Academic literature on the topic 'Breeding tools'

The high cost of cultivar development encourages efficiencies to reduce time and costs to develop cultivars. Doubled haploid (DH) technology and marker assisted breeding (MAB) are two such tools that improve efficiencies. Since 1997, twenty five wheat cultivars in seven market classes, developed using DH methods, have been registered by the Canadian Food Inspection Agency. These DH cultivars accounted for more than one third of the Canadian wheat acreage in 2009. The DH cultivar Lillian, eligible for grades of Canada Western Red Spring class and currently the most widely grown wheat cultivar in Canada, was developed using MAB to improve grain protein content with the Gpc-B1/Yr36 on chromosome 6BS introgressed from Triticum turgidum L. (Zhuk.) dicoccoides (Körn. Ex Asch. & Graebn). AC Andrew, a Canada Western Soft White spring DH cultivar, was the most widely grown cultivar in its class for the last two years. The new market class, Canada Western Hard White Spring wheat, is based entirely on DH cultivars. Goodeve, one of the first Canada Western Red Spring cultivars released with the gene Sm1 on chromosome 2BS for resistance to the orange wheat blossom midge (Sitodiplosis mosellana (Géhin)) was selected by application of the DNA marker WM1. Glencross, the first cultivar in the Canada Western Extra Strong wheat class with Sm1, was selected using the WM1 marker on haploid plants prior to doubling. Development of the durum wheat cultivars CDC Verona and Brigade involved the use of a marker for Cdu1, a major gene on chromosome 5B that regulates grain cadmium concentration. Marker technology permits a more strategic and integrated approach to breeding by quantifying the introgression of various key genes into advanced breeding material, identifying targeted loci in parents and following up with MAB in the progeny.

Plant breeding is facing simultaneous challenges from a changing climate, increasing prices for nonrenewable inputs, and evolving consumer demands. Biotic and abiotic stresses are increasing with climate change; sustainable and stable production will require higher yields with lower inputs; consumers expect health-promoting, safe and traceable food. To meet these challenges, breeding requires more efficient tools with which to unlock and apply existing genetic diversity, to understand the relationship between genotype and phenotype, and to apply the approaches of biotechnology where appropriate. The growing genomics toolbox, based on genome projects for crop plants, offers much promise for acceleration of plant breeding. Here, these approaches will be explored with an emphasis on barley and wheat, which are the key cereal crops of Europe.

As climate changes and a growing global population continue to escalate the need for greater production capabilities of food crops, technological advances in agricultural and crop research will remain a necessity. While great advances in crop improvement over the past century have contributed to massive increases in yield, classic breeding schemes lack the rate of genetic gain needed to meet future demands. In the past decade, new breeding techniques and tools have been developed to aid in crop improvement. One such advancement is the use of speed breeding. Speed breeding is known as the application of methods that significantly reduce the time between crop generations, thereby streamlining breeding and research efforts. These rapid-generation advancement tactics help to accelerate the pace of crop improvement efforts to sustain food security and meet the food, feed, and fiber demands of the world’s growing population. Speed breeding may be achieved through a variety of techniques, including environmental optimization, genomic selection, CRISPR-Cas9 technology, and epigenomic tools. This review aims to discuss these prominent advances in crop breeding technologies and techniques that have the potential to greatly improve plant breeders’ ability to rapidly produce vital cultivars.

Maize will continue to expand and diversify as an industrial resource and a feed and fuel crop in the near future. The United Nations estimate that in 2050 the global population will reach 9.7 billion people. In this context, food security is increasingly being discussed. Additionally, another threat to food security is global warming. It is predicted that both the quantity and the quality of crops will be seriously affected by climate change in the near future. Scientists and breeders need to speed up the process of creating new maize cultivars that are resistant to climate stress without diminishing yield or quality. The present paper provides a brief overview of some of the most important genomics tools that can be used to develop high-performance and well-adapted hybrids of maize and also emphasizes the contribution of bioinformatics to an advanced maize breeding. Genomics tools are essential for a precise, fast, and efficient breeding of crops especially in the context of climate challenges. Maize breeders are able now to develop new cultivars with better traits more easily as a result of the new genomic approaches, either by aiding the selection process or by increasing the available diversity through precision breeding procedures. Furthermore, the use of genomic tools may in the future represent a way to accelerate the processes of de novo domestication of the species.

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.

Abstract This book, which is selected in nature to Agaricus bisporus, presents fundamental guidelines for mushroom production together with the advances in research in this field. The first chapter presents the history of button mushroom cultivation, mushroom classification, distribution, and nutritional and medicinal value. The methods of composting for substrate preparation, their basics, application, and innovation are discussed in Chapter 2. The basic principles and methods to improve compost quality are shown in the third chapter. Moving to the fourth chapter, the genetics, breeding approaches, and selection of new mushroom strains are discussed in detail. Chapter 5 addresses the stages of casing and cropping by focusing on the tools and methods to optimize production during these stages. Chapter 6 details the management of pests and control of diseases at a mushroom farm, with a special focus on the ideal farm design to avoid the spread of infesting agents. The last chapter of the book shows the advances in harvest and postharvest technologies, applied to maximize the postharvest benefits from button mushroom cultivation.

Abstract This chapter discussed the factors which affect responses to within-breed selection, and some of the tools and technologies used, especially for more effective within-breed selection. Highlights focused on the current reproductive technologies in animal breeding, molecular genetic tools, and modern data capture tools.

Abstract This chapter highlights the application of genetic principles such as strategies for genetic improvements, selection response within breeds, tools and technologies in animal breeding, genetic analysis, and predicting values in beef cattle.

Abstract This chapter discussed the effects of applying the different principles in animal breeding such genetic analysis, predicting breeding values, use of tools and breeding technology, selection response within breeds, and strategies for genetic improvements in dairy cattle.

The economic-productive activity of the Sarazm population in the Eneolithic period was mainly based on the agricultural way of life, accompanied by cattle breeding, fishing and hunting, which were of secondary importance. Livestock breeding allowed the involvement of various types of domestic crafts - leatherworking, wickerwork and weaving. The planigraphy of the finds of weaving and weaving tools reflects these activities in each dwelling complex. Stone tools associated with farming are defined - tillage, reaping, and grain processing. The types of domestic production also include: woodworking, pottery, metallurgy and metalworking. Woodworking is represented by household objects. Ceramic is represented by molded vessels of various shapes and purposes. Metalworking is represented by the items associated with the processing of ore, as well as with the manufacture and processing of metal products.

Meiotic recombination contributes to the generation of biodiversity as well as to genome stability, ensuring the proper segregation of homo logs during meiosis. It is also an essential step in the process of plant breeding. It generates the diversity needed by the breeder for selection of novel varieties. In this project, we have collaborated towards the goals to identify and characterize key genes involved in meiotic recombination. In addition we have shown how some of these genes can be used, through loss of function, or through overexpression, to enhance homologous recombination in Arabidopsis and in maize. Our main achievements can be summarized as follows: 1- To establish a collection of mutants, in Arabidopsis and in maize for candidate genes. In Arabidopsis, insertion mutants were isolated in the following genes: AtMSHI, AtMSH4, AtMSH5, AtMLH3, AtPHSl, and mutants in the Mre11/Rad50/Nbs1 complex. In maize, the TILLING system was established and enabled to isolate mutants in candidate genes, such as Rad2l-4a, Sgo2, and Aml. In addition, we generated phs 1 mutant alleles as well as mutants in the Mre11/Rad50/Nbs1 complex. No mus8l mutants have been found thus far. 2- We showed that mutants in AtMLH3 have decreased rates of crossover, suggesting that overexpression of these genes may enhance crossover. Mutants in AtMSHlhad no effect and mutants in AtMSH4 and 5 showed sterility. Overexpression of these genes might also enhance crossover. The effect of other mutations on crossovers in maize is still being investigated. 3- We showed that overexpression of AtMLH1, RecG and RuvC under a meioticspecific promoter enhances meiotic crossover in Arabidopsis. The effect of PHSloverexpression on crossover is expected to be determined soon. 4- New tools have been developed and perfected to study the recombination genes effect on meiotic crossovers. This includes antibodies, cDNAs and fusion proteins.

The goals of this project were to develop essential genomic tools for modern chickpea genetics and genomics research, map the genes and quantitative traits of importance to chickpea production and generate DNA markers that are well-suited for enhanced chickpea germplasm analysis and breeding. To achieve these research goals, we proposed the following research objectives in this period of the project: 1) Develop an ordered BAC library with an average insert size of 150 - 200 kb (USA); 2) Develop 300 simple sequence repeat (SSR) markers with an aid of the BAC library (USA); 3) Develop SSR marker tags for Ascochyta response, flowering date and grain weight (USA); 4) Develop a molecular genetic map consisting of at least 200 SSR markers (Israel and USA); 5) Map genes and QTLs most important to chickpea production in the U.S. and Israel: Ascochyta response, flowering and seed set date, grain weight, and grain yield under extreme dryland conditions (Israel); and 6) Determine the genetic correlation between the above four traits (Israel). Chickpea is the third most important pulse crop in the world and ranks the first in the Middle East. Chickpea seeds are a good source of plant protein (12.4-31.5%) and carbohydrates (52.4-70.9%). Although it has been demonstrated in other major crops that the modern genetics and genomics research is essential to enhance our capacity for crop genetic improvement and breeding, little work was pursued in these research areas for chickpea. It was absent in resources, tools and infrastructure that are essential for chickpea genomics and modern genetics research. For instance, there were no large-insert BAC and BIBAC libraries, no sufficient and user- friendly DNA markers, and no intraspecific genetic map. Grain sizes, flowering time and Ascochyta response are three main constraints to chickpea production in drylands. Combination of large seeds, early flowering time and Ascochyta blight resistance is desirable and of significance for further genetic improvement of chickpea. However, it was unknown how many genes and/or loci contribute to each of the traits and what correlations occur among them, making breeders difficult to combine these desirable traits. In this period of the project, we developed the resources, tools and infrastructure that are essential for chickpea genomics and modern genetics research. In particular, we constructed the proposed large-insert BAC library and an additional plant-transformation-competent BIBAC library from an Israeli advanced chickpea cultivar, Hadas. The BAC library contains 30,720 clones and has an average insert size of 151 kb, equivalent to 6.3 x chickpea haploid genomes. The BIBAC library contains 18,432 clones and has an average insert size of 135 kb, equivalent to 3.4 x chickpea haploid genomes. The combined libraries contain 49,152 clones, equivalent to 10.7 x chickpea haploid genomes. We identified all SSR loci-containing clones from the chickpea BAC library, generated sequences for 536 SSR loci from a part of the SSR-containing BACs and developed 310 new SSR markers. From the new SSR markers and selected existing SSR markers, we developed a SSR marker-based molecular genetic map of the chickpea genome. The BAC and BIBAC libraries, SSR markers and the molecular genetic map have provided essential resources and tools for modern genetic and genomic analyses of the chickpea genome. Using the SSR markers and genetic map, we mapped the genes and loci for flowering time and Ascochyta responses; one major QTL and a few minor QTLs have been identified for Ascochyta response and one major QTL has been identified for flowering time. The genetic correlations between flowering time, grain weight and Ascochyta response have been established. These results have provided essential tools and knowledge for effective manipulation and enhanced breeding of the traits in chickpea.

Chickpea is the third most important pulse crop in the world and ranks first in the Middle East; however, it has been subjected to only limited research in modern genomics. In the first period of this project (US-3034-98R) we constructed two large-insert BAC and BIBAC libraries, developed 325 SSR markers and mapped QTLs controlling ascochyta blight resistance (ABR) and days to first flower (DTF). Nevertheless, the utilities of these tools and results in gene discovery and marker-assisted breeding are limited due to the absence of an essential platform. The goals of this period of the project were to use the resources and tools developed in the first period of the project to develop a BAC/BIBAC physical map for chickpea and using it to identify BAC/BIBACcontigs containing agronomic genes of interest, with an emphasis on ABR and DTF, and develop DNA markers suitable for marker-assisted breeding. Toward these goals, we proposed: 1) Fingerprint ~50,000 (10x) BACs from the BAC and BIBAC libraries, assemble the clones into a genome-wide BAC/BIBAC physical map, and integrate the BAC/BIBAC map with the existing chickpea genetic maps (Zhang, USA); 2) fine-map ABR and DTFQTLs and enhance molecular tools for chickpea genetics and breeding (Shahal, Sherman and DaniShtienberg, Israel; Chen and Muehlbauer; USA); and 3) integrate the BAC/BIBAC map with the existing chickpea genetic maps (Sherman, Israel; Zhang and Chen, USA). For these objectives, a total of $460,000 was requested originally, but a total of $300,000 was awarded to the project. We first developed two new BAC and BIBAC libraries, Chickpea-CME and Chickpea- CHV. The chickpea-CMEBAC library contains 22,272 clones, with an average insert size of 130 kb and equivalent to 4.0 fold of the chickpea genome. The chickpea-CHVBIBAC library contains 38,400 clones, with an average insert size of 140 kb and equivalent to 7.5 fold of the chickpea genome. The two new libraries (11.5 x), along with the two BAC (Chickpea-CHI) and BIBAC (Chickpea-CBV) libraries (7.1 x) constructed in the first period of the project, provide libraries essential for chickpea genome physical mapping and many other genomics researches. Using these four libraries we then developed the proposed BAC/BIBAC physical map of chickpea. A total of 67,584 clones were fingerprinted, and 64,211 (~11.6 x) of the fingerprints validated and used in the physical map assembly. The physical map consists of 1,945 BAC/BIBACcontigs, with each containing an average of 39.2 clones and having an average physical length of 559 kb. The contigs collectively span ~1,088 Mb, being 1.49 fold of the 740- Mb chickpea genome. Third, we integrated the physical map with the two existing chickpea genetic maps using a total of 172 (124 + 48) SSR markers. Fourth, we identified tightly linked markers for ABR-QTL1, increased marker density at ABR-QTL2 and studied the genetic basis of resistance to pod abortion, a major problem in the east Mediterranean, caused by heat stress. Finally, we, using the integrated map, isolated the BAC/BIBACcontigs containing or closely linked to QTL4.1, QTL4.2 and QTL8 for ABR and QTL8 for DTF. The integrated BAC/BIBAC map resulted from the project will provide a powerful platform and tools essential for many aspects of advanced genomics and genetics research of this crop and related species. These includes, but are not limited to, targeted development of SNP, InDel and SSR markers, high-resolution mapping of the chickpea genome and its agronomic genes and QTLs, sequencing and decoding of all genes of the genome using the next-generation sequencing technology, and comparative genome analysis of chickpea versus other legumes. The DNA markers and BAC/BIBACcontigs containing or closely linked to ABR and DTF provide essential tools to develop SSR and SNP markers well-suited for marker-assisted breeding of the traits and clone their corresponding genes. The development of the tools and knowledge will thus promote enhanced and substantial genetic improvement of the crop and related legumes.

Global food security is increasingly threatened by numerous challenges, including widespread malnutrition. Nutritional deficiency, particularly in essential micronutrients such as Zinc (Zn) and Iron (Fe), affects over three billion people worldwide, with pregnant women and children being the most vulnerable. Wheat, as principal cereal crop, provides more than 50% of daily caloric for many populations, contributing essential nutrients such as proteins, vitamins, minerals, and phytochemicals. Wheat’s adaptability to diverse growing conditions and its wide production and consumption make it the most important staple crop globally, and a strong candidate for addressing nutritional deficiencies. However, enhancement of wheat’s quality traits is constrained by the limited genetic diversity within modern cultivars. In contrast, wild relatives and ancient cereals harbor significant genetic variation that can be exploited for crop improvement. Identifying, characterizing, and deploying key genetic loci for quality traits, facilitated by modern breeding tools such as marker-assisted selection (MAS),—offer pathways for the enhancement of both functional and nutritional qualities in wheat. This review examines the current understanding and advancements in wheat quality improvement, with an emphasis on ancient cereals and alien germplasms, and highlights the role of advanced breeding methodologies for optimizing the nutrition, sensory, and end-use qualities of wheat across different growing environments.

The Cucurbitaceae family includes a broad array of economically and nutritionally important crop species that are consumed as vegetables, staple starches and desserts. Fruit of these species, and types within species, exhibit extensive diversity as evidenced by variation in size, shape, color, flavor, and others. Fruit size and shape are critical quality determinants that delineate uses and market classes and are key traits under selection in breeding programs. However, the underlying genetic bases for variation in fruit size remain to be determined. A few species the Cucurbitaceae family were sequenced during the time of this project (cucumber was already sequenced when the project started watermelon and melon sequence became available during the project) but functional genomic tools are still missing. This research program had three major goals: 1. Develop whole genome cucumber and melon SNP arrays. 2. Develop and characterize cucumber populations segregating for fruit size. 3. Combine genomic tools, segregating populations, and phenotypic characterization to identify loci associated with fruit size. As suggested by the reviewers the work concentrated mostly in cucumber and not both in cucumber and melon. In order to develop a SNP (single nucleotide polymorphism) array for cucumber, available and newly generated sequence from two cucumber cultivars with extreme differences in shape and size, pickling GY14 and Chinese long 9930, were analyzed for variation (SNPs). A large set of high quality SNPs was discovered between the two parents of the RILs population (GY14 and 9930) and used to design a custom SNP array with 35000 SNPs using Agilent technology. The array was validated using 9930, Gy14 and F1 progeny of the two parents. Several mapping populations were developed for linkage mapping of quantitative trait loci (QTL) for fruit size These includes 145 F3 families and 150 recombinant inbred line (RILs F7 or F8 (Gy14 X 9930) and third population contained 450 F2 plants from a cross between Gy14 and a wild plant from India. The main population that was used in this study is the RILs population of Gy14 X 9930. Phenotypic and morphological analyses of 9930, Gy14, and their segregating F2 and RIL progeny indicated that several, likely independent, factors influence cucumber fruit size and shape, including factors that act both pre-anthesis and post-pollination. These include: amount, rate, duration, and plane of cell division pre- and post-anthesis and orientation of cell expansion. Analysis of F2 and RIL progeny indicated that factors influencing fruit length were largely determined pre-anthesis, while fruit diameter was more strongly influenced by environment and growth factors post-anthesis. These results suggest involvement of multiple genetically segregating factors expected to map independently onto the cucumber genome. Using the SNP array and the phenotypic data two major QTLs for fruit size of cucumber were mapped in very high accuracy (around 300 Kb) with large set of markers that should facilitate identification and cloning of major genes that contribute to fruit size in cucumber. In addition, a highly accurate haplotype map of all RILS was created to allow fine mapping of other traits segregating in this population. A detailed cucumber genetic map with 6000 markers was also established (currently the most detailed genetic map of cucumber). The integration of genetics physiology and genomic approaches in this project yielded new major infrastructure tools that can be used for understanding fruit size and many other traits of importance in cucumber. The SNP array and genetic population with an ultra-fine map can be used for future breeding efforts, high resolution mapping and cloning of traits of interest that segregate in this population. The genetic map that was developed can be used for other breeding efforts in other populations. The study of fruit development that was done during this project will be important in dissecting function of genes that that contribute to the fruit size QTLs. The SNP array can be used as tool for mapping different traits in cucumber. The development of the tools and knowledge will thus promote genetic improvement of cucumber and related cucurbits.

Tomato yellow leaf curl virus (TYLCV) is one of the most devastating viruses of cultivated tomatoes. Although first identified in the Mediterranean region, it is now distributed world-wide. Sequence analysis of the virus by the Australian group has shown that the virus is now present in Australia. Despite the importance of the disease and extensive research on the virus, very little is known about the resistance genes (loci) that determine host resistance and susceptibility to the virus. A symptom-less resistant line, TY-172, was developed at the Volcani Center which has shown the highest resistance level among all tested varieties. Preliminary results show that TY-172 is a good candidate to confer resistance to both TYLCV and to Tomato leaf curl virus (ToLCV) in Queensland conditions. Furthermore, Segregation analysis has previously indicated that the resistance is determined by 2-3 genes. In this proposal we aimed to substantiate that TY-172 can contribute to resistance breeding against TYLCV in Queensland, to develop DNA markers to advance such resistance breeding in both Israel and Queensland, and to exploit these markers for resistant breeding in Australian and Israeli lines. To map quantitative trait loci (QTLs) controlling TYLCVresistance in TY172, appropriate segregating populations were analyzed using 69 polymorphic DNA markers spanning the entire tomato genome. Results show that TYLCV resistance in TY172 is controlled by a previously unknown major QTL, originating from the resistant line, and four additional minor QTLs. The major QTL, termed Ty-5, maps to chromosome 4 and accounts for 39.7-to-46.6% of the variation in symptom severity among segregating plants (LOD score: 33-to-35). The minor QTLs, originated either from the resistant or susceptible parents, were mapped to chromosomes 1, 7, 9 and 11, and contributed 12% to the variation in symptom severity in addition to Ty-5. Further analysis of parental lines as well as large F₁, BC₁F₁, F₂ and BC₁F₂ populations originating from crosses carried out, in reciprocal manner, between TY172 and the susceptible processing line M-82 (LA3475) during spring-summer 2010, indicated that: (1) the minor QTLs we have previously identified are in effect not reproducible, (2)Ty-5 alone can yield highly resistant plants with practically no extra-chromosomal effects, and (3) the narrow-sense heritability estimate of resistance levels, attributed to additive factors responsive to selection, does not significantly deviate from 1. All of these results point to Ty-5 as the sole resistance locus in TY172 thus significantly increasing the likelihood of its successful molecular dissection. The DNA markers developed during the course of this study were transferred together with the TY172 genotype to Queensland. TY172 was crossed to a panel of Australian genotypes and the resulting populations were subjected to segregation analysis. Results showed that resistant locus, Ty-5, is highly reproducible in the Australian conditions as well. The Australian group was also able to make improvements to the marker assays by re-designing primer pairs to provide more robust PCR fragments. The Ty-5 locus has now been introgressed into elite Australian germplasm and selection for TYLCV resistance has begun. Cumulatively, our results show that Ty-5 can be effectively used, together with the TY172 genotype to expedite TYLCV resistance breeding and improve our understanding of the genetics that underline the response of tomato to TYLCV. Contributions to agriculture include: (1) the development of tools for more efficient resistance breeding, allowing the incorporation of resistance to local tomato varieties in Australia, Israel and elsewhere; and (2) establish a solid framework for a future attempt to clone the genes that encode such resistance. The latter will enable to decipher the resistance mechanisms that could be applied to other geminiviruses in tomato and possibly in other plant species.

Mango is one of the most important fruit crops. However, the biology of this fruit tree is under studied. The lack of genetic and genomic resources has limited progress in mango research and breeding. Several research groups have recently started developing genomic tools for mango by creating transcriptome and genomic data. Sexual reproduction in plants is the main pathway for the creation of new genetic combinations. In modern agriculture, breeders exploit the genetic diversity generated through sexual reproduction to develop elite cultivars; however, these cultivars require genetic stabilization before they are suitable for mass propagation for uniform crop production. In heterozygous plants such as fruit trees, vegetative propagation (cloning) is the primary path for the propagation of genetically uniform plants. Another natural plant mechanism that can create genetically uniform plants (clones) is apomixes. Apomixis is defined as asexual reproduction through seeds that lead to the production of clonal progeny whose genotype is identical to that of the mother plant. In fruit crops like citrus and mango, sporophytic apomixes result in polyembryony, where seeds contain multiple embryos, one of which is sexually originated, and the others are clones of the mother tree. As part of this research, the reference genome of mango was established as a basic platform for mango breeding and research. It was used to map two important mango traits fruit size and polyembryony. The draft genome 'Tommy Atkins' sequence was generated using NRGene de-novo Magic on high molecular weight DNA of 'Tommy Atkins,' supplemented by 10X Genomics long read sequencing to improve the initial assembly. The final 'Tommy Atkins' genome assembly was a consensus sequence that included 20 pseudomolecules representing the 20 chromosomes of mango. The availability of a genome enables the genetic dissection of important traits. We demonstrated the utility of the genome assembly and the 'Tommy Atkins' x 'Kensington Pride' map by analyzing fruit weight phenotypic data and identifying two QTLs for this trait.

Background and objectives: Manipulation of plant organ growth is one of the primary reasons for the success of mankind allowing increasing amounts of food for human and livestock consumption. In contrast with the successful selection for desirable growth characteristics using plant breeding, transgenic manipulations with single genes has met limited success. While breeding is based on accumulation of many small alterations of growth, usually arise from slight changes in expression patterns, transgenic manipulations are primarily based on drastic, non-specific up-regulation or knock down of genes that can exert different effects during different stages of development. To successfully harness transgenic manipulation to attain desirable plant growth traits we require the tools to subtly regulate the temporal and spatial activity of plant growth genes. Polar morphology along the adaxial/abaxial axis characterizes lateral organs of all plants. Juxtaposition of two cell types along this axis is a prerequisite of laminar growth induction. In the study summarized here, we addressed the following questions: Can we identify and harness components of the organ polarity establishment pathway for prolonged growth? Can we identify specific regulatory sequences allowing spatial and temporal manipulation in various stages of organ development? Can we identify genes associated with YABBY-induced growth alterations? Major conclusions and implications: We showed that regulated expression, both spatially and temporally of either organ polarity factors such as the YABBY genes, or the organ maturation program such as the CIN-TCPs can stimulate substantial growth of leaves and floral organs. Promoters for such fine manipulation could be identified by comparison of non-coding sequences of KAN1, where a highly conserved domain was found within the second intron, or by examination of multiple 5” regions of genes showing transient expression along leaf ontogeny. These promoters illustrate the context dependent action of any gene we examined thus far, and facilitate fine tuning of the complex growth process. Implications, both scientific and agricultural. The present study was carried out on the model organism Arabidopsis, and the broad application of its findings were tested in the tomato crop. We learned that all central regulators of organ polarity are functionally conserved, probably in all flowering plants. Thus, with minor modifications, the rules and mechanisms outlined in this work are likely to be general.

Background The tomato yellow leaf curl disease (TYLCV) has been a major deterrent to tomato production in Israel for the last 20 years. This whitefly-transmitted viral disease has been found in the Caribbean Island in the early 1990s, probably as an import from the Middle East. In the late 1990s, the virus has spread to the US and is now conspicuous in Florida and Georgia. Objectives Because of the urgency facing the TYLCV epidemics, there was a compelling need to mobilize scientists to develop tomato variety resistant to TYLCV. The major goal was to identify the virus movement protein (MP) and to express a defective from of MP in a cultivar that contained the natural Ty-1 resistance gene. The research included 1. cloning of the TYLCV isolate from the Dominican Republic (DR) which is (or a close variant) also present in the continental USA; 2. ddefining the role of the MP; 3. mutating the putative MP gene; 4. introducing the modified gene into an advance Ty-1 line; 5. testing the transgenic plants in the field. The pressing threat to tomato production in the US resulted in an extension of the objectives: more emphasis was placed on characterization of TYLCV i the DR, on determination of the epidemiology of the virus in the DR, and on using new TYLCV resistance sources for tomato breeding. Achievements and signification 1. The characterization of TYLCV-DR allowed for more effective TYLCV management strategies that are now implemented in the DR. 2. The identification of the TYLCV MPs and, more importantly, insight into their function has provided a model for how these proteins function in TYLCV movement and support the targeting of one or more of these proteins in a dominant lethal strategy to engineer plants for TYLCV resistance. 3. The transgenic plants that are being generated with wild-type and mutated TYLCV MPs will serve to test the hypothesis that interference with one or more of the TYLCV movement proteins will be a strategy for generating TYLCV-resistant plants. 4. The fine mapping of the resistance Ty-1 gene allowed eliminating deleterious chromosome segments from the wild tomato genitor L. chilense. It may in a near future allow the cloning of the first geminivirus resistance gene. 5. Another resistance source from the wild tomato species L. hirsitum was introgressed into the domesticated tomato, resulting in the production of resistant breeding lines. Implications 1. The monitoring of TYLCV in whiteflies has been applied in the DR. These tools are presently being used to assist in the evaluation of the host-free period and to help select the appropriate locations for growing tomatoes in the DR. 2. An overall strategy to obtain resistance against TYLCV has been used. The expression of wild-type or mutated TYLCV MPs in transgenic tomato is another addition to the arsenal used to fight TYLCV, together with marker assisted breeding and mobilization of additional resistant genes from the wild.

The general goals of the BARD research grant US-4423-11 are to understand how Or regulates carotenoid accumulation and to reveal novel strategies for breeding agricultural crops with enhanced β-carotene level. The original objectives are: 1) to identify the genes and proteins in the Or regulatory network in melon; 2) to genetically and molecularly characterize the candidate genes; and 3) to define genetic and functional allelic variation of these genes in a representative germplasm collection of the C. melo species. Or was found by the US group to causes provitamin A accumulation in chromoplasts in cauliflower. Preliminary genetic study from the Israeli group revealed that the melon Or gene (CmOr) completely co-segregated with fruit flesh color in a segregating mapping population and in a wide melon germplasm collection, which set the stage for the funded research. Major conclusions and achievements include: 1). CmOris proved to be the gene that controls melon fruit flesh color and represents the previously described gflocus in melon. 2). Genetic and molecular analyses of CmOridentify and confirm a single SNP that is responsible for the orange and non-orange phenotypes in melon fruit. 3). Alteration of the evolutionarily conserved arginine in an OR protein to both histidine or alanine greatly enhances its ability to promote carotenoid accumulation. 4). OR promotes massive carotenoid accumulation due to its dual functions in regulating both chromoplast biogenesis and carotenoid biosynthesis. 5). A bulk segregant transcriptome (BSRseq) analysis identifies a list of genes associated with the CmOrregulatory network. 6). BSRseq is proved to be an effective approach for gene discovery. 7). Screening of an EMS mutation library identifies a low β mutant, which contains low level of carotenoids due to a mutation in CmOrto produce a truncated form of OR protein. 8). low β exhibits lower germination rate and slow growth under salt stress condition. 9). Postharvest storage of fruit enhances carotenoid accumulation, which is associated with chromoplast development. Our research uncovers the molecular mechanisms underlying the Or-regulated high level of carotenoid accumulation via regulating carotenoidbiosynthetic capacity and storage sink strength. The findings provide mechanistic insights into how carotenoid accumulation is controlled in plants. Our research also provides general and reliable molecular markers for melon-breeding programs to select orange varieties, and offers effective genetic tools for pro-vitamin A enrichment in other important crops via the rapidly developed genome editing technology. The newly discovered low β mutant could lead to a better understanding of the Or gene function and its association with stress response, which may explain the high conservation of the Or gene among various plant species.