Dissertations / Theses on the topic 'Breeding tools'

The increasing availability of molecular tools is facilitating marker-assisted selection (MAS) in plant improvement programs. The objectives of this research were to: 1) populate the framework buffelgrass genome map with additional molecular markers, 2) develop polymerase chain reaction (PCR)-based markers from selected, informative restriction fragment length polymorphism (RFLP) markers on the buffelgrass genome map, and 3) increase marker resolution near the locus conferring apomixis (PApo1). Buffelgrass [Pennisetum ciliare (L.) Link syn. Cenchrus ciliaris L.] (2n=4x=36), a highly polymorphic, apomictic, perennial forage grass, is well-suited for genetic linkage analyses. One hundred and seventy one probes from an apomictic, spikelet-specific, complementary deoxyribonucleic acid (cDNA) library and 70 expressed sequence tag simple sequence repeats (EST-SSRs) from apomictic pistil cDNAs were evaluated and added to the framework buffelgrass genome map. The improved linkage map contains 851 markers from 11 grass species and covers approximately 80-85% of the buffelgrass genome. Two RFLPs from the buffelgrass genome map were converted to PCR-based markers for both the identification of hybrids and quantification of sexual versus apomictic reproduction. A gel-free, high-throughput technique was developed to analyze these markers directly in 96-well plates. Five additional markers were placed onto the buffelgrass linkage group with the PApo1 apomixis locus through comparative mapping of candidate orthologs from the sorghum genome map and bulked-segregant analysis of amplified-fragment-lengthpolymorphisms (BSA-AFLP). Increasing the mapping population size did not increase map resolution in the PApo1 region. Association mapping revealed that the recombination suppression near PApo1 is moderate and would complicate comparative map-based cloning efforts of the orthologous region in sorghum.

Doctor of Philosophy<br>Genetics Interdepartmental Program<br>Jesse Poland<br>The Green Revolution is credited with saving billions of lives by effectively harnessing new genetic resources and breeding strategies to create high-yielding varieties for countries lacking adequate food security. To keep the next billion people in a state of food security, plant breeders will need to rapidly incorporate novel approaches and technologies into their breeding programs. The work presented here describes new genomic and phenomic strategies and tools aimed at accelerating genetic gain in plant breeding. Plant breeders have long relied on regional testing networks to evaluate new breeding lines across many locations. These are an attractive resource for both retrospective and contemporary analysis due to the vast amount of data available. To characterize genetic progress of plant breeding programs in the Central Plains, entries from the Southern Regional Performance Nursery dating back to 1992 were evaluated in field trials. The trend for annual improvement was 1.1% yr⁻¹, matching similar reports for genetic gain. During the same time period, growth of on-farm yields stagnated. Genomic selection, a promising method to increase genetic gain, was tested using historical data from the SRPN. A temporal-based model showed that, on average, yield predictions outperformed a year-to-year phenotypic correlation. A program-based model found that the predictability of a breeding program was similar when using either data from a single program or from the entire regional collection. Modern DNA marker platforms either characterize a small number of loci or profile an entire genome. Spiked genotyping-by-sequencing (sGBS) was developed to address the need in breeding programs for both targeted loci and whole-genome selection. sGBS uses a low-cost, integrated approach that combines targeted amplicons with reduced representation genotyping-by-sequencing. This approach was validated using converted and newly-designed markers targeting known polymorphisms in the leaf rust resistance gene Lr34. Plant breeding programs generate vast quantities of data during evaluation and selection of superior genotypes. Many programs still rely on manual, error-prone methods to collect data. To make this process more robust, we have developed several open-source phenotyping apps with simple, intuitive interfaces. A contemporary Green Revolution will rely on integrating many of these innovative technologies into modern breeding programs.

El blat fariner (Triticum aestivum L.) és el principal cultiu arreu del món, però la seva producció es veurà afectada per l’impacte del canvi climàtic, sobretot a la conca Mediterrània. L’objectiu final d’aquesta tesi és oferir coneixement científic i les eines necessàries pel desenvolupament les noves varietats de blat fariner resilients a l’esperat increment de la sequera en les següents dècades com a conseqüència del canvi climàtic. Per aconseguir aquest objectiu, l’existència d’estructures genètiques, fenotípiques i/o geogràfiques en les col•leccions de germoplasma va ser explorada i els QTLs controlant caràcters relacionats amb la resistència terminal a la sequera usant l’anàlisi GWAS van ser identificats. La col•lecció MED6WHEAT del IRTA, amb 170 varietats tradicionals originàries de 24 països mediterranis i 184 varietats modernes cultivades a 19 països de la regió va ser caracteritzada amb més de deu mil marcadors SNP. Un clar patró geogràfic associat a l’estructura genètica es va trobar en les varietats tradicionals, amb 3 subpoblacions (SPs) representant l’oest, el nord i l’est del Mediterrani, mentre que les modernes van ser estructurades d’acord amb els programes de millora genètica de la regió: CIMMYT/ICARDA, França/Itàlia i països balcànics/est-europeus. Les varietats tradicionals van ser utilitzades per investigar l’arquitectura del sistema radicular seminal (RSA) adaptat a les condicions de secà de la regió mediterrània. Les varietats dels països Nord-Mediterranis van mostrar el màxim nombre d’arrels seminals amb un angle radical no significativament diferent al dels de l’oest del Mediterrani, mentre que les varietats tradicionals Est-Mediterrànies van mostrar el mínim nombre d’arrels, major angle radical, les tiges més llargues i el mínim pes de llavor. L’estudi GWAS va detectar MTAs relacionats amb arrels i es van identificar 31 gens candidats vinculats a RSA, mida de la llavor, i tolerància a estrès abiòtic en 15 regions genòmiques. La col•lecció completa va ser avaluada a un assaig a camp de dos anys mitjançant tecnologies de fenotipat d’alt rendiment (HTP) amb l’objectiu de predir caràcters agronòmics. La millor estimació de l’índex d’àrea foliar (LAI) es va assolir amb l’índex MTVI2, i els índexs de vegetació RGB mesurats manualment a camp van mostrar millors prediccions dels caràcters agronòmics. Els resultats basats en un estudi de tres anys consecutius van mostrar diferències significatives pels caràcters agronòmics entre SPs, ressaltant la divisió en varietats tradicionals i varietats modernes. Les SPs modernes van mostrar majors valors de rendiment i dels seus components, de l’índex de collita i de biomassa i una durada de l’ompliment del gra més llarga que les SPs de les varietats tradicionals, que van ser més altes. El major rendiment va ser observat en les varietats modernes de França i Itàlia. El GWAS va identificar 2579 marcadors associats a caràcters agronòmics i relacionats amb els VIs que, finalment, van quedar simplificats en 11 regions genòmiques. L’anàlisi in silico de gens candidats va detectar 12 gens sobre-expressats en condicions d’estrès abiòtic en 6 d’aquestes regions. Cinc d’aquests gens ja havien estat descrits prèviament per estar relacionats amb la tolerància a l’estrès abiòtic. Globalment, aquests resultats van constatar que les varietats tradicionals Mediterrànies són una font important de variabilitat per introduir nous al•lels de caràcters desitjats en programes de millora genètica al voltant de la conca Mediterrània. L’ús de noves tecnologies de teledetecció és una eina eficient i ràpida per la mesura dels caràcters agronòmics. Finalment, el GWAS ha resultat ser un enfoc molt útil per la identificació de regions genòmiques que controlen caràcters importants del germoplasma del blat fariner Mediterrani.<br>El trigo harinero (Triticum aestivum L.) es el principal cultivo en el mundo, pero su producción se verá seriamente afectada por el cambio climático, principalmente en la cuenca Mediterránea. El objetivo final de esta tesis es desarrollar el conocimiento científico y las herramientas necesarias para el desarrollo de las nuevas variedades de trigo harinero resilientes al incremento de la sequía en las próximas décadas como consecuencia del cambio climático. Para ello se ha explorado la existencia de las estructuras genética y fenotípica, así como geográfica en la colección de germoplasma utilizada y se han identificado QTLs que controlan caracteres relacionados con la resistencia a la sequía terminal mediante análisis GWAS. Se caracterizó la colección MED6WHEAT del IRTA, con 170 variedades tradicionales de 24 países mediterráneos y 184 variedades modernas cultivadas en 19 países de la región, con más de diez mil marcadores SNP. Se encontró un patrón geográfico asociado a la estructura genética con 3 subpoblaciones (SPs) de variedades tradicionales que representan el oeste, el norte y el este de la cuenca Mediterránea, mientras que las variedades modernas fueron estructuradas de acuerdo con los programas de mejora de la región: CIMMYT/ICARDA, Francia/Italia y países del este de Europa. Las variedades tradicionales se usaron para investigar la arquitectura del sistema radicular (RSA) adaptado a las condiciones de secano del Mediterráneo. Las variedades de los países del norte mostraron el mayor número de raíces seminales con un ángulo radical no significativamente diferente al de los del oeste, mientras que las variedades tradicionales del este mostraron el menor número de raíces, un mayor ángulo radical, unos tallos más largos y el menor peso de la semilla. El análisis GWAS detectó la presencia de marcadores asociados a los distintos caracteres y se identificaron 31 genes candidatos vinculados a RSA, el tamaño de la semilla, y la tolerancia a estrés abiótico en 15 regiones genómicas. La colección completa fue evaluada en un ensayo en campo de dos años mediante tecnologías de fenotipado masivo (HTP) con el objetivo de predecir caracteres agronómicos. La mejor estimación del LAI se logró con el índice MTVI2, y los índices de vegetación (VI) RGB medidos manualmente en campo mostraron mejores predicciones para los caracteres agronómicos. Los resultados basados en un estudio de tres años consecutivos mostraron diferencias significativas para los caracteres agronómicos entre SPs, resaltando la división en variedades tradicionales y variedades modernas. Las SPs modernas mostraron mayores valores de rendimiento y de sus componentes, mayor índice de cosecha, biomasa y duración de llenado de grano que las SPs de las variedades tradicionales, que fueron a su vez más altas. El mayor rendimiento fue observado en las variedades modernas de Francia e Italia. El análisis GWAS identificó 2579 marcadores asociados a caracteres agronómicos y relacionados con los VIs, que finalmente quedaron simplificados en 11 regiones genómicas. El análisis in silico de genes candidatos detectó 12 genes en condiciones de estrés abiótico en 6 de estas regiones. Cinco de estos genes ya se han descrito previamente relacionados con la tolerancia a estrés abiótico. Estos resultados constataron que las variedades tradicionales Mediterráneas son una fuente importante de variabilidad para introducir nuevos alelos en programas de mejora genética en la cuenca Mediterránea. El uso de nuevas tecnologías de teledetección es una herramienta eficiente y rápida para estimar caracteres agronómicos. Finalmente, el análisis GWAS ha resultado ser un enfoque muy útil para la identificación de regiones genómicas que controlen caracteres importantes del germoplasma del trigo harinero Mediterráneo.<br>Bread wheat (Triticum aestivum L.) is the main crop cultivated around the world, but climate change will significantly affect its production, with special impact in the Mediterranean basin. The ultimate purpose of this PhD thesis is to provide scientific knowledge and useful tools for the development of the next generation of superior bread wheat varieties resilient to the increased drought expected in the next decades as consequence of climate change. To achieve this objective, the existence of genetic, phenotypic and/or geographic structures in the germplasm collections was explored and quantitative trait loci (QTLs) controlling traits related to terminal drought resistance using a genome wide association study (GWAS) were identified. The MED6WHEAT IRTA-panel, with 170 landraces from 24 Mediterranean countries and 184 to modern varieties cultivated in 19 countries in the region, was characterized with more than 10K single nucleotide polymorphism (SNP) markers. A clear geographical pattern was found for the landraces, with three subpopulations (SPs) representing the western, northern, and eastern Mediterranean, whereas the modern cultivars were structured according to the breeding programmes operating in the region: CIMMYT/ICARDA, France/Italy, and Balkan/eastern European countries. Landraces were used to investigate their seminal root system architecture (RSA) adapted to rainfed Mediterranean conditions. Those from northern Mediterranean countries showed the highest number of seminal roots with a root angle not statistically different from the western Mediterranean ones, whereas eastern Mediterranean landraces showed the lowest number of roots but the widest angle, the longest shoots, and the lowest seed weight. A GWAS detected marker-trait associations (MTAs) linked to root-related traits and 31 candidate genes related to RSA traits, seed size, root development and abiotic stress tolerance were found within 15 QTL hotspots. The whole panel was evaluated on a two-year field trial using high-throughput phenotyping (HTP) technologies with the aim to predict agronomic traits. The best estimation of LAI was achieved through the modified triangular vegetation index (MTVI2), and ground-based RGB vegetation indices (VIs) showed better predictions of agronomic traits. The predictive value of the models developed for modern genotypes increased when the data of more than one growing season were aggregated to build them. Results based on a three consecutive year study found significant differences for agronomic traits between subpopulations, pointing out the division of the whole set into landraces and modern cultivars. Modern SPs showed higher values of grain yield and components, harvest index and biomass and longer grain filling duration than landrace SPs, which were taller. The highest grain yield was observed for modern cultivars from France and Italy. A GWAS identified 2579 markers associated with agronomic and VIs–related traits that were simplified to 11 QTL hotspots. In silico analysis of candidate genes detected 12 differentially expressed genes (DEG) upregulated under abiotic stress within 6 QTL hotspots. Among them, five genes were previously reported to be involved in abiotic stress tolerance. Overall, these results proved that Mediterranean wheat landraces are a valuable source of variability to introgress new alleles for desirable traits in the breeding programs in the Mediterranean Basin. The use of remote sensing technology is an efficient and rapid tool for the assessment of agronomic traits. Finally, GWAS have resulted a useful approach for the identification of genomic regions controlling important traits in the bread wheat Mediterranean germplasm.

During the recent years, the amount of fish for human consumption provided by aquaculture has impressively increased, and this led to a growing attention towards the development and the improvement of production techniques, including selective breeding programs, that are seen as principal drivers in the improvement of production traits (growth, morphology) and efficiency traits (processing yield, feed efficiency, product quality and disease resistance) of the farmed species. Disease outbreaks in aquaculture may have severe consequences, interfering with the progress and sustainability of the farm and causing huge production and economic losses. For these reasons, the interest towards the genetic improvement of disease resistance has significantly increased, thanks to the availability of new and more advanced genomic tools and techniques, the development of genome-wide sequencing and genotyping technologies and the generation of high-density marker data. In aquaculture species, selective breeding with the aim to establish disease resistant lines is an effective approach, in particular for those viral and bacterial infections that are not treatable with chemotherapeutics and antibiotics or for which vaccines are not currently available on a commercial scale. Selective breeding allows a long-term control of the disease and a cumulative and permanent improvement of the trait over generation at the population level. Betanodaviruses (nervous necrosis viruses, NNV) cause one of the major and severe infections (viral nervous necrosis, VNN) in more than 50 fish species, including the European sea bass (Dicentrarchus labrax L.). The effects of VNN outbreaks in sea bass hatcheries and farms are serious, with mortality rates up to 100% in the larval and juvenile stages. The first aim of the project was to estimate the genetic and genomic parameters for VNN resistance traits. A sea bass experimental population derived from a commercial breeding stock (N = 650) was subjected to a VNN challenge test and a genome-wide SNP dataset for sea bass was generated through a high-throughput sequencing approach (2b-RAD). Estimates of heritability for mortality as a binary trait (0/1) and survival time were moderate or low (genetic h2mort = 0.14-0.23, genomic h2mort = 0.06; genetic h2surv = 0.07-0.14, genomic h2surv = 0.03). Due to the possible application of indirect approaches to improve VNN resistance, we investigated the variation in a physiological parameter (post-stress cortisol concentration) and an immunological parameter (antibody titer to NNV) to determine the additive genetic variation of these traits, and the genetic correlation with disease resistance. Both traits showed a significant genetic and genomic variability (genetic h2HC = 0.19-0.23, genomic h2HC = 0.19; genetic h2AT = 0.28-0.39, genomic h2AT = 0.26). Cortisol concentration showed a null genetic correlation with VNN mortality, while the genetic relationship between antibody titer and mortality was negative (ra mort/AT = -0.39). The genetic and genomic heritability estimates for body weight (548 d post-hatching) were confirmed high (genetic h2BW = 0.45-0.60, genomic h2BW = 0.45); moreover, body weight was positively genetically correlated with cortisol concentrations and antibody titer (ra BW/HC = 0.12 and ra BW/AT = 0.49), while negatively correlated with mortality (ra BW/mort = -0.39). The second aim of the project was to investigate the potential and the effectiveness of genomic tools in predicting the genetic merit for the traits of concern. Five Bayesian models (BayesA, BayesB, BayesC, Bayesian LASSO, Bayesian Ridge Regression) were compared and their accuracy (in terms of correlation between observed value and prediction) when predicting the phenotype or the EBV (estimated breeding value) for VNN mortality, post-stress cortisol concentrations, antibody titer or body weight was estimated, without observing any differences between the five models. The prediction accuracy of the EBV for VNN mortality reached values equal to 0.89; the prediction accuracies of the EBV and the phenotype for post-stress cortisol concentration were 0.88 and 0.22, respectively, for antibody titer 0.76 and 0.26, respectively, and for body weight 0.69 and 0.39, respectively. Different metrics (the area under the ROC curve, the proportion of true results among the total number of samples, the Matthew’s correlation coefficient) were employed to evaluate the classification performance of prediction of the EBV for VNN mortality when used to classify the phenotype for the same trait, resulting in better classification performance, as measured by the AUC metric, in comparison with the classification based on genomic predictions of the phenotype. Overall, the results indicate that genomic prediction could have a great potential for traits like VNN mortality, body weight, cortisol concentration and antibody titer in European sea bass, leading to the reduction of the need of routine phenotyping and outperform traditional approaches in the enhancement of disease resistance, although further investigations on a larger experimental population are needed. Moreover, further studies should elucidate the effects of the method of infection used in challenge tests (immersion or injection) on the variation in the infection response observed and genetic parameters thereof. The third part of the project addressed the genetic architecture of sex determination in European sea bass. Sea bass sex determination model is complex and consistent with a polygenic model, where phenotypic sex is determined by the sum of small individual gene effects, but it is significantly affected by environmental temperature, as well. The comprehension of the genetic architecture of sex tendency in sea bass could be interesting even in the context of aquaculture. Actually, the strong bias towards males under aquaculture conditions has been recognised by farmers as problematic for different reasons (lower growth rates of males compared to females, reduced flesh quality and general decrease of the commercial values of the product). Because polygenic sex determination is considered evolutionarily unstable, the genetic architecture could be different in populations belonging to various geographical areas and exposed to different environmental/thermal conditions. Experimental progeny crosses (N = 927) were produced using wild dams from the Western Mediterranean and wild sires from four geographical areas (North Atlantic, Western Mediterranean, North-Eastern Mediterranean, South-Eastern Mediterranean), consistent with the natural range of the species in North Atlantic and Mediterranean Sea and characterized by different environmental conditions. The sex-ratio was strongly skewed towards males both in the global dataset and within each paternal group, with significant variation among populations, dams and sires. Sex, body weight and body length showed moderate heritability (h2sex =0.52 ± 0.17, h2BW =0.46 ± 0.17, h2BL = 0.34 ± 0.15) and sex tendency was genetically correlated with body weight and length (ra sex/BW = 0.69 ± 0.12, ra sex/BL = 0.66 ± 0.13). A weighted genome-wide association study (wGWAS) performed both on the global dataset and within each paternal group revealed a different genetic architecture of sex determination between Atlantic and Mediterranean populations, with a more similar genetic architecture among geographically close populations compared to geographically distant populations, consistent with the hypothesis of a population-specific evolution of the polygenic sex determination system in different environments.<br>Negli ultimi anni la richiesta di prodotti derivanti dall’acquacoltura è aumentata considerevolmente, comportando una maggiore attenzione volta allo sviluppo ed al miglioramento delle tecniche di produzione, compresi programmi di selezione genetica volti al miglioramento di caratteri produttivi (peso, morfologia) e di efficienza delle specie allevate (resa produttiva, efficienza alimentare, qualità del prodotto, resistenza alle malattie). In particolare, la comparsa di infezioni virali e batteriche in avannotteria ed in allevamento, può avere gravi conseguenze, sia in termini economici e produttivi, sia dal punto di vista del progresso e della sostenibilità dell’allevamento stesso. Per questo motivo, l’interesse verso il miglioramento genetico della resistenza alle malattie in specie ittiche è notevolmente aumentato, anche grazie al costante progresso di strumenti e tecniche di biologia molecolare che consentono la genotipizzazione e la generazione di panel di marcatori genome-wide. La selezione per produrre lotti di animali geneticamente migliorati risulta una strategia particolarmente efficace e di specifico interesse per patologie virali o batteriche per le quali le comuni misure di gestione come farmaci ed antibiotici non sono contemplate, presentano scarsa efficacia, o come i vaccini, risultano essere al momento soltanto in fase di sviluppo sperimentale. La selezione genetica per la resistenza alle malattie consente invece un controllo a lungo termine della patologia attraverso un cambiamento permanente delle caratteristiche genetiche degli animali che può essere trasmesso di generazione in generazione. L’encefalopatia e retinopatia virale (o viral nervous necrosis, VNN) è una delle maggiori e più gravi patologie riscontrata in più di 50 specie ittiche, tra cui il branzino (Dicentrarchus labrax L.). Le conseguenze di focolai di VNN in branzino sono decisamente significative, con tassi di mortalità fino al 100% negli stadi larvali e giovanili. Il primo obiettivo del progetto è stato quello di valutare i parametri genetici e genomici per i caratteri di resistenza a VNN. 650 animali prodotti in un allevamento commerciale sono stati quindi sottoposti ad infezione sperimentale con Betanodavirus (nervous necrosis virus, NNV) ed è stato prodotto un dataset di marcatori genome-wide (SNP) mediante approcci di sequenziamento high-throughput (tecnica 2b-RAD). L’ereditabilità per la mortalità intesa come carattere binario (0/1) o come tempo di sopravvivenza ha mostrato valori moderati o lievi (genetica: h2mort = 0,14-0,23, h2surv = 0,07-0,14; genomica: h2mort = 0,06, h2surv = 0,03). Visto l’interesse verso possibili indicatori da utilizzare come criteri di selezione indiretta, sono stati studiati anche parametri fisiologici (livello di cortisolo ematico post-stress) ed immunitari (titolo anticorpale a Betanodavirus) con lo scopo di determinarne la variabilità genetica additiva e la correlazione genetica con i caratteri di resistenza a Betanodavirus. Entrambi i parametri hanno mostrato valori di ereditabilità genetica e genomica interessanti (genetica: h2HC = 0,19-0,23, h2AT = 0.28-0.39, genomica: h2HC = 0,19, h2AT = 0,26). La correlazione genetica tra concentrazione di cortisolo e probabilità di morte è risultata nulla, mentre quella tra titolo anticorpale e probabilità di morte è risultata negativa (ra mort/AT = -0,39). L’ereditabilità genetica e genomica per il peso (548 giorni post-hatching) si è confermata elevata (genetica: h2BW = 0,45-0,60; genomica: h2BW = 0,45); il peso, inoltre, è risultato positivamente correlato dal punto vista genetico ai livelli di cortisolo ed al titolo anticorpale (ra BW/HC = 0,12 e ra BW/AT = 0,49), mentre la correlazione genetica tra questo carattere e la mortalità è risultata negativa (ra BW/mort = -0,39). Il secondo obiettivo del progetto è stato quello di investigare il potenziale predittivo di strumenti genomici per i caratteri considerati. Sono stati confrontati cinque modelli Bayesiani (BayesA, BayesB, BayesC, Bayesian LASSO, Bayesian Ridge Regression) e ne è stata stimata l’accuratezza (in termini di correlazione tra valori osservati e predetti) quando i modelli sono stati utilizzati per predire il fenotipo o l’EBV (estimated breeding value) per mortalità a VNN, livelli di cortisolo, titolo anticorpale o peso, senza però osservare differenze significative tra i modelli. L’accuratezza nel predire gli EBV per la mortalità ha raggiunto valori di 0,89; l’accuratezza nel predire gli EBV ed il fenotipo per i livelli di cortisolo è risultata 0,88 e 0,22, rispettivamente, per il titolo anticorpale 0,76 e 0,26, rispettivamente, per il peso 0,69 e 0,39, rispettivamente. Diverse metriche (area sottesa alla curva ROC, proporzione di risultati corretti sul numero totale di casi, coefficiente di correlazione di Matthew) sono state impiegate per valutare le performance di predizione degli EBV per la mortalità con lo scopo di classificare il fenotipo del carattere stesso, risultando in una migliore performance di classificazione, come indicato dalla metrica AUC, rispetto alla classificazione basata sulle predizioni genomiche del fenotipo. Nel complesso, questi risultati indicano che la predizione genomica potrebbe avere un enorme potenziale per caratteri come la mortalità dovuta a VNN, peso, concentrazione di cortisolo e titolo anticorpale in branzino, utile anche per ridurre la necessità di raccolte periodiche di dati fenotipici e fornendo migliori risultati rispetto agli approcci tradizionali per il miglioramento della resistenza alle patologie. Studi ulteriori su un maggior numero di animali sono comunque indispensabili, così come un approfondimento sulle variazioni nella risposta all’infezione, e quindi nei parametri genetici, dovute ai diversi metodi di infezione usati nei challenge test (immersione, iniezione). Il terzo obiettivo di questo progetto è stato invece rappresentato dallo studio dell’architettura genetica del meccanismo di determinazione del sesso in branzino. Il determinismo sessuale in branzino è un meccanismo poligenico complesso, in quanto risultato dell’interazione tra numerosi geni (con effetto limitato ma aventi azione uguale e cumulativa sul valore fenotipico) e temperatura ambientale. Capire i meccanismi genetici complessi che stanno alla base del determinismo sessuale è comunque interessante anche per una eventuale applicazione in acquacoltura, dal momento che l’alta percentuale di produzione di maschi rispetto alle femmine rappresenta un problema per diversi motivi (i maschi presentano precoce maturazione sessuale osservata in allevamento, con conseguente tasso di crescita inferiore, qualità inferiore del prodotto e, in generale, inferiore valore commerciale rispetto alle femmine). Partendo dal presupposto che un meccanismo di determinazione sessuale poligenico è evolutivamente instabile, l’architettura genetica potrebbe essere differente in popolazioni appartenenti a distinte aree geografiche caratterizzate da condizioni ambientali e termiche diverse. A questo scopo sono stati studiati 927 individui prodotti incrociando riproduttori femmine provenienti dalla zona Ovest del Mar Mediterraneo e riproduttori maschi provenienti da quattro diverse aree geografiche caratterizzate da condizioni ambientali differenti (Nord Atlantico, Ovest Mediterraneo, Nord-Est Mediterraneo e Sud-Est Mediterraneo) e corrispondenti alla distribuzione ed alla struttura genetica delle popolazioni naturali di branzino in Atlantico e Mar Mediterraneo. In generale, la percentuale di femmine nel dataset globale ed entro ciascun gruppo di diversa origine è sempre risultata inferiore rispetto alla percentuale di maschi, con differenze significative tra popolazioni, madri e padri. I valori di ereditabilità per sex tendency, peso e lunghezza (180 giorni post-hatching) sono risultati moderati (h2sex = 0,52 ± 0,17, h2BW = 0,46 ± 0,17, h2BL = 0,34 ± 0,15) e la sex tendency è risultata geneticamente correlata con peso e lunghezza, mostrando valori significativi (ra sex/BW = 0,69 ± 0,12, ra sex/BL = 0,66 ± 0,13). Uno studio di associazione (weighted GWAS) è stato effettuato sia sul dataset globale sia su ciascun gruppo di diversa origine con lo scopo di individuare marcatori SNP potenzialmente associati al determinismo sessuale. Lo studio ha rivelato una diversa architettura genetica per le popolazioni Atlantiche e Mediterranee, ma con affinità per individui appartenenti ad aree geografiche adiacenti, consistenti con l’ipotesi di evoluzione popolazione-specifica del meccanismo di determinazione sessuale in aree caratterizzate da condizioni ambientali differenti.

Arapaima gigas is the largest scaled freshwater fish in the world reaching over 250 kg. With growth rates of 10 kg+ within 12 months, A. gigas is considered as a promising candidate species for aquaculture development in South America. However, the lack of reproductive control in captivity is hindering the industry expansion. The work carried out in this doctoral thesis therefore aimed to better understand the species’ reproductive physiology, develop tools to identify gender and monitor gonad development, test hormonal therapies to induce ovulation and spawning and characterise the cephalic secretion for its potential roles in pheromone release and during parental care. Initially, a genomic study investigated the overall extent of polymorphism in A. gigas, which was found to be surprisingly low, with only 2.3 % of identified RAD-tags (135 bases long) containing SNPs. Then, a panel with 293 single nucleotide polymorphism (SNP) was used to characterise the genetic diversity and structure of a range of Amazon populations. Results revealed populations from the Amazon and Solimões appeared to be genetically different from the Araguaia population, while Tocantins population comprised individuals from both stocks. This data provided a tool for broodstock identification and future management. The PhD then aimed to evaluate the effects of slow-release mGnRH implants and different broodstock size pairings on maturation and spawning. Results showed that the implants stimulated the brain-pituitary-gonad axis resulting in increased plasma levels of testosterone (females) and 11-ketotestosterone in males, respectively regardless of pairing sizes. However, no spawning was observed. Results also showed the release of sex steroids with potential pheromonal action through the cephalic secretion, a biological fluid released from the adult head along the reproductive period. Thereafter, a non-surgical field endoscopy method was developed and validated for ovarian assessment and gender identification. The method was then used to describe the female gonopore and obtain biopsy of the ovary through cannulation which allowed the description of oogenesis in A. gigas. Importantly, oocytes obtained by cannulation confirmed that adult females under investigation were maturing with oocytes in final maturation stage but failed to ovulate/spawn. Another hormonal induction trial was therefore performed in which a combination of GnRHa (mGnRHa/sGnRHa) was used by injection to induce ovulation and spawning in selected maturing females with effects on oocyte maturation monitored post-induction through biopsy. However, this trial appeared to not be successful at inducing ovulation or spawning. Finally, the peptidome and proteome of the cephalic secretion was further characterised through the comparison between parental and non-parental fish. Results highlighted the complex role of this biological fluid including potential roles on the developing offspring during the parental care period. Overall, this doctoral thesis provided new basic and applied data on A. gigas reproduction and tools that can be used in future studies to better understand the environmental and hormonal control of oogenesis and spawning.

Cette étude analyse la quasi-totalité du cycle de vie de deux espèces de puffins sympatriques, le puffin tropical (Puffinus bailloni, PT) et le puffin du Pacific (P. pacificus, PP) nichant sur Aride Island (Seychelles). L’objectif est de comprendre les interactions existantes entre ces espèces, ainsi que l’influence de l’environnement, au sein de la colonie et dans le milieu marin. Le nombre de couples ont était estime à 15,000 pour le PP et 30,000 pour le PT (où la population est probablement en déclin). Ils sélectionnent davantage des zones escarpées avec des végétations basses. Les PP montrent également une préférence pour les zones rocheuses avec un sol profond. Les PT nichent pendant toute l’année mais de manière plus intensive en fin de saison de reproduction des PP et le taux d'échec de la reproduction est maximal durant la période où les deux espèces nichent en même temps. Dans le milieu marin, il-y-a une ségrégation spatiale entre les deux espèces, exploitant des zones d’alimentation bien distinctes hors saison de reproduction et se distinguant également dans leur comportement alimentaire (profondeur de plonge). Dans la colonie les deux espèces sont en concurrence sur le site de reproduction mais les PP semblent être de meilleurs compétiteurs. Nous proposons la mise en place de mesures de conservation pour les deux espèces, mais favorisant spécialement les PT<br>The procellariiformes are one of the most endangered orders in the world. In addition, information on population estimates, trends, movements at sea and population dynamic is lacking for many species. We analysed almost the full life cycle of two sympatric species: the tropical (Puffinus bailloni, TS) and the wedge-tailed shearwater (P. pacificus, WS) breeding at Aride Island (Seychelles), in order to understand the interactions between them and the environment both at sea and in the colony. We assessed habitat selection, abundance, distribution and breeding success of the two species at the colony and the habitat selection and distribution at sea. The population was 15,000 and 30,000 pairs for WS and TS respectively; the TS is possibly in decline. Both species selected steep and lowly vegetated areas; the WS also selected boulder-like zones. The TS nests year-round but it has a peak of breeding at the end of the WS breeding season and its failure rate is higher when the two species co-exist. We found spatial, rather than ecological, segregation between the two species at sea in both foraging behaviour (diving depth) and inter-breeding areas. On land they are likely to compete for breeding sites with the WS often over competing the TS. We then suggest future conservation plans focused on both species but especially on the TS

La malaltia de l’oïdi o cendrosa del presseguer, causada pel fong ascomicet Podosphaera pannosa, pot arribar a causar pèrdues greus en la producció i en la qualitat del fruit. Actualment, el control de l’oïdi es basa en aplicacions periòdiques de fungicides. Els efectes derivats de l’ús d’aquests productes en la salut humana i en el medi ambient han provocat una gran conscienciació de la societat, tot afavorint el desenvolupament de noves estratègies basades en el maneig integrat de plagues i malalties. Aquestes estratègies han de comptar necessàriament amb un coneixement profund dels agents implicats en la malaltia; és a dir, del patogen, de l’hoste i de les condicions ambientals. En aquesta tesi doctoral s’han estudiat algunes variables ambientals que descriuen el progrés de la malaltia de l’oïdi i s’ha dissenyat una estratègia per a retardar l’inici de l’aplicació dels tractaments fitosanitaris (Capítol 3). En concret, l’inici retardat del programa de protecció, a partir dels 220 graus-dia acumulats després de la floració, ha permès reduir el nombre de tractaments fitosanitaris en un 33%, sense perdre eficàcia en el control de la malaltia. També s’han estudiat aspectes relacionats amb l’inòcul primari de P. pannosa i l’aerobiologia del fong durant l’època infectiva (Capítol 4). Mitjançant tècniques moleculars, s’ha confirmat que l’inòcul primari es troba principalment en forma de miceli hivernant en la superfície dels branquillons afectats. També s’ha pogut quantificar els propàguls de P. pannosa presents en l’aire durant el període infectiu, d’abril a juliol. Una alternativa al control de l’oïdi que requereix més temps en la seva aplicació, però que evitaria el tractament amb fungicides, és el desenvolupament de cultivars resistents de presseguer. En aquesta tesi s’ha caracteritzat el gen de resistència Vr3, procedent de l’ametller (Capítol 5). Ha estat possible localitzar-lo en una regió molt específica del genoma, que inclou 27 gens candidats a conferir la resistència. Mitjançant una anàlisi d’expressió dels gens candidats i l’anàlisi dels polimorfismes de les reseqüències dels parentals, s’ha pogut determinar que el gen de resistència RGA2 podria ser el gen candidat Vr3, a falta d’una futura validació funcional. Finalment, i per tal d’obtenir noves varietats resistents a P. pannosa, s’ha introgressat la resistència Vr3 en cultivars comercials de presseguer (Capítol 6), mitjançant els creuaments d’individus amb una o dues introgressions d’ametller que incloïen el gen Vr3.<br>El oídio del melocotonero, enfermedad debida al hongo ascomiceto Podosphaera pannosa, puede llegar a causar pérdidas graves en la producción y en la calidad del fruto. Actualmente, el control del oídio se basa en aplicaciones periódicas de fungicidas. Los efectos derivados del uso de estos productos en la salud humana y en el medio ambiente han tenido como consecuencia una mayor concienciación social, lo que ha llevado al desarrollo de nuevas estrategias basadas en el control integrado de plagas y enfermedades. Este tipo de estrategias deben contar con un conocimiento profundo de los agentes implicados en la enfermedad; esto es, del patógeno, el huésped y el ambiente. En esta tesis doctoral se han estudiado algunas variables ambientales que describen el progreso de la enfermedad del oídio y se ha diseñado una estrategia para retardar el inicio de las aplicaciones fitosanitarias (Capítulo 3). En concreto, el retardo en el inicio del programa de protección, a partir de los 220 grados-día acumulados después de la floración, ha permitido reducir hasta un 33% el número de tratamientos fitosanitarios sin perder la eficacia en el control de la enfermedad. También se ha estudiado el inóculo primario de P. pannosa y la aerobiología del mismo durante la época infectiva (Capítulo 4). Mediante el uso de técnicas moleculares se ha confirmado que el inóculo primario se encuentra principalmente en forma de micelio latente en la superficie de las ramillas afectadas. También se ha logrado cuantificar los propágulos de P. pannosa presentes en el aire durante el período infectivo, que se extiende principalmente de abril a julio. Una técnica alternativa en el control de la enfermedad, que requiere más tiempo en su aplicación pero que puede evitar el uso de fungicidas, es el desarrollo de cultivares resistentes de melocotonero. En esta tesis se ha caracterizado el gen de resistencia Vr3 procedente del almendro (Capítulo 5). Ha sido posible localizarlo en una región muy específica del genoma, que incluye 27 genes candidatos. Mediante un análisis de expresión de los genes candidatos y un análisis de los polimorfismos de las resecuencias de los parentales, se ha podido determinar que el gen de resistencia RGA2 podría ser el gen candidato Vr3, aunque ello depende de una futura validación funcional. Finalmente, y con el fin de obtener nuevas variedades resistentes a P. pannosa, se ha introgresado la resistencia Vr3 en cultivares comerciales de melocotonero (Capítulo 6), mediante el cruce de individuos con una o dos introgresiones de almendro que incluían dicho gen.<br>Peach powdery mildew (PPM), caused by the ascomycete fungus Podosphaera pannosa, is one of the major diseases of peach, which may cause significant decreases in yield and fruit quality. Powdery mildew is currently managed through calendar-based fungicide applications. Adverse effects resulting from pesticide applications on human health and the environment have raised a great social awareness, which has driven the development of new integrated strategies more respectful to both environment and human health status. These disease management strategies are strongly depending on a deep knowledge of key aspects of the disease, which includes the pathogen and the host, and their relationship with the environment. In this thesis, the influence of some environmental variables on the disease progress of PPM have been studied. Furthermore, a strategy was designed to delay the onset of fungicide applications (Chapter 3). Specifically, the delayed initiation of fungicide programs at 220 accumulated degree-days after flowering reduced the number of fungicide applications by 33% while keeping effectiveness in the disease control. Several traits related to the latency of the pathogen primary inoculum and the aerobiology of the pathogen propagules during the infectious period were also studied (Chapter 4). Thus, by using molecular techniques, it was confirmed that the primary inoculum is mainly present as overwintering mycelium on the surface of the affected twigs. The detection and quantification of airborne P. pannosa propagules during the growing season confirmed that those propagules can be detected from April to July. The development of resistant cultivars is considered an alternative to disease control that is only based on fungicide treatments. In this thesis, the resistance gene Vr3, inherited from almond, was characterized (Chapter 5). We were able to locate it in a very specific region of the genome spanning 27 candidate genes. Through the expression analysis of candidate genes and an analysis of polymorphisms from parental resequences, it was concluded that the RGA2 resistance gene could be the best Vr3 candidate gene, assuming that a future functional validation is still required. Finally, in order to obtain resistant varieties to P. pannosa, the Vr3 resistance is currently being introgressed into high-quality peach varieties (Chapter 6), by crossing individuals with one or two almond introgressions which included the Vr3 gene.

Cleaner fish, including ballan wrasse (Labrus bergylta) have been proposed as a sustainable solution to sea lice infestations affecting farmed Atlantic salmon (Salmo salar) globally. However, in order to become sustainable, ballan wrasse need to be farmed. This thesis investigated the establishment of captive broodstock and protocols to optimise hatchery performance and productivity of ballan wrasse. High throughput sequencing was used to develop a panel of novel single nucleotide polymorphic markers (SNPs). These SNPs were used to investigate the phylogeographic structuring of ballan wrasse populations within northern geographic ranges including the UK and Norway. Results indicated fine scale population structuring within the UK suggesting that founder broodstock should be sourced locally to minimise the risk of genetic introgression with wild ballan wrasse. Secondly, captive breeding was benchmarked from harems to determine total egg production over the spawning season. Data quantified the spawning periodicity and seasonal changes in egg quality parameters. In addition, microsatellite markers identified the parental contribution to each spawning event of captive broodstock. Results confirmed, for the first time, the repeat-batch spawning behaviour and suggested that spawning events were single-paired matings. Furthermore, bottlenecks in commercial production were investigated including the benthic adhesive eggs and complex spawning behaviours of ballan wrasse within broodstock tanks. Experiments were conducted to optimise the spawning dynamics and egg productivity using fragmented spawning zones and coloured substrates. Finally, an effective bath treatment for removal of the adhesive gum layer of eggs using the proteolytic enzyme alcalase® was found to assist in egg disinfection and incubation. Overall, this research provides important baseline data on the management of broodstock and the optimisation of hatchery protocols to improve the commercial productivity and performance of ballan wrasse for use as a biological control of sea lice of farmed Atlantic salmon.

Il Breeding Blanket (BB) del reattore DEMO rappresenta un sistema complesso in un ambiente pericoloso. Infatti, esso deve soddisfare diversi requisiti e vincoli ingegneristici sia di tipo nucleare, termo-strutturale che di sicurezza. Per questi motivi, è necessaria una progettazione omnicomprensiva che preveda l'applicazione di strumenti avanzati di simulazione basati su approcci multi-fisici. Questi strumenti devono eseguire simultaneamente diversi tipi di analisi. Tre di esse possono essere considerate prioritarie e propedeutiche per lo studio di tutti gli altri fenomeni riguardanti il BB, vale a dire l´analisi nucleare, termo-fluidodinamica e strutturale. In questa tesi, è proposto un innovativo approccio multi-fisico che copre i tre pilastri principali su cui è basato il progetto del BB (la neutronica, la termoidraulica e la termo-meccanica). Queste analisi devono essere condotte in maniera integrata, consentendo una valutazione olistica dei carichi volumetrici di potenza, delle prestazioni termiche sia del fluido di raffreddamento che delle strutture, nonché dei campi di tensione e deformazione. La strategia seguita per il conseguimento di questa sfida consiste nella creazione di una procedura “CAD-centric” e “loosely-coupled” (debolmente accoppiata) per la progettazione dei concetti di BB utilizzando una tecnica di analisi basata su sotto-modelli. Questa procedura prende il nome di Multi-physics Approach for Integrated Analysis (MAIA). Essa basa la sua architettura sull'uso di codici validati e sulla minimizzazione del loro numero. In particolare, MAIA è articolata in 10 fasi principali che vanno dalla creazione di un modello per le analisi nucleari generato dalla decomposizione in geometrie semplici di un generico CAD alla valutazione della potenza volumetrica, dal calcolo dei campi di temperatura e velocità nella struttura e nel refrigerante alla valutazione dei campi di spostamento, deformazione e stress, dalla stima dei tassi di produzione degli isotopi dell´azoto prodotti dall'attivazione dell'ossigeno presente nell'acqua al calcolo della loro distribuzione spaziale di concentrazione tenendo conto degli effetti del trasporto convettivo. Tutti i vari passaggi condividono gli stessi dettagli geometrici. In particolare, MAIA differisce dagli approcci convenzionali usati nell´accoppiamento multi-fisico su tre aspetti chiave. Innanzitutto, non introduce omogeneizzazioni dei modelli e dei carichi. In secondo luogo, MAIA permette di determinare, con un’alta risoluzione spaziale, i gradienti dei carichi per tutte le analisi coinvolte senza richiedere sforzi computazionali proibitivi. In terzo luogo, MAIA permette di mantenere la coerenza tra le tre analisi garantendo la congruenza tra gli input e gli output. Tuttavia, l´onere computazionale richiesto dall´approccio CAD-centric, su cui si basa la procedura MAIA, non permette di rappresentare il BB nel suo complesso ma solo alcune sue porzioni (una slice, per esempio). Ciò impone la definizione e, conseguentemente, la validazione di opportune condizioni al contorno per ogni sotto-modello utilizzato e per ogni analisi eseguita. A tal proposito, per quanto riguarda le analisi nucleari, le condizioni al contorno utilizzate nel modello locale della slice sono: definizione di una sorgente locale neutronica/fotonica per tener in conto l´effetto albedo dell´intero reattore, rappresentazione del Vacuum Vessel (VV) per simulare il back scattering verso il BB, e l´applicazione di condizioni di riflessione (“mirror”, specchio/simmetria, nella direzione poloidale e “white”, riflessione isotropica, in quella toroidale) per simulare la presenza delle slice adiacenti a quella analizzata. I risultati ottenuti mostrano una variazione della potenza depositata del -0.48 % tra il modello di riferimento DEMO e quello locale (slice). Inoltre, è stata eseguita un'analisi di sensibilità sulla distribuzione angolare della sorgente neutronica/fotonica locale determinando una discretizzazione ottimale in 10 suddivisioni poloidali. Questa suddivisione rappresenta un buon compromesso sia in termini di fedeltà dei risultati ottenuti, rispetto a quelli del modello di riferimento (DEMO), che di minimizzazione dell´onere computazionale. Per quanto riguarda l'analisi delle condizioni al contorno termo-idrauliche usate nel modello locale della slice, è stata applicata una condizione di simmetria termica poloidale. Assumendo una variazione delle portate comprese tra ~ -1.3% e ~ 0.6% e una fluttuazione della densità di potenza fino a ~ 6% tra slice vicine, è stata ottenuta una variazione della distribuzione delle temperature del ± 2.4% dimostrando, quindi, l'applicabilità di tali condizioni. Per quanto riguarda le analisi termo-meccaniche, le condizioni al contorno identificate per il modello locale della slice sono: simmetria sul piano inferiore della slice, Generalised Plane Strain su quello superiore e spostamenti radiali e toroidali impediti ai nodi che giacciono nella parte posteriore della back supporting structure lungo la direzione toroidale e poloidale. Queste condizioni, applicate al sotto-modello, producono una variazione compresa tra il -6% e il 4% tra gli spostamenti calcolati nella slice e quelli nel modello di riferimento DEMO, nonché una stima conservativa delle tensioni primarie e secondarie sia di membrana che di flessione. Inoltre, è stato anche studiato l'impatto della variazione (± 2.4%) di temperatura dimostrando che le fluttuazioni sulle deformazioni totale sono comprese tra il -0.3% e l’1.7%, fino a un massimo del 15% sulle tensioni equivalenti di membrana e tra il -7% e il 5% su quelle di flessione. Infine, la procedura MAIA è stata utilizzata per valutare l'impatto sul design del BB. La sua applicazione ha dimostrato la presenza di alcune criticità nel progetto. In particolare, i risultati fluidodinamici mostrano una violazione dei limiti di temperatura che non sono stati risolti introducendo soluzioni progettuali adeguate. Inoltre, queste violazioni producono, a loro volta, valori molto intensi delle tensioni equivalenti di Von Mises che potrebbero indicare un pericolo per l'integrità strutturale del BB. L´applicazione di MAIA al design del BB a permesso di dimostrare il valore aggiunto di questa procedura la quale potrebbe diventare uno strumento fondamentale e di riferimento per la progettazione del BB. Inoltre, la procedura MAIA ha permesso di mappare localmente variabili importanti come flussi neutronici e temperature, nonché le tensioni primarie e secondarie che sono utilizzate per la determinazione delle tensioni ammissibili applicate per la verifica dei criteri di progettazione. Al fine di dimostrare ulteriormente la versatilità e l'adattabilità della procedura MAIA, è stato studiato il problema di attivazione dell'acqua del sistema di trasferimento di calore primario (Primary Heat Transfer System, PHTS). Utilizzando la procedura MAIA, è stato possibile prendere in considerazione gli effetti dell´efflusso sulla concentrazione degli isotopi dell´azoto e fornire informazioni utili per lo sviluppo sia del design del BB che del suo PHTS.<br>The Breeding Blanket (BB) of the DEMO reactor represents a harsh system in a dangerous environment. It has to satisfy engineering requirements and constraints that are of nuclear, thermo-structural, material and safety kind. For these reasons, the application of advanced simulation tools, based on a multi-physics approach, is required for its comprehensive design. These tools have to simultaneously perform different kind of analyses among which three, and namely nuclear, thermofluid-dynamic and thermo-mechanical, can be prioritized and considered as propaedeutic for the investigation of all the other issues related to the BB. In this dissertation, a multi-physic approach, covering the three pillars of the BB design (the neutronics, thermal-hydraulics and thermo-mechanics), is proposed. These analyses have to be conducted in a strongly integrated way, allowing a holistic assessment of volumetric heat loads, thermal performances of coolant and structures as well as their stress and deformation states. The strategy, followed for the achievement of this challenge, consists of creating a CAD-centric and loosely-coupled procedure for the BB concepts design adopting a sub-modelling technique, named Multi-physics Approach for Integrated Analysis (MAIA). The MAIA procedure bases its architecture on the use of validated codes and on the minimisation of their number. It is articulated in 10 main steps that go from the decomposition of generic CAD in a format suitable for neutron/photon transport analysis to the nuclear analysis for the assessment of volumetric heating, from the assessment of temperature and velocity fields within coolant and structure to the evaluation of their displacement, deformation and stress fields, from the evaluation of nitrogen isotopes production rates from water oxygen activation to the calculation of their concentration spatial distribution taking into account the effects of passive convective transport. All the steps share the same geometry details and the consistency between input and output parameters. The new MAIA procedure differs from the conventional coupling approach with respect to three key aspects. First, it does not introduce homogenisations of models and loads. Second, MAIA can capture load gradients at high resolution in the three directions for all the analysis involved without requiring prohibitive computational efforts. And third, MAIA keeps the consistency between the three analyses maintaining the congruence between inputs and outputs. However, the computational effort required by the CAD-centric feature of MAIA procedure imposes the representation of BB portions and, therefore, the definition and validation of boundary conditions for each performed calculation. Regarding the nuclear analysis, it has been found that the set of reflecting and white conditions in the poloidal and toroidal directions, respectively, together with the presence of Vacuum Vessel (VV) and the definition of local neutron and photon source, produces a mismatch of -0.48 % in terms of power deposition between the DEMO and the local (e.g. slice) models. It has been demonstrated that the neutronic symmetry conditions are valid in the entire module up to the last slices nearby the caps. Furthermore, a sensitivity analysis on the angular distribution of local neutron and photon source has been performed indicating in 10 cosine bins the optimal discretisation choice in terms of compromise between the fidelity of the results obtained respect to those of the reference model and the relevant computational effort. Concerning the analysis of thermal-hydraulic boundary conditions, it has been found that the variation on mass flow rates (comprised between the ~-1.3 % and the ~0.6 %) as well as power density fluctuation (up to the ~6 % in the neighbouring domains) affect the temperature distribution for less than ±2.4 % demonstrating the applicability of poloidal symmetry conditions. As far as the thermo-mechanical analyses are concerned, it has been identified the set of boundary conditions (radial and toroidal displacements prevented to the nodes lying in the rear of the back supporting structure along the toroidal and poloidal direction, symmetry at the lower cut surface and Generalised Plane Strain to the top one) that produce a discrepancy in terms of displacement in the sub-model comprised between the -6 % and the 4 % as well as a conservative assessment of membrane and bending stresses both for primary and secondary stresses. The impact of the temperature variation has also been investigated showing that the fluctuations on total deformation are comprised between -0.3 % and the 1.7 %, on equivalent membrane stress up to 15 % while on equivalent bending stress between the -7 % and the 5 %. As a proof-of-concept, the MAIA procedure has been then used to evaluate the impact on the BB design, demonstrating that some criticalities are present in the design. In particular, the fluid-dynamic results show a violation of the temperature requirement limits that have not been solved introducing proper design solutions. Furthermore, these violations of thermal-hydraulic requirements produce very intense values of Von Mises equivalent stresses that could jeopardize the structural integrity of the segment box. This demonstrates that MAIA procedure can become the reference tool for the design of the BB. Moreover, the MAIA procedure has proven the possibility to locally map important variables such as the neutron flux and the temperature as well as the primary and secondary stress that are used for the determination of the allowable stress and applied for compering with design criteria. In order to further demonstrate the versatility and adaptability of the MAIA procedure, the water activation issue occurring within the blanket Primary Heat Transfer System (PHTS) has been studied. Using MAIA procedure, it has been possible to take into account the effects of the flow on the nitrogen concentration and to provide useful information for the development of both BB design and its PHTS.

Breeding waders have suffered widespread population declines on lowland wet grassland. Conservation efforts on reserves are concentrated on the provision of optimum foraging and nesting conditions to improve wader productivity. This thesis aimed to investigate the potential offarmyard manure (FYM) application to increase soil invertebrate availability (a component of wader diet) in spring, thereby enhancing foraging habitat quality. Fieldwork was conducted in 2004-2006 on lowland wet grassland RSPB reserves to assess. the effects of FYM at rates of 5, 10 and 15 tonnes ha-1 on soil invertebrates and other factors of relevance to wader conservation and wider management objectives. Additional studies were carried out to investigate the response of earthworms to FYM on a finer scale and establish the role of earthworms in the diet, habitat choices and foraging efficiency of lapwing adults and chicks. An increase in earthworm availability in the breeding season was seen following an autumn application ofFYM spread at rates of 10 and 15 tonnes ha-1 Ã?Â? There was a strong aggregative response of earthworms to FYM on a small scale. No effect of FYM application at these rates was seen on epigeal invertebrate numbers, but the average body mass of Coleoptera increased on FYM treatments. There was no evidence to suggest a detrimental effect on either soil or vegetation properties on reserves. Earthwonn availability was an important factor in foraging choice and rates in female adult lapwing and for older chicks, with chicks close to fledging showing significantly greater levels of earthworms in their diet. An assessment of the gross energy content of common earthworm species suggests the increases in earthworm availability seen at these rates of FYM application are sufficient to be biologically meaningful to waders in the Breeding waders have suffered widespread population declines on lowland wet grassland. Conservation efforts on reserves are concentrated on the provision of optimum foraging and nesting conditions to improve wader productivity. This thesis aimed to investigate the potential offarmyard manure (FYM) application to increase soil invertebrate availability (a component of wader diet) in spring, thereby enhancing foraging habitat quality. Fieldwork was conducted in 2004-2006 on lowland wet grassland RSPB reserves to assess. the effects of FYM at rates of 5, 10 and 15 tonnes ha-1 on soil invertebrates and other factors of relevance to wader conservation and wider management objectives. Additional studies were carried out to investigate the response of earthworms to FYM on a finer scale and establish the role of earthworms in the diet, habitat choices and foraging efficiency of lapwing adults and chicks. An increase in earthworm availability in the breeding season was seen following an autumn application ofFYM spread at rates of 10 and 15 tonnes ha-1 Ã?Â? There was a strong aggregative response of earthworms to FYM on a small scale. No effect of FYM application at these rates was seen on epigeal invertebrate numbers, but the average body mass of Coleoptera increased on FYM treatments. There was no evidence to suggest a detrimental effect on either soil or vegetation properties on reserves. Earthwonn availability was an important factor in foraging choice and rates in female adult lapwing and for older chicks, with chicks close to fledging showing significantly greater levels of earthworms in their diet. An assessment of the gross energy content of common earthworm species suggests the increases in earthworm availability seen at these rates of FYM application are sufficient to be biologically meaningful to waders in the field.

Марченко, І. О. Регулювання розвитку галузі козівництва в Україні : дис. ... канд. екон. наук : 08.00.08 / І. О. Марченко. - Чернігів, 2020. - 220 с.<br>Дисертація присвячена обґрунтуванню теоретичних підходів та розробленню науково-практичних рекомендацій, спрямованих на удосконалення заходів, інструментів, механізмів і методів регулювання розвитку галузі козівництва в Україні. У роботі систематизовано методичні підходи при обґрунтуванні концептуальних основ удосконалення інструментів регулювання у тваринництві. Запропоновано застосування «адміністративно-процедурного принципу компартмелізації» як нового інструменту прямого методу регулювання з метою розроблення послідовної системи інструкцій для виробників козлятини, нішевих безлактозних продуктів із молока кіз при плануванні, узгодженні та прийнятті нормативних актів, прогнозів і програм розвитку галузі козівництва. Удосконалено організаційно-економічний механізм регулювання через внесення доповнень до Класифікатора видів економічної діяльності ДК 009:2010 у частині групи 01.4 «Тваринництво». Запропоновано з групи 01,45 «розведення овець» виокремити групу 01.48 «розведення кіз» як окремого виду економічної діяльності та інтеграції його у аграрне виробництво. Здійснено аналіз стану та тенденцій розвитку козівництва в Україні і світі з оцінкою впливу регулювання галузі в умовах продовольчих викликів. Доведено, що важливу роль в розвитку галузі козівництва відіграють галузеві резерви. Зокрема досягнення до 2025 р. прогнозних обсягів виробництва молока кіз до 23,32 тис. т, зокрема у фермерських господарствах до 18,0 тис. т відбудеться за умови нарощування поголів’я кіз до 900 тис. голів. Результати досліджень можуть бути використані як складова частина науково-методичної бази при плануванні системних змін із орієнтацією як на виробничу діяльність, так і на її соціальну складову, щоб прогнозувати економічні наслідки розвитку галузі в сучасних умовах<br>Диссертация посвящена обоснованию теоретических подходов и разработке научно-практических рекомендаций, направленных на улучшение инструментов, механизмов и методов регулирования развития отрасли козоводства в Украине. В работе систематизированы методические подходы при обосновании концептуальных основ совершенствования инструментов регулирования в животноводстве. Расширены теоретические представления о закономерностях функционирования отраслей животноводства и предложено новый инструмент прямого метода регулирования «административно-процедурный принцип компартмелизации» с целью разработки системы инструкций для производителей нишевых безлактозных продуктов из молока коз, козлятины, при планировании, согласовании и принятии нормативных актов, прогнозов и программ развития отрасли козоводства. Усовершенствован организационно-экономический механизм регулирования путем внесения дополнений в Классификатор видов экономической деятельности ДК 009: 2010 в части группы 01.4 «Животноводство». Предложено из группы 01.45 «разведение овец» выделить группу 01.48 «разведение коз» как отдельного вида экономической деятельности и интеграции его в аграрное производство. Также осуществлен анализ состояния и тенденций развития козоводства в Украине и мире с учетом оценки воздействия регулирования отрасли в условиях продовольственных вызовов. Обоснованы приоритетные направления по обеспечению внутреннего спроса на продукцию овцеводства и козоводства отечественного производства на основе комплексной оценки тенденций его развития в 1991-2019 гг. с анализом уровня производства, балансов спроса и предложения. Путем осуществления системного анализа состояния отрасли определено, что страна является импортозависимой по кодам товарных позиций 0301-0307, 1604-1605 генетические ресурсы, сыры и молочные продукты, детское питание согласно УКТ ВЭД. Изучение и оценка инструментов государственного регулирования отрасли животноводства показали тенденцию к увеличению предложения нишевых безлактозных молочных продуктов, включая сыры, йогурты, на базе первичной и глубокой переработки козьего молока как продукта с высокой добавленной стоимостью. Доказано, что использование на практике усовершенствованного в работе механизма регулирования позволило систематизировать предложения к совершенствованию формы отчетности № 24-сг (месячная) «Отчет о производстве продукции животноводства и количество сельскохозяйственных животных». Это позволит на основе достоверных данных и действующих методических основ разрабатывать прогнозы и планы развития козоводства, согласовывать их с системой программных документов экономического и социального развития Украины, освоить требования инвестиционно-инновационной модели функционирования животноводства, обеспечивать обмен такой информацией с международными организациями. Разработаны новые методические подходы к функционированию отрасли козоводства через эффективность производства продукции на основе модели, реализационной цены, композиционных уровней новизны групп безлактозных продуктов из молока коз, рассчитанных методом парных сравнений. Доказано, что важную роль в развитии отрасли козоводства играют отраслевые резервы. Достижение до 2025 г. прогнозных объемов производства молока коз до 23,32 тыс. гол., в том числе в фермерских хозяйствах до 18,0 тыс. гол. будет при условии наращивания поголовья коз до 900 тыс. гол. Результаты исследований могут быть использованы в качестве составной части научно-методической базы при планировании системных изменений с ориентацией как на производственную деятельность, так и на ее социальную составляющую, чтобы прогнозировать экономические последствия развития отрасли козоводства в Украине в условиях трансформационных процессов<br>The thesis is devoted to substantiation of theoretical approaches and development of scientific and practical recommendations aimed at improving measures, tools, mechanisms and methods of regulating the development of the goat industry in Ukraine. The methodical approaches in substantiation of conceptual bases of improvement of tools of regulation in animal husbandry are systematized in the work. It is proposed to use the "administrative-procedural principle of compartmentalization" as a new tool of direct regulation to develop a consistent system of instructions for producers of niche lactose-free products from goat's milk, goat when planning, agreeing and adopting regulations, forecasts and programs for goat farming. The organizational and economic mechanism of regulation has been improved through amendments to the Classifier of Economic Activities DK 009: 2010 in part of group 01.4 "Livestock". It is proposed to separate the group 01.48 "goat breeding" from the group 01.45 "sheep breeding" as a separate type of economic activity and its integration into agricultural production. An analysis of the state and trends in the development of goat breeding in Ukraine and the world with an assessment of the impact of industry regulation in terms of food challenges. It is proved that industry reserves play an important role in the development of the goat breeding industry. In particular, the achievement of the projected volumes of goat milk production up to 23.32 thousand tons by 2025, including in farms up to 18.0 thousand tons, will take place under the condition of increasing the number of goats to 900 thousand heads. The results of research can be used as part of the scientific and methodological framework in planning systemic changes with a focus on both production activities and its social component to predict the economic consequences of development in modern conditions

Thesis (MSc)--Stellenbosch University, 2012.<br>ENGLISH ABSTRACT: The decline of Haliotis midae (perlemoen) populations together with the ensuing collapse of commercial abalone fisheries in South Africa have shifted the responsibility to abalone farms to meet the demand for perlemoen. Attention has recently turned to the genetic enhancement of cultured abalone in order for the farms to remain competitive in the international aquaculture market. To develop a successful breeding programme it is imperative to draw on a good foundation of high levels of genetic diversity and to successfully maintain these levels in order to create an enhanced strain of cultured abalone. A Performance Recording Scheme (PRS) was established as the first breeding programme for Haliotis midae to utilise molecular tools. This programme was aimed at enhancing the growth rate of abalone in order to shorten the production times on farms. The current study made use of 12 species-specific microsatellite markers to assign parentage to a group of faster-growing PRS animals, as selected by the abalone farms, in order to select a diverse on-farm generation of broodstock. Additionally, the influence of standard selection practises on the genetic diversity of a population compared to genotypic selection was investigated. This data was also used to study the differentiation and levels of genetic diversities within and between cultured and wild populations. Selection based on genotypic traits successfully retained genetic diversity while some diversity was lost in phenotypically selected populations. These phenotypic populations differed significantly from each other and wild populations, while the genotypic populations were similar in genetic composition to each other and wild populations of the West coast. The broodstock populations used in the PRS spawning event were representative of the wild populations from where they were sourced, with no significant differentiation between the broodstock and West coast population. When these broodstock populations were compared to their corresponding offspring populations, only two populations displayed a significant loss in diversity; although all of the offspring populations showed significant differentiation with their corresponding broodstock populations. This was attributed to the differential contribution of broodstock and the effect of artificial selection. It was established that the cultured populations of the participating abalone farms should be used with caution in ranching and reseeding programmes. These populations differed significantly from both the East and West coast wild populations. This study concluded that it is possible to retain genetic diversity by selecting breeding animals based on genotypic traits. The loss of diversity in some cultured populations and significant differentiation from the wild populations indicate that animals are exposed to different selection pressures in the cultured environment. The results found in this study highlight the need for the effective management of hatchery practices and the genetic monitoring of the breeding animals.<br>AFRIKAANSE OPSOMMING: Die afname in Haliotis midae (perlemoen) populasies en die daaropvolgende ineenstorting van die kommersiële perlemoen bedryf in Suid-Afrika het die verantwoordelikheid om in die aanvraag na perlemoen te voorsien, na perlemoen plase verskuif. Die genetiese verbetering van verboude perlemoen geniet tans aandag in ‘n poging om kompeterend te bly in die internasionale mark. Dit is noodsaaklik vir die sukses van ‘n broeiprogram om gebruik te maak van ‘n goeie genetiese basis met hoë vlakke van genetiese diversiteit en die suksesvolle behoud van die vlakke om so ‘n verbeterde lyn te skep. ‘n Groeiprestasie aanteken stelsel [Performance Recording Scheme (PRS)] is gestig as die eerste broeiprogram vir Haliotis midae wat gebruik maak van molekulêre tegnieke. Die doel van hierdie program was om die groeitempo van verboude perlemoen te verbeter om produksie tye te verkort. Die huidige studie het gebruik gemaak van 12 spesie-spesifieke mikrosatelliet merkers om ouerskap toe te ken aan ‘n groep vinnig-groeiende PRS-diere, soos geselekteer deur die perlemoen plase, om ‘n diverse generasie gekultiveerde diere te selekteer wat as broeidiere kan dien. Die invloed van standaard seleksie metodes op die genetiese diversitiet van ‘n populasie in vergelyking met genotipiese seleksie is ook ondersoek. Die ouerskap data is ook gebruik om differensiasie en vlakke van genetiese diversiteit tussen verboude perlemoene en wilde populasies vas te stel. Seleksie gebasseer op genetiese eienskappe het daarin geslaag om genetiese diversiteit te behou, terwyl diversiteit verlore gegaan het in die fenotipies geselekteerde populasies. Hierdie fenotipiese populasies het ook beduidend met mekaar sowel as met die wilde populasies verskil, terwyl genotipiese populasies soortgelyk was in hul genetiese samestelling en nie van die wilde populasies van die Weskus verskil het nie. Die broeidiere wat in die PRS broeiprogram gebruik is, was verteenwoordigend van die wilde populasies vanwaar hulle oorspronlik gekom het, met geen beduidende differensiasie tussen die broeidiere en die Wes kus populasies nie. Met die vergelyking van die broeidiere en hul ooreenstemmende nageslag, het dit geblyk dat slegs twee populasies ‘n beduidende verlies aan genetiese diversiteit getoon het, alhoewel al die nageslag beduidende populasie differensiasie met hul ouers getoon het. Hierdie bevindinge is toegeskryf aan oneweredige bydraes van die broeidiere tydens gameetvrystelling en die invloed van kunsmatige seleksie. Hierdie studie het ook vasgestel dat die verboude perlemoen populasies met sorg gebruik moet word om wilde populasies te herstel, aangesien hierdie populasies beduidend verskil het van wilde populasies van beide die Oos en Wes-kus. Hierdie studie het gevind dat dit moontlik is om genetiese diversiteit te behou deur diere te selekteer op grond van genotipiese eienskappe. Die verlies van diversiteit in sommige van die verboude perlemoen populasies en die beduidende verskil met die wilde populasies dui daarop dat diere in die gekultiveerde omgewing blootgestel word aan verskillende tipes seleksiedruk. Hierdie bevindinge beklemtoon die belang vir effektiewe bestuur van broeiery praktyke en genetiese monitering van broeidiere.

This study conducted an evaluation of farmer and shepherd perceptions on Anatolian Shepherd dogs, used as livestock guarding dogs to mitigate farmer–wildlife conflict and meet conservation ends. The case study involved Anatolian Shepherd dogs bred at the Anatolian Shepherd Dog Breeding Project in the Namaqua National Park, and placed mostly on farms and at stock posts near the Namaqua National Park in the Northern Cape. The data were collected during structured and semi-structured interviews with livestock farmers and shepherds that received Anatolian Shepherd dogs from the Breeding Project. Anatolian Shepherd dogs were evaluated in terms of their contribution to reduce livestock losses and conservation of wildlife species. In terms of effectiveness in preventing or reducing livestock losses, 84% of the dogs eliminated or reduced livestock losses. Respondent satisfaction with the dogs was high, with 95% of respondents willing to recommend the Breeding Project and the use of Anatolian Shepherd dogs. Of the respondents, 95% perceived their dogs to be economically beneficial. Another 48% of respondents reported some form of behavioural problems at least once during the placement of the dogs. The most common reported problem was resting in the shade rather than accompanying the livestock. However, corrective training was effective in all cases where training was undertaken immediately. In terms of conservation, fewer respondents used lethal predator control methods in the years after placement of the dogs than before their placement. The overall perception regarding the use of lethal control methods (e.g. gin traps, shooting and poison) was that such control is cruel and that it is better to use non-lethal control methods only. However, a few respondents reported that lethal control measures were occasionally necessary to make a living when no other control measures were available, especially when the herd was relatively large. The effects of Anatolian Shepherd dogs on non-predator species in this study were minimal. The presence of the dogs therefore aided predator conservation and improved farmer tolerance of predators, especially by effectively reducing livestock losses.

With the advent of high-throughput genotyping modern plant breeding has reached a new frontier of high-volume, high-density, yet low-cost, genomic data. Previously the acquisition of this data has been a logistical bottleneck within breeding programmes, yet with genomic data now abundantly available to breeding programmes, it has been speculated that the collection of phenotype data will become the next operational bottleneck. That being the inability to phenotype all material for all desired traits within a programme . The journey to improve the collection of phenotypic data is well underway, with focus being placed upon next generation phenotyping (NGP) technologies, such as high-throughput field phenotyping systems, to aid in the pairing of genotype to phenotype. Numerous sensors and methods of deployment have been investigated for application within small-plot field trials and suggested as tools for wheat and other field-crop breeding programmes, though few have explored how these can be deployed at scale or the suitability of collected data for use by breeders. This thesis investigates the deployment of commercially available digital cameras and LiDAR sensors within large-scale wheat breeding field trials, assessing the suitability of collected data for its application within the analytical pipelines of breeding programmes. Digital cameras were deployed opportunistically within large-scale wheat breeding trials, and through basic open-source image analysis methods, were capable of objectively assessing colour-based traits traditionally scored with visual assessment, producing levels of heritability similar to or greater than traditional methods. As part of this process a tractor-based high-throughput phenotyping platform was developed for the deployment of digital cameras, leveraging upon infrastructure present within the breeding programme and enabling images to be captured at a speed of 7,400 plots per hour. Given the success of digital cameras to measure colour-based traits, digital cameras were also deployed manually at a small scale to measure above ground biomass, plant height and harvest index, using photogrammetric techniques. Though data capture and processing methods were low-throughput, correlations between digital and manually collected measurements were strong (up to r = 0.94), highlighting the potential of the three-dimensional point cloud data type. To further this investigation LiDAR sensors were deployed on the high-throughput phenotyping platform to collect point cloud data of wheat plots from multiple field sites and collection dates. Processed point cloud data correlated strongly to traditional measurements of above ground biomass and canopy height and was shown to be highly repeatable and suitable for integration in routine breeding analyses. The findings of this work demonstrate that commercially available digital cameras and Li- DAR sensors can be deployed within large-scale wheat breeding trials, in a high-throughput, non-destructive and non-disruptive manner, for the accurate and repeatable measurement of traits which are traditionally subjective, laborious and/or destructive. Investigation of these measurements showed their suitability for inclusion within routine breeding analyses, giving breeders confidence in the data collected by next generation phenotyping technologies. The findings of this work are not only relevant to wheat breeders, but also to breeders of other field-crops and scientists conducting field research at a large scale.<br>Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2020

Development of a winter-hardy spring wheat breeding platform could increase the gain in selection per year over traditional winter wheat breeding programs. To make use of spring wheat being able to produce three generations per year, an indoor cold tolerance screen using chlorophyll fluorescence (Fv/Fm) and visual assessment two weeks after freezing as evaluation parameters was developed. Evaluation of Ontario-adapted winter and spring wheat varieties demonstrated that the test was able to differentiate between winter and spring wheat. Specific varieties from this data set were used to develop an indoor freezing survival index (IFSI) to normalize data for effective ranking of germplasm in further experiments. Indepth analysis of a Froid (winter) x Siete Cerros (spring) wheat population using molecular markers indicated that a significant level of cold tolerance is preserved when the Vrn-B1 spring allele is used compared to the Vrn-A1 allele. Generation means analysis of the same cross indicated that the cold tolerance was due to additive genetic effects. Multiple populations with at least one spring parent were advanced to the F3:4 generation. IFSI analysis indicated that several lines from the populations had cold tolerance similar to Ontario-adapted winter wheats and better than several winter barley varieites. Further testing found a significant 5% improvement in cold tolerance was noted for spring wheat varieties treated with Cruiser Maxx seed treatment. Finally, a Norstar (winter) x Bergen (spring) doubled haploid wheat population was analysed and a significant correlation to LT50 data from an independent laboratory validated the methods used in these experiments. In a separate experiment, multiple indicies calculated from spectral reflectance measurements taken on the Ontario winter wheat performance trial at Elora and Harriston in 2008-09 were found to be significantly correlated to winter survival ratings. Fall reflectance measurements indicate non-random plant density or vigour effects in the trials. To adjust winter survival ratings accordingly, linear and non-linear approaches were used and found the non-linear model to be statistically superior. Large differences between locations illustrated that for complete modelling of winter survival, more data from locations of differing soil types, plant density and plant growth stage is required.

The African wild dog (AWD; Lycaon pictus) is endangered with the current population estimated at 6,600 animals, scattered over several subpopulations in Southern and Eastern Africa. They show a complex social structure including a separate male and female hierarchy and a cooperative breeding system where subdominants usually do not breed but help in raising the pups. To maintain a viable captive population and genetic diversity, animals are often translocated between institutions to form a new breeding pack. Similarly, a metapopulation management plan has been introduced in South Africa, involving the reintroduction of AWDs in small protected areas and regular translocations of individuals between subpopulations. However, due to their complex social structure, new pack formations can often lead to aggression between animals resulting in injury or even mortality. Sperm freezing, and development of artificial insemination (AI) techniques, can aid species management and conservation of the AWD. The use of semen cryopreservation and AI could overcome problems of intra-pack aggression associated with new pack formations by supplementing genetic diversity without disrupting existing pack structure; and thereby facilitating captive breeding and metapopulation management. In addition, transporting spermatozoa instead of live animals reduces the risk of disease transmission and has ecological and economic benefits. Sperm from free-roaming males could be used to increase genetic diversity in captivity, avoiding the removal of animals from the wild. Lastly, establishing a sperm bank of genetically valuable animals will provide a genetic back-up of the remaining population, providing a buffer against possible threats. Therefore, the aim of this thesis was to develop assisted breeding and behavioural management techniques to enable the application of AI in this species, through the following objectives: (i) determine the effect of social rank on subordinate male fertility (Chapter 2); (ii) develop a sperm freezing protocol (Chapter 3); (iii) determine if Dog Appeasing Pheromone (DAP) can reduce stress and aggression associated with temporary pack separation (Chapter 4); and (iv) validate the use of behaviour and faecal steroid hormone profiles as a non-invasive way to time the fertile period in AWD females for timed AI (Chapter 5). The study included n=15 males from 5 packs housed in zoological institutions in the US (ABQ, Albuquerque BioPark, Albuquerque, NM; TOP, Topeka Zoo, Topeka, KS; BRK, Brookfield Zoo, Chicago, IL; BIN, Binder Park Zoo, Battle Creek, MI; and OKC, Oklahoma City Zoo, Oklahoma City, OK) and n=13 males and n=3 females from 3 packs housed at Harnas Wildlife Foundation, Gobabis, Namibia (BRU, Brutus pack; PLA, Platform pack; SAN, San pack). Males were immobilised during the pre-breeding season (n=12; ABQ, BRK, BIN, TOP) and breeding season (n=24; ABQ, BRK, BIN, OKC, BRU, PLA, SAN) and male fertility parameters including hormones (faecal androgen - fAM and glucocorticoid metabolite - fGCM concentrations), prostate and testes volume, preputial gland size, semen collection success, and multiple measures of sperm quality were recorded (objective 1). Sperm samples of sufficient quality collected in the breeding season were split and frozen using 2 canine freezing protocols: Protocol 1: a one-step dilution in TRIS-20% egg yolk containing 8% glycerol; and Protocol 2: a two-step dilution in TRIS-20% egg yolk containing a final extender concentration of 5% glycerol and 0.5% Equex STM, coupled with a TRIS-citrate-fructose thawing solution (objective 2). In addition, males from US packs were treated topically either with DAP (n=11; 4 packs) or placebo solution (n=12, 4 packs), applied at the end of anaesthesia prior to reintroduction into the pack (objective 3). Behavioural interactions as well as fGCM and fAM were examined from 3 days before (objective 1) until 4-6 days after immobilisation (objective 3). Moreover, behavioural interactions, faecal progesterone (fPM) and estrogen (fEM) metabolite concentrations were examined for n=3 alpha females during their periovulatory period. Furthermore, each female was immobilised on 2 or 3 occasions at this time to evaluate vulvar size, and serum progesterone and oestrogen levels as well as perform vaginal cytology, vaginoscopy, and ovarian ultrasound (objective 4). Semen could be collected successfully from all alpha males but from only half the subordinate males in the pre-breeding season, with higher urine contamination in lower ranking animals. Fertility parameters did not differ between social ranks, except for a lower sperm progressive motility and normal morphology in subordinates. In the breeding season, preputial gland size increased with social rank, but no difference between ranks was observed in any other parameter, including sperm quality (objective 1). Eight ejaculates of sufficient quality were frozen in the breeding season. Sperm motility persisted for up to 8h after thawing for Protocol 2, while it dropped to nearly 0% after 2h incubation for Protocol 1. In addition, viability and acrosome integrity of spermatozoa were higher for Protocol 2 throughout post-thaw incubation (objective 2). The application of DAP to males at the end of anaesthesia and prior to reintroduction, did not alter the rise in fGCM levels after intervention. However, fAM increased in placebo but was prevented in DAP treated animals. On the day of reintroduction, DAP treated packs tended to show lower rates of contact-dominance and active-submission behaviour, but higher rates of non-contact dominance behaviour (objective 3). In females, late oestrus (fertile period) could be clearly distinguished from early oestrus by an increase in frequency of certain types of behavioural interactions between the alpha male and female (affiliative behaviour, sexual follow, male initiating behaviour, ride-up, and copulation). One female exhibited an anovulatory cycle while the remaining 2 showed a significant 2.5- to 3-fold increase in fPM levels and similar declining fEM levels (after a rise in pro-oestrus) compared to baseline. However, vaginal cytology and vaginoscopy results deviated from patterns seen in domestic dogs, and blood progesterone showed lower levels. Moreover, ovarian structures were difficult to visualise by ultrasound. As such, without frequent sampling, these invasive diagnostic techniques are unsuitable to determine the fertile phase in wild canids such as the African wild dog (objective 4). In conclusion, reproductive suppression of subordinate males appears to be behaviourally mediated, given that AWD males of all social ranks produce semen of similar quality during the breeding season, making them suitable candidates for sperm banking (objective 1). In addition, a two-step dilution in TRIS-egg yolk-glycerol extender containing Equex STM yields significantly improved post-thaw quality and longevity of AWD spermatozoa, making it appropriate for sperm banking and artificial insemination initiatives (objective 2). The observed effects of DAP on AWD hormones and behaviour could decrease the risk of agonistic interactions, making it a useful tool to help manage temporary pack separation, needed when performing semen freezing and AI (objective 3). Lastly, distinctive behaviours during late oestrus, together with an increase in faecal progesterone and decline in faecal oestrogen in AWD females, could potentially be used to determine the timing of the fertile period (objective 4). These results combined facilitate sperm banking and the application of AI in the African wild dog, thereby assisting management and conservation of the species.

Tese de Doutoramento em Biociências, especialização em Biotecnologia, apresentada à Faculdade de Ciências e Tecnologia da Universidade de Coimbra.<br>Arbutus unedo L. (medronheiro, Ericaceae) é uma árvore perene com uma distribuição circum-mediterrânica. Devido à sua resiliência a stresses abióticos e bióticos, tem grande relevância ecológica nas florestas do sul da Europa. As suas bagas vermelhas são comestíveis e usadas na produção de produtos tradicionais, incluindo um destilado de grande valor económico. Além disso, diversos compostos com atividade biológica têm sido usados na indústria farmacêutica e cosmética. De uma espécie negligenciada, o medronheiro tem vindo gradualmente a converter-se numa espécie de grande valor, com uma crescente área de plantação. Devido ao aumento da procura de plantas com características melhoradas por parte dos produtores, a implementação de um programa de melhoramento para a espécie é crucial. Apesar da intensa atividade de investigação realizada nos últimos anos em medronheiro, existem ainda muitas lacunas no conhecimento que terão de ser preenchidas, tais como o conhecimento da biologia da planta, otimização de protocolos de propagação e dos mecanismos por detrás da resistência aos stresses abiótico e biótico do medronheiro. Assim, três linhas principais de investigação foram seguidas nesta tese: i) desenvolvimento e otimização de protocolos de micropropagação (embriogénese somática, proliferação de meristemas e organogénese em meio líquido), avaliação da capacidade de tolerância ao stress hídrico das plantas micropropagadas e estudo do microbioma das plantas in vitro; ii) estudo da biologia reprodutiva, cruzamento e seleção de genótipos resistentes ao stress hídrico, e estudo dos mecanismos de tolerância em condições de stress hídrico; e iii) caracterização do microbioma, caracterização química, e a sua relevância na defesa da planta. A indução de embriogénese somática revelou-se particularmente eficaz com 2 mg L-1 de 6-benzilaminopurina (BAP) e 2 mg L-1 de ácido 1-naftalenoacético (NAA), com taxas de indução de 97,22% ± 4,81. No entanto, as taxas de indução variam em função do genótipo. Apesar de a fase de indução ser eficaz, a conversão de embriões em plantas é ainda limitada, o que poderá estar relacionado com a ocorrência de anomalias. A proliferação de meristemas em meio líquido revelou-se um método bastante eficaz assim como através de organogénese. Apesar de apresentaram sinais de vitrificação, as plantas recuperam o seu fenótipo normal e a sua performance em stresse hídrico, avaliada através de parâmetros fisiológicos, não é afetada. No que diz respeito à análise do microbioma, 79 géneros de bactérias pertencentes a 7 filos e apenas um género de Archaea foram identificados. O filo Actinobacteriota foi o mais abundante e diverso (48%), seguido pelo filo Proteobacteria (43%). Foram encontradas diferenças entre genótipos na composição do microbioma nas plantas in vitro. No entanto, essas diferenças diluíram-se quando as plantas foram aclimatizadas e um aumento significativo de diversidade foi igualmente observado. Um estudo morfológico das estruturas reprodutoras e da fenologia da planta permitiu a realização de cruzamentos controlados e obtenção de híbridos, que quando submetidos a condições de défice hídrico revelarem grande plasticidade fenotípica. Além da importância do genótipo na tolerância ao stresse hídrico, verificou-se que a proveniência das plantas poderá também ter a sua contribuição. Este trabalho confirma o fato de o medronheiro apresentar um comportamento tipicamente isohídrico em condições de stresse hídrico, através de um rigoroso controlo dos estomas. Um perfil metabólico específico foi encontrado num genótipo tolerante e várias hormonas chave (e.g., ácido abscísico e salicílico) mostraram estar regulados positivamente em plantas sob stresse hídrico. No que diz respeito à comunidade de endófitos cultivável, foram isoladas e identificadas diversas espécies de fungos (e.g., Aureobasidium pullulans e Trichoderma atroviride) e bactérias (ex. Bacillus cereus e Paenibacillus humicus). Alguns destes microrganismos produzem várias enzimas, tais como celulases e proteases, sideróforos, e outros compostos que podem desempenhar um papel essencial nos mecanismos de defesa da planta e reduzir o efeito dos mais relevantes patógenos do medronheiro, entre eles Phytophthora cinnamomi. Em particular, Trichoderma atroviride e Bacillus cereus revelaram ser antagonistas de vários fitopatógenos. Finalmente, através de análises químicas, foram identificados 54 compostos na folha, e o perfil químico mostrou variações entre genótipos e também sazonais. O extrato foliar da planta, assim como alguns compostos fenólicos isolados (arbutina e hidroquinona), mostraram ter atividade antifúngica, o que revela um complexo e intrincado mecanismo de defesa contras patógenos. Na sua globalidade, este trabalho interdisciplinar, realizado com a colaboração de diferentes laboratórios, representa um considerável avanço no conhecimento sobre a biologia de A. unedo. Assim, foi possível otimizar protocolos de micropropagação, compreender aspetos relacionados com a reprodução sexuada, analisar o papel de metabolitos secundários em termos de resposta ao stresse e perceber a interação da espécie com microrganismos. Além disso, os resultados obtidos abriram também caminho para novas linhas de investigação que poderão ajudar a promover a fileira do medronho e tornar ainda mais percetível a passagem desta espécie de negligenciada a espécie de interesse agrícola, especialmente em zonas onde outras espécies arbóreas ou arbustivas são difíceis de cultivar. As áreas de investigação consideradas prioritárias no futuro são também destacadas, tais como a seleção de plantas, o desenvolvimento de métodos rápidos de seleção através de marcadores metabólicos, a avaliação da introdução de endófitos em plantas produzidas em viveiro ou diretamente no campo, assim como o desenvolvimento de projetos de sequenciação genómica que permitam realizar estudos de biologia fundamental e a seleção com base em marcadores genómicos.<br>Arbutus unedo L. (strawberry tree, Ericaceae) is a perennial tree with a circum-Mediterranean distribution. It has a great ecological relevance in south Europe forests due to its resilience against abiotic and biotic stresses. The edible red berries are used in the production of traditional products, including a high-value spirit. Furthermore, several compounds with bioactive properties have been used by the cosmetic and the pharmaceutical industries. From a neglected species, strawberry tree has been gradually converted into a high valuable crop with an increased cultivation area in Southern European and Northern African countries. Due to an increasing demand for plants with improved features by farmers and other stakeholders, it is crucial to establish a breeding program for this species. Despite the intense research activity carried out in recent years in strawberry tree, some major knowledge gaps still need to be filled, such as our understanding of plant biology, optimization of propagation protocols and the mechanisms behind abiotic and biotic stress resistance. Thus, three main lines of investigation were followed in this thesis: i) development and optimization of micropropagation protocols (somatic embryogenesis, shoot proliferation and organogenesis in liquid medium), evaluation of the water stress tolerance capacity of micropropagated plants and study of the microbiome of plants in vitro; ii) study of reproductive biology, breeding and selection of genotypes resistant to water stress, and study of tolerance mechanisms under water stress conditions; and iii) microbiome characterization, chemical fingerprint, and their relevance for plant defence. Induction of somatic embryogenesis was particularly effective with 2 mg L-1 of 6-benzylaminopurine (BAP) and 2 mg L-1 of 1-naphthaleneacetic acid (NAA), with induction rates of 97.22% ± 4.81. However, induction rates vary depending on the genotype. Although the induction phase is effective, the conversion of embryos into plants is still limited, which could be related to the anomalies observed in several somatic embryos. The proliferation of meristems in liquid medium proved to be a very effective method as well as through organogenesis. Despite showing signs of hyperhydricity, the plants recover their normal phenotype and their performance under water stress, evaluated through physiological parameters, is not affected. Regarding the microbiome analysis, 79 bacterial genera belonging to 7 phyla and only one Archaea genus were identified. The phylum Actinobacteriota was the most abundant and diverse (48%), followed by the phylum Proteobacteria (43%). Differences between genotypes were found in the composition of the microbiome in plants in vitro. However, these differences were diluted when the plants were acclimatized and a significant increase in diversity was also observed. A morphological study of the reproductive structures and of the plant's phenology allowed the realization of controlled crossings and the obtainment of hybrids, which when subjected to conditions of water deficit reveal great phenotypic plasticity. In addition to the importance of the genotype in tolerance to water stress, it was found that the origin of the plants could also play an important role. This work confirms the fact that the strawberry tree presents a typical isohydric behaviour in water stress, through a rigorous control of the stomata and adds new scientific knowledge. A specific metabolic profile was found in the tolerant genotype and several key hormones (e.g., abscisic and salicylic acid) were up-regulated in plants under water stress. With regard to the cultivable endophyte community, several species of fungi (e.g., Aureobasidium pullulans and Trichoderma atroviride) and bacteria (e.g., Bacillus cereus and Paenibacillus humicus) were isolated and identified. Some of these microorganisms produce various enzymes, such as cellulases and proteases, siderophores, and other compounds that can play an essential role in the plant defence mechanisms and reduce the effect of the most relevant pathogens of strawberry tree, such as Phytophthora cinnamomi. In particular, Trichoderma atroviride and Bacillus cereus have been shown to be antagonists of several phytopathogens. Finally, through a chemical analysis, 54 compounds were identified in the leaf, and the chemical profile showed variations between genotypes and also seasonal. The plant leaf extract, as well as some isolated phenolic compounds (arbutin and hydroquinone) were shown to have antifungal activity, which reveals a complex and intricate defence mechanism against pathogens. Overall, this interdisciplinary work, carried out in collaboration with different laboratories, represents a considerable advance in the knowledge about the biology of A. unedo. Thus, it was possible to optimize micropropagation protocols, understand aspects related to sexual reproduction, analyze the role of secondary metabolites in terms of stress response and understand the interaction of the species with microorganisms. In addition, the results obtained also opened the way for new lines of research that could help to promote even further the conversion of this once neglected species into a plant of agricultural interest, especially in areas where other tree or shrub species are difficult to cultivate. Research areas considered to be priorities for the future are also highlighted, such as plant selection, the development of rapid selection methods using metabolic markers, the evaluation of the introduction of endophytes in nursery or directly on the field, as well as the development of genomic sequencing projects that allow for fundamental biology studies and selection based on genomic markers.<br>ReNATURE (CENTRO-01-0145-FEDER-000007), co-financed by the Regional Operational Programme Centro 2020, Portugal 2020, and the European Union, through the European Regional Development Fund (ERDF).

Increasingly, National Park Managers have begun to recognize the importance of understanding, assessing and managing cumulative effects. In Jasper National Park, the rarest habitat, namely the montane, contains the greatest intensity of human use and development in the park. I developed the Ecosite Representation and Breeding Bird Habitat Effectiveness Models to contribute to an established framework for assessing and managing cumulative effects in the high use area of the park. The Breeding Bird Model integrates call-count survey results, data delineating habitat types and quantifying human use with parameters developed from the literature in an Arc/info GIS. Similarly, the Ecosite Representation Model integrates habitat and human use data with a set of parameters derived from the literature. To assess cumulative effects on breeding bird habitat, I describe a functional relationship depicting the response of breeding bird species detected in the surveys to human activity and development. The relationship for ecosite representation assumes that within a disturbance distance of a human use feature, habitat is degraded. Through these relationships, data layers are integrated to predict cumulative effects, expressed as a change in the effectiveness of habitat for the indicators. This method tracks how the area lost and degraded changes over time and in response to different land use scenarios. Prior to using the models, I conducted a sensitivity analysis identifying the sources and influence of ecological uncertainty on model results. Following this, I completed a cumulative effects analysis which indicates that failure to assess and act on cumulative effects has resulted in impacts on both indicators concentrated in a group of montane habitat types. Development in Three Valley Confluence has predominantly been concentrated in eight habitat types, some of which are rare in abundance and the most important in the park for supporting breeding bird richness. Therefore, I recommend strategic land use planning to ensure new development and expansion does not continue within these habitat types and restoration efforts be undertaken to improve conditions for both indicators. I present several realistic options including planning based on clustering development, reducing access points, restoration and continuing development of a framework for cumulative effects assessment and management.

Thesis (Ph. D.)--University of Georgia, 2009.<br>Directed by John Ruter. Includes an article published in Journal of the American Society for Horticultural Science, articles accepted by HortScience and Native plants journal, and articles submitted to Scientia horticulturae, Journal of the American Society for Horticultural Science, and HortScience. For abstract see https://getd.libs.uga.edu/pdfs/contreras%5Fryan%5Fn%5F200912%5Fphd.pdf. Includes bibliographical references.

<p>Accurate assessments of behavior and welfare are needed to evaluate the state of domestic dogs maintained in commercial breeding (CB) and other types of kennels. Field assessments of dogs’ states of being must be reliable, valid and efficient. However, observer subjectivity and situational variation in dogs’ responses pose a challenge to incorporating behavioral metrics into welfare assessment tools. The published Field Instantaneous Dog Observation (FIDO) tool, designed to capture the immediately observable physical and behavioral status of dogs in kennels, was thus evaluated on its reliability and validity. Specifically, the main goals were to determine 1) reliability of the behavioral scoring when used by novice raters, 2) whether and to what extent dogs’ behavioral responses to stranger-approach changed during a 30-second observation period, and 3) the predictive power of the FIDO scoring on behavioral responses of dogs placed within a standardized arena with a stranger. Behavioral responses to stranger-approach were organized into three categories: red, indicating a fearful response to approach, green, indicating an affiliative or neutral response, and yellow, indicating an ambivalent response. In study one, behavior assessment was conducted by two novice raters with 50 dogs housed at two US shelters. A stranger approached the home pen of each dog in a non-threatening manner, stood quietly, extended a hand to the dog and scored the response while the test was video-recorded. Intra-rater reliability was assessed by comparing each rater’s live observation scores with their scores of the same dogs using video recordings. Inter-rater agreement between scores from video recordings was also calculated. In study two, 81 commercial breeding dogs maintained at four USDA-licensed CB facilities in the US were approached by one observer and scored once every five seconds for 30 seconds. Of the 81 subject dogs, 56 met the criteria for full sampling. In study three, 40 pairs of dogs were scored using the FIDO tool in their home pens at four USDA-licensed CB facilities and assessed on responses to an unfamiliar observer in a field test conducted in an outdoor arena. Behavioral measures from the field test such as latencies to approach the stranger and durations in different areas of the arena were captured from video recordings and subjected to a principal components analysis [PCA] for reduction of variables. A multivariate multiple regression analysis was thereafter used, with principal component scores obtained from the PCA as outcome variables, and FIDO scores as predictor variables, accounting for other factors such as breed and group composition. Results from study one indicated that raters showed almost perfect agreement between their own scores of live and video-recorded shelter dog responses (kappa = 0.83, 0.89) and between each other’s video-recorded scores (kappa = 0.87), indicating high intra- and inter-rater reliability. Results from study two indicated that over a 30-second time frame with five-second increments, 91% of the dogs showed no change in their behavioral response to approach. This suggests that the first five seconds of scoring provide a reliable time point for assessing behavior using the FIDO tool and indicates no benefit to extending the FIDO scoring period to gauge dogs’ immediate responses to stranger-approach. Results from study three showed that dogs scored as red, as determined by the FIDO scoring, also showed higher scores associated with avoidance, indicated by greater time spent away and more time taken to approach the unfamiliar person in the field test (p = 0.039). FIDO scoring was, however, not significantly predictive of other behavioral responses such as interaction with the unfamiliar person. How dogs were housed was also significantly associated with incidences of escape attempts from the arena; dogs paired as a mixed sex (i.e. male-female) showed lower scores on incidences of escape attempts than dogs paired as a same sex (i.e. female-female) (p = 0.003). Taken together, these results suggest that the FIDO tool can be used by individuals without much expertise in canine behavior, and also attest to the practicality of the tool via a reliable five-second approach. Further, results also indicate that the FIDO scoring can help to identify fearful dogs in need of greater socialization towards unfamiliar people. Future research would, however, be valuable in validating behavioral scores obtained using the tool against long-term indicators of overall welfare.</p>