Citrus is the main fruit crop in the world and Spain is the 6th producer and the major exporter for the fresh fruit market. Seedlessness is one of the most important fruit quality traits for this market since consumers do not accept seedy fruits. Recovery of triploid hybrids has become an important breeding strategy to develop new seedless citrus varieties and several of them have been already released from citrus breeding programs worldwide. Despite the undisputable importance of polyploidy in plant species, their genetics are much less well known than those of their diploid counterparts. Citrus triploid hybrids can be routinely recovered from sexual polyploidization (2x ¿ 2x) or interploid crosses (2x ¿ 4x and 4x ¿ 2x). In 2x ¿ 2x sexual crosses, spontaneous triploid hybrids arise from the union of an unreduced (2n) megagametophyte with haploid pollen. In the case of interploid sexual crosses (2x ¿ 4x and 4x ¿ 2x), triploid hybrids result from the fecundation of a diploid gamete arising from the tetraploid parent and a haploid gamete arising from the diploid parent. The genetic and phenotypic structures of triploid populations greatly depend on the parental heterozygosity restitution (HR) in the diploid gamete at each locus, which is mainly affected by the triploid recovery strategy. In 2x ¿ 2x crosses, HR depends on the underlying mechanism leading to the unreduced gamete formation, which are genetically equivalent to First Division Restitution (FDR) or Second Division Restitution (SDR) mechanisms. Moreover, under each restitution mechanism, HR also depends on the locus-centromere genetic distance. In the case of interploid crosses, parental heterozygosity restitution from tetraploid parents depends on the double reduction frequency. In citrus, the unreduced gamete formation mechanism is still controversial; FDR has been the mechanism proposed for sweet orange, whereas SDR has been proposed for clementine. On the other hand, inferring the allelic configurations of genetic markers is a main challenge in polyploidy crops to infer genotypic and gametic structures with the objective to analyze meiosis and inheritance mechanisms. According to this scientific context, the objectives of the thesis where: (i) to develop a new approach for allele dosis assignation when using co-dominant markers, (ii) to implement and apply methods for the analysis of 2n gametes origin and locate centromeres, and (iii) to take advantage of this knowledge to locate a major gene of resistance to Alternaria Brown Spot (ABS) which is a major constraint for triploid mandarin breeding. For microsatellite (SSR) markers, we have demonstrated that triploid progeny genotyping can be successfully performed using the microsatellite allele-counting peak ratio (MAC-PR) method. However, SSR analysis remains relatively costly and time consuming compared with actual SNP genotyping methods. Moreover, with the increasing availability of EST databases and whole genome sequences, SNPs have become the most abundant and powerful polymorphic markers that can be selected along the entire genome. In this thesis, a new method based on competitive allele-specific PCR has been developed to assign SNP allele dosage in an accurate, simple, and cost effective way. Combining the MAC-PR and the new developed SNP genotyping methods offers the possibility to utilize a broad range of molecular markers in genotyping triploid genotypes. Both methods have been used in further works included in this thesis. SDR has been demonstrated as the mechanism underlying unreduced gamete production in `Fortune¿ mandarin by genotyping triploid progenies with SSR markers. In addition, a new method to locate the centromere, based on the best fit between observed heterozygosity restitution within a linkage group and theoretical functions under either partial or no chiasma interference hypotheses has been developed and successfully applied. To expand the knowledge of the mechanism underlying unreduced gamete formation to other citrus genotypes besides clementines and `Fortune¿ mandarin, a maximum likelihood method based on parental heterozygosity restitution of centromeric loci was developed and successfully applied in sixteen mandarin cultivars. The new method developed in the study allows inferring the restitution mechanism both at population level and even at individual level. Maternal origin of 2n gametes was confirmed for all triploid hybrids and SDR was proposed as the restitution mechanism for all analyzed progenies. The information acquired from the mode of heterozygosity restitution in citrus was useful to determine the genetic and phenotypic structures of new triploid populations arising from different breeding strategies. We studied these structures for the resistance to Alternaria brown spot (ABS), a serious fungal disease producing necrotic lesions on fruits and young leaves in susceptible citrus genotypes. In the present work, different approaches were combined taking advantage of the particular genetic structures of 2n gametes resulting from SDR to map a genome region linked to ABS resistance in triploid citrus progeny. The monolocus dominant inheritance of the susceptibility, proposed on the basis of diploid population studies, was corroborated in triploid progeny. A 3.3 Mb genomic region linked to ABS resistance was located near the centromere on chromosome III, which includes clusters of resistance genes. SSR and SNP markers were developed for an efficient early selection of ABS resistant hybrids and they are currently used in our breeding program to perform marker assisted selection. The knowledge obtained in this thesis on the mechanism of sexual polyploidization and inheritance of concrete traits in citrus will allow implementing much more efficient triploid breeding programs on the basis of current and future needs. Indeed, applied outcomes of this PhD are already routinely used in the IVIA triploid breeding program. / Cuenca Ibáñez, J. (2013). Mechanisms of sexual polyploidization and inheritance in triploid citrus populations [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/34671 / Premios Extraordinarios de tesis doctorales

Tesis por compendio / This thesis aimed to study three main aspects: (i) the mechanisms underlying unreduced pollen gamete formation in the diploid `CSO¿ tangor hybrid used as male parent in 4x x 2x triploid breeding programs, (ii) the frequencies and mechanisms involved in the unreduced 2n female gametes production for `Eureka Frost¿ and `Fino¿ lemon genotypes, and (iii) the interspecific recombination and the resulting diploid gamete structures of doubled-diploid `Mexican¿ lime to evaluate the possibility that natural interploid hybridization maybe the origin of C. latifolia (`Tahiti¿ lime type) and C. aurantifolia (`Tanepao¿ lime type) triploid varieties. The production of 54 tetraploid hybrids from 4x x 2x sexual hybridizations allowed the analysis of the mechanisms underlying unreduced pollen gamete formation. SSR and SNP molecular markers revealed that the majority of these plants were obtained from 2n pollen of the diploid tangor parent. Then, the maximum-likelihood method based on parental heterozygosity restitution (PHR) of centromeric loci revealed that both FDR (First Division Restitution ) with predominant occurrence and to a lower extend SDR (Second Division Restitution) were the mechanisms leading to unreduced male gamete formation in the tangor studied. These observations were confirmed by the analysis of PHR pattern along the linkage group (LG) 2. To our knowledge, this is the first report of tetraploid citrus progenies arising from unreduced pollen and the first description of the coexistence of two meiotic restitution mechanisms (SDR and FDR) producing unreduced pollen in citrus. In order to study the frequencies and the mechanisms involved in the unreduced 2n female gametes production in two different genotypes of lemon, we produced 43 triploid and tetraploid hybrids from 2x x 2x and 2x x 4x sexual hybridizations using `Eureka Frost¿ and `Fino¿ as female parents. The frequencies of 2n gamete production were respectively 4.9% and 8.3%. The maximum-likelihood analysis and pattern of PHR along LG1 revealed that SDR is the main mechanism of unreduced female lemon gametes (88%), followed by FDR or pre-meiotic doubling (PRD) (7%) and post-meiotic genome doubling (PMD) mechanisms (5%). This is the first report of the production of a large number of lemon progenies from 2n gametes and the first identification of a new mechanism, PMD that has never been observed in citrus and has rarely been described in other herbaceous or woody species. Across both studies, we demonstrated at the methodological level the effectiveness of using two complementary approaches, the analysis of the PHR pattern in one LG and the maximum-likelihood method based on centromeric loci for distinguishing between the different mechanisms of unreduced gamete production. We analyzed the meiotic mechanisms of a doubled diploid `Mexican¿ lime, the interspecific recombination and the resulting diploid gamete structures combining a segregation analysis of SSR and SNPs markers, a cytogenetic study and pollen viability evaluation. We concluded that the doubled-diploid `Mexican¿ lime had a predominantly disomic segregation for three LGs, intermediate inheritance with disomic tendency was found for five LGs and intermediate models for one LG. The resulting interspecific diploid gamete structures displayed high C. medica / C. micrantha heterozygosity. The revealed genetic structures of the diploid gametes produced by the doubled-diploid `Mexican¿ lime are compatible with the hypothesis that `Tahiti¿ and `Tanepao¿ triploid varieties results from interploid hybridization involving a doubled-diploid `Mexican¿ like lime. This disomic tendency limits the recombination and the diversity of the diploid gamete population; however the observed pollen viability restoration at tetraploid level could be advantageous for intensive breeding projects. / Esta tesis estudia tres aspectos principales: (i) los mecanismos de la formación de gametos 2n de polen originados por un híbrido diploide (tangor `CSO¿) utilizado como parental masculino en hibridaciones sexuales 4x x 2x, (ii) las frecuencias y los mecanismos de la producción de gametos 2n femeninos en dos genotipos de limón, `Eureka Frost¿ y `Fino¿, y (iii) el análisis de la recombinación interespecífica y las estructuras de los gametos diploides originados por la lima `Mejicana¿ doble diploide para evaluar el origen de las las variedades triploides de lima C. latifolia (`Tahiti¿) y C. aurantifolia (`Tanepao¿). La obtención de 54 híbridos tetraploides a partir de hibridaciones 4x x 2x permitió analizar los mecanismos de la formación de gametos 2n de polen. El análisis molecular reveló que la mayoría de estas plantas se obtuvieron a partir de gametos 2n de polen del parental `CSO¿. A continuación, el análisis mediante el método de máxima verosimilitud indicó que FDR (Restitución de la Primera División meiótica) y SDR (Restitución de la Segunda División meiótica) son los mecanismos implicados con una mayor dominancia de FDR respecto SDR. Estos resultados se confirmaron mediante el análisis de la restitución de la heterocigosidad en el grupo de ligamiento (LG) 2. Con los datos publicados hasta la fecha, es la primera vez que se han obtenido progenies tetraploides de cítricos mediante gametos 2n de polen y es la primera descripción en cítricos de la coexistencia de dos mecanismos SDR y FDR. Para estudiar las frecuencias y los mecanismos de la producción de gametos 2n en dos genotipos de limón, se obtuvieron 43 híbridos triploides y tetraploides a partir de hibridaciones sexuales 2x x 2x y 2x x 4x utilizando los limones `Eureka Frost¿ y `Fino¿ como parentales femeninos. Las frecuencias de producción de gametos 2n fueron respectivamente 4,9% y 8,3%. El análisis de máxima verosimilitud y el patrón de PHR a lo largo del LG1 reveló que SDR es el mecanismo principal de formación de gametos 2n femeninos (88%), seguido por FDR o duplicación del genoma pre-meiosis (PRD) (7%) y se identificó un nuevo mecanismo originado a partir de la duplicación del genoma post-meiosis (PMD) (5%). En este trabajo se describe por primera vez en cítricos la producción de un elevado número de híbridos de limón a partir de gametos 2n y es la primera vez que se identifica un nuevo mecanismo PMD que nunca se ha observado en cítricos. En ambos estudios se demostró la efectividad del uso de dos métodos complementarios, el análisis del patrón de PHR a lo largo de un LG y el método de máxima verosimilitud para distinguir entre los diferentes mecanismos implicados en la formación de gametos 2n. También se ha analizado el modelo de segregación cromosómica de la lima `Mejicana¿ doble diploide (DD) así como la recombinación interespecífica y las estructuras de los gametos diploides resultantes. Se ha realizado análisis de la viabilidad del polen junto con un análisis citogenético y con marcadores SSRs y SNPs. Estos trabajos nos han permitido concluir que la lima `Mejicana¿ DD presenta una segregación predominantemente disómica para tres LGs, herencia intermedia con tendencia disómica para cinco LGs y un tipo de segregación intermedia para un LG. Las estructuras de los gametos diploides mostraron una alta heterocigosis en C. medica/C. micrantha, parentales de la lima `Mejicana¿. Las estructuras genéticas de los gametos diploides de la lima `Mejicana¿ DD son compatibles con la hipótesis de que las variedades triploides `Tahiti¿ y `Tanepao¿ se obtuvieran a partir de una hibridación interploide a partir de la lima `Mejicana¿ DD. El tipo de segregación disómico conlleva una limitación de la recombinación y la diversidad genética de la población de gametos 2n. Sin embargo la viabilidad del polen de la lima `Mejicana¿ DD en comparación con la lima `Mejicana¿ diploide permite / Aquesta tesi ha tingut com a objectius l'estudi de (i) els mecanismes subjacents a la formació de pol·len de gàmetes 2n al tangor híbrid diploide `CSO', utilitzat com a progenitor masculí en els programes de millora de triploides 4x x 2x (ii) les freqüències i els mecanismes implicats en la producció gàmetes 2n en la femella als genotips de llimona `Eureka Frost¿ i Fina' (iii) la recombinació interespecífica i les estructures resultants de gàmetes diploides del doble-diploide (DD) de `llima Mèxicana' per avaluar la possibilitat que la hibridació interploid natural potser l'origen de les varietats triploides C. latifolia (llima tipus `Tahiti') i C. aurantifolia (llima tipus `Tanepao'). La producció de 54 híbrids tetraploides obtinguts d¿hibridacions sexuals 4x x 2x va permetre l'anàlisi dels mecanismes de formació de gàmetes 2n de pol·len. Els marcadors moleculars SSR i SNP van revelar que la majoria d'aquestes plantes es van obtenir de pol·len 2n del parental diploide tangor. Llavors, el mètode de màxima probabilitat va revelar que tant FDR (Restitució en la Primera Divisió), amb ocurrència predominant, com SDR (Restitució en la Segona Divisió) van ser els mecanismes que condueixen a la formació de gàmetes masculins 2n en aquest tangor. Aquestes observacions van ser confirmades per l'anàlisi de patró de RHP al llarg del cromosoma 2. Des del nostre coneixement, aquest és el primer estudi de progènies de cítrics tetraploides derivats de pol·len no reduït i la primera descripció de la coexistència de dos mecanismes de restitució meiòtiques (FDR i SDR) produint pol·len no reduït en els cítrics. Per tal d'estudiar les freqüències i els mecanismes implicats en la producció de gàmetes 2n sense reduir de la femella, en dos genotips diferents de llimona, vam obtenir 43 híbrids triploides i tetraploides d¿hibridacions sexuals 2x x 2x i 4x x 2x utilitzant `Eureka Frost ' i `Fino' com progenitors femenins. Les freqüències de la producció de gàmetes 2n van ser, respectivament, 4,9% i 8,3%. L'anàlisi de màxima probabilitat i el patró de RHP al llarg del cromosoma 1 van revelar que SDR és el principal mecanisme de gàmetes 2n en la llimona utilitzada com parent femení (88%), seguit pels mecanismes FDR o duplicació pre-meiòtica (PRD) (7%) i la duplicació del genoma post-meiòtica (PMD) (5%). Per primera volta en els cítrics s¿ha obtingut un gran nombre de progènie de llimona a partir de gàmetes 2n i s¿ha identificat un nou mecanisme, el PMD que poques vegades s'ha descrit en altres espècies herbàcies o llenyoses. A través dels dos treballs, hem demostrat, a nivell metodològic, l'eficàcia d'utilitzar dos enfocaments complementaris, és a dir, l'anàlisi del patró de RHP en un cromosoma amb el mètode de màxima probabilitat basat en loci centromèrics per distingir entre els diferents mecanismes de la producció de gàmetes 2n . Es van analitzar els mecanismes meiòtics d'un DD `llima Mèxicana', la recombinació interespecífica i les estructures resultants de gàmetes diploides combinant una anàlisi de segregació de marcadors SSR i SNP, un estudi citogenètic i l'avaluació de la viabilitat del pol·len. Hem arribat a la conclusió que el DD de `llima Mèxicana' tenia una segregació predominantment disómica en tres cromosomes, herència intermèdia amb tendència disómica en cinc cromosomes i els models intermedis per a un altre. Les estructures resultants de gàmetes diploides interespecífiques mostren alta heterozigositat C. medica / C. micrantha. Les estructures genètiques revelades dels gàmetes diploides produïts pel DD de `llima Mèxicana' són compatibles amb la hipòtesi que les varietats triploides `Tahiti¿ i `Tanepao¿ resulten de la hibridació interploide que impliquen un DD de tipus `llima Mèxicana'. Aquesta tendència disómica limita la recombinació i la diversitat de la població de gàmetes diploides, però, la restauració de la viabilitat / Houssem, R. (2017). Genetic Structure of Diploid Gametes for the Production of Triploid Citrus Hybrids [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90634 / Compendio

Contact zones of cytotypes harbour a unique opportunity to study the dynamics and evolution of mixed-ploidy systems. Butomus umbellatus (flowering rush) is one of a few species in which diploid and triploid cytotype is present in nature. The first known mixed-ploidy populations of B. umbellatus were discovered in south-eastern Slovakia during the previous studies. Common occurrence of the species in an area with substantially natural dynamics of wetland ecosystems represents a unique model system for unbiased comparisons of traits and genetic diversity between diploid and triploid individuals along with their dispersal capabilities on a landscape level. The main aim of of this thesis is to uncover the truth behind evolutionary processes that are taking place in the contact zone of diploid and triplioid cytotype of B. umbellatus in south-eastern Slovakia. Using flow cytometry and molecular methods (microsatellites) we will test whether this is a primary or secondary contact zone and assess the level of genetic variability between individuals and populations of both cytotypes. Using the analysis od microsatellite loci we discovered that the contact zone of cytotype is of both primary and secondary character and gene flow between cytotypes was also detected. These results were supported by high production and...

Submitted by Verena Pereira (verenagoncalves@uefs.br) on 2018-07-03T23:23:34Z No. of bitstreams: 1 TESE 2018 - Doutorado RGV_UEFS - Carla Maria de Jesus Silva.pdf: 1687907 bytes, checksum: 3e9fe14e189c00f951079ca2a71e3c66 (MD5) / Made available in DSpace on 2018-07-03T23:23:34Z (GMT). No. of bitstreams: 1 TESE 2018 - Doutorado RGV_UEFS - Carla Maria de Jesus Silva.pdf: 1687907 bytes, checksum: 3e9fe14e189c00f951079ca2a71e3c66 (MD5) Previous issue date: 2018-03-29 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The watermelon (Citrullus lanatus) is a very important vegetable for a region of Northeast Brazil due to its adaptation as the natural conditions and the good characteristics of the fruit that are obtained. The aims of the current study are to assess watermelon genotype responses to calluses in anthers by using growth and temperature regulators, and to induce polyploidy through colchicine use (at different concentrations), exposure time, mechanical scarification and application methods. Anthers of Smile and Sugar Baby lines were inoculated in MS medium at different concentrations of 2.4-D or of BAP with 2.4-D, in combination with the pre-treatment (low temperature). Crimson Sweet cultivar seedlings were treated with different colchicine concentrations at two different times, with and without scarification, in order to induce polyploidy. Line LDRO was subjected to different colchicine concentrations at two different times and application methods: a) direct in the seed method (with, and without, scarification) (DSM, WE and WOE), b) Radicle emission method (ERM), c) Hypocotyl and root insertion point method (HRIM), d) At the apex of the seedling method (ASM) and e) Inverted hypocotyl method (IHM). Crimson Sweet flower buds were subjected to colchicine, at two different times, in order to induce polyploidy. Results have shown that 2.4-D often induced callus formation in both lines, but BAP/2.4-D interaction and pre-treatment did not increase the induction frequency. Crimson Sweet showed higher induction rate at 0.2% colchicine for 48h, WE. Line LDRO presented plants with tetraploid cells at 0.2% colchicine for 24 and 48h. The method 0.2% DSM, WE and WOE also generated plants with tetraploid cells. The diameter of treated pollen grains in flower buds have increased; the higher rate of non-reduced gametes induction was 16.07% and flower bud diameter (1.5mm) was estimated as adequate for induction / A melancia (Citrullus lanatus) ? uma hortali?a muito importante para a regi?o Nordeste do Brasil por sua adapta??o as condi??es clim?ticas e pelas boas caracter?sticas de fruto que se obt?m. Os objetivos desse trabalho foram avaliar as respostas de gen?tipos de melancia quanto ? indu??o de calos em anteras utilizando reguladores de crescimento e temperatura; e induzir a poliploidia mediante o uso da colchicina em diferentes concentra??es, tempos de exposi??o, escarifica??o mec?nica e m?todos de aplica??o. Anteras das linhagens de Smile e Sugar Baby foram inoculadas em meio MS sob diferentes concentra??es de 2,4-D ou BAP com 2,4-D, associado ao pr?-tratamento (baixa temperatura). Para indu??o de poliploidia, sementes da cultivar Crimson Sweet foram tratadas com diferentes concentra??es de colchicina, em dois tempos, com e sem escarifica??o. Para a linhagem LDRO, utilizaram-se diferentes concentra??es de colchicina em dois tempos e m?todos de aplica??o: a) M?todo direto na semente (com escarifica??o e sem escarifica??o) (MDS, CE e SE), b) M?todo da semente com emiss?o da rad?cula (MER), c) M?todo no ponto de inser??o do hipoc?tilo e raiz (MIHR), d) M?todo no ?pice da pl?ntula (MAP) e e) M?todo do hipoc?tilo invertido (MHI). Na indu??o de poliploidia em bot?es florais de Crimson Sweet utilizou-se colchicina em dois tempos. Os resultados mostraram que o 2,4-D induziu a maior frequ?ncia de calos nas duas linhagens, a intera??o BAP com 2,4-D e o pr?-tratamento, n?o aumentaram a frequ?ncia de indu??o. Para Crimson Sweet, o maior percentual de indu??o foi obtido com colchicina a 0,2% por 48 h, SE. Para LDRO, observou-se uma frequ?ncia de plantas com c?lulas tetraploides com colchicina a 0,2% por 24 h e 48 h. Nos m?todos 0,2% MDS CE e MHI observou-se tamb?m plantas com c?lulas tetraploides. Em bot?es florais, o di?metro dos gr?os de p?len tratados aumentou; o maior percentual de indu??o de gametas n?o reduzidos foi de 16,07% e; o di?metro em torno de 1,5 mm do bot?o floral foi estimado como adequado para indu??o