 1 2011_dis_mejbezerra.pdf: 651481 bytes, checksum: 65790caefa4c6c14d290eab82d44af9c (MD5) / Approved for entry into archive by José Jairo Viana de Sousa(jairo@ufc.br) on 2014-09-23T21:37:21Z (GMT) No. of bitstreams: 1 2011_dis_mejbezerra.pdf: 651481 bytes, checksum: 65790caefa4c6c14d290eab82d44af9c (MD5) / Made available in DSpace on 2014-09-23T21:37:21Z (GMT). No. of bitstreams: 1 2011_dis_mejbezerra.pdf: 651481 bytes, checksum: 65790caefa4c6c14d290eab82d44af9c (MD5) Previous issue date: 2011 / The state of Ceará has been highlighted in the production of flowers and ornamental plants in recent years and has been growing in many ways influencing in the developing the economy of Ceará. However, despite the great economic importance of roses in Ceará as well as in other regions of Brazil, there is a lack of research on these flowers in the country. Information is scarce as, for example, the nutritional requirements of roses under the conditions of production in Brazil, as well as the possible beneficial associations established with edaphic microorganisms. The objective of this work was to evaluate the effect of arbuscular mycorrhizal colonization on the development of in a Neosoil Quartzarenic in the Eusébio county in Ceará state. The experiment was initially conducted under greenhouse conditions and was continued in field conditions after transplanting. The study was conducted at Exotic Landscaping company in the Eusebio county. The soil collected was used for chemical and microbiological analysis. The cuttings rose miniature, white variety, were planted in trays containing sterile soil or natural soil, where they were kept for a period of 30 days for rooting. These cuttings also received sterile water or natural water during the formation of roots. The rooting phase was conducted in greenhouse conditions. After this period, the seedlings were transplanted to plastic pots containing only natural soil. These plants after transplanting began to receive only natural lake water and two different levels of phosphorus. The experiment was destructive, with two samplings periods, to 60 and 90 days after transplanting, and three replicates for each treatment. A factorial completely randomized design 2 (two phosphorus levels) x 2 (roots in sterile soil or natural) x 2 (irrigation with natural pond water or sterile pond water) with four replicates was adopted. The parameters evaluated were: shoot dry matter, plant height, diameter of stalk, number of roses, determination of P in the plant, arbuscular mycorrhizal colonization, spore density and diversity of AMF species and basal soil respiration. To 90 days after transplanting, the plants that received suboptimal levels of P (P2), the early colonization significantly increased the production of shoot dry matter of plants rooted in natural soil when compared to plants rooted in sterile soil. The FMA may have been stimulated by the low supply of phosphorus. To 90 days after transplanting, increased plant height, influenced by the rooting conditions, may have been promoted by the roots in natural soil, or by the presence of AMF during the formation of roots. To 60 days after transplanting, the plants that received the treatments T5 and T6 were the only plots that there were no roses at the this period both treatments were made up by a factor of rooting in sterile soil, eg. in the absence of AMF. To 90 days after transplanting period, the plants that received the treatments T3 and T4 and therefore were rooted in natural soil (pre-colonized) had the largest number of roses at the time of sampling and subsequent counting of roses. Related to mycorrhizal colonization, in both periods, the P factor was, statistically, the only factor to influence the results. Rooting in natural soil promoted the further development of the rose miniature in soil with less P available. The cuttings of rose miniature when rooted in soil had less lost cuttings, increased survival to transplanting and better growth and vigor when compared to plants grown commercially in powder coconut and rice shell. / O Estado do Ceará vem se destacando na área de produção de flores e plantas ornamentais nos últimos anos e tem apresentado crescimento em vários aspectos, influenciando, dessa maneira, no desenvolvimento da economia cearense. No entanto, apesar da grande importância econômica das rosas no Ceará como também em outras regiões do Brasil, há carência de pesquisas sobre essas flores no país. As informações são escassas quanto, por exemplo, às exigências nutricionais de roseiras nas condições de produção no Brasil, bem como sobre as possíveis associações benéficas estabelecidas com microrganismos edáficos. Objetiva-se neste trabalho avaliar o efeito da colonização micorrízica arbuscular sobre o desenvolvimento de minirosa em um Neossolo Quartzarênico do município de Eusébio no Estado do Ceará. O experimento foi conduzido, inicialmente, em estufa e teve continuidade em condições de campo após o transplantio. O trabalho foi realizado na empresa Exotic Paisagismo, no município de Eusébio-Ce. O solo utilizado foi um Neossolo Quartzarênico o qual foi coletado para análises químicas e microbiológicas. As estacas de minirosa, variedade branca, foram plantadas em bandejas contendo solo estéril ou natural onde foram mantidas por um período de 30 dias para o enraizamento. Estas estacas também receberam água de lagoa estéril ou natural durante o período de formação de raízes. A fase de enraizamento ocorreu em estufa. Após este período, as mudas foram transplantadas para vasos plásticos contendo somente solo natural. Estas plantas, após transplantio, passaram a receber água da lagoa apenas natural e dois níveis de fósforo. O experimento foi destrutivo, com duas épocas de coleta, aos 60 (coleta 1) e 90 (coleta 2) DAT, onde foram retiradas três repetições de cada tratamento para a realização de análises. O experimento obedeceu a um delineamento experimental inteiramente casualizado, em esquema fatorial 2 ( dois níveis de fósforo) x 2 (enraizamento em solo estéril ou natural) x 2 (irrigação com água da lagoa natural ou água da lagoa estéril), com 3 repetições. Os parâmetros a serem avaliados foram: massa da matéria seca da parte aérea MSPA, altura da planta, diâmetro do caule, número de rosas, determinação de P da parte aérea, colonização micorrízica arbuscular, densidade de esporos e diversidade de FMA no solo e respiração basal do solo. Na coleta 2, nas plantas que receberam o nível subótimo de P (P2), a colonização precoce aumentou de forma significativa a produção de MSPA de plantas enraizadas em solo natural em relação às plantas enraizadas em solo estéril. Os FMA podem ter sido estimulados pelo menor suprimento de fósforo. Na coleta 2, o aumento da altura das plantas, influenciado pelas condições de enraizamento, pode ter sido promovido pelo enraizamento em solo natural, ou seja, pela presença de FMA durante o período de formação de raízes. Na coleta 1, as plantas que receberam os tratamentos T5 e T6 foram as únicas parcelas a não apresentarem rosas aos 60 DAT, sendo que, ambos os tratamentos foram compostos pelo fator enraizamento em solo estéril, ou seja, na ausência de FMA. Na coleta 2, as plantas que receberam os tratamentos T3 e T4 e que, portanto, foram enraizadas em solo natural (pré-colonizadas) apresentaram o maior número de rosas no momento da coleta e, subseqüente, contagem das rosas. Em relação a colonização micorrízica, tanto na coleta 1 quanto na coleta 2 o fósforo foi, estatísticamente, o único fator a influenciar nos resultados. O enraizamento em solo natural promoveu um maior desenvolvimento da minirosa no solo com menos P disponível. As estacas de minirosas quando enraizadas em solo apresentaram um menor número de estacas perdidas, maior sobrevivência ao transplantio e melhor crescimento e vigor do que as plantas cultivadas, comercialmente, em pó de coco e casca de arroz carbonizada.