Studio e caratterizzazione del colore rosso dell'epicarpo nella pera Max Red Bartlett, mutante della cv William

Durante il secolo scorso sono state individuate alcune mutazioni per il colore della buccia della variet�� William che invece di essere giallo arriva a maturazione con diverse tonalit�� di colore rosso. L���intensit�� e la tipologia del fenotipo dovuto a questa mutazione mostra una variabilit�� all���interno dei diversi cloni rossi di questa cultivar: Max Red Bartlett, Rosired e Sensation. Questa mutazione �� ereditabile e usando come genitore uno dei sopra-citati mutanti per il rosso sono state prodotte altre cultivar caratterizzate da buccia rossa come Cascade. Max Red Bartlett presenta una intensa colorazione rossa nelle prime fasi di maturazione per poi striarsi perdendo di lucentezza e non ricoprendo totalmente la superficie del frutto. Max Red Bartlett ha inoltre il problema di regressione del colore. Questa mutazione infatti non �� stabile e dopo qualche anno pu�� regredire e presentare il fenotipo di William. Diverso �� invece lo sviluppo per esempio di Rosired che durante le prime fasi di accrescimento del frutto �� identica a Williams (di colore verde con la parte del frutto rivolta verso il sole leggermente rossastra) per poi virare e mantenere un vivo colore rosso su tutta la superficie del frutto. Questa tesi si �� proposta di caratterizzare questa mutazione che coinvolge in qualche modo la via biosintetica per la sintesi del colore. In particolare si �� cercato di investigare sui probabili geni della via degli antociani coinvolti e in quale modo vengono espressi durante la maturazione del frutto, inoltre si �� cercato di trovare quali specifiche molecole venissero diversamente sintetizzate. Le cultivar utilizzate sono state William e Max Red Bartlett. Di quest���ultima era gi�� disponibile una mappa molecolare, ottenuta sulla popolazione di���incrocio di Abate Fetel (gialla) x MRB (rossa) con AFLP e SSR, quest���ultimi hanno permesso di denominare i diversi linkage group grazie alla sintenia con le altre mappe di pero e di melo. I semenzali appartenenti a questa popolazione, oltre a dimostrare l���ereditariet�� del carattere, erano per il 50% gialli e 50% rossi. Questo ha permesso il mappaggio di questo carattere/mutazione che si �� posizionato nel linkage group 4. Una ricerca in banca dati eseguita in parallelo ha permesso di trovare sequenze di melo dei geni coinvolti nella via biosintetica degli antociani (CHS, CHI, F3H, DFR, ANS e UFGT), sulle quali �� stato possibile disegnare primer degenerati che amplificassero su DNA genomico di pero. Le amplificazioni hanno dato frammenti di lunghezza diversa. Infatti nel caso di F3H e DFR l���altissima omologia tra melo e pero ha permesso l���amplificazione quasi totale del gene, negli altri casi invece �� stato necessario utilizzare primer sempre pi�� vicini in modo da facilitare l���amplificazione. I frammenti ottenuti sono stati clonati sequenziati per confermare la specificit�� degli amplificati. Non sono stati evidenziati polimorfismi di sequenza in nessuna delle sei sequenze tra William e Max Red Bartlett e nessun polimorfismo con Abate, per questo motivo non �� stato possibile mapparli e vedere se qualcuno di questi geni era localizzato nella medesima posizione in cui era stato mappato il ���colore/mutazione���. Sulle le sequenze ottenute �� stato possibile disegnare altri primer, questa volta specifici, sia per analisi d���espressione. Inizialmente �� stato sintetizzato il cDNA dei geni suddetti per retrotrascrizione da RNA estratto sia da bucce sia da foglie appena germogliate (le quali presentano solo in questa fase una colorazione rossastra in MRB ma non in William). Al fine di osservare come varia l���espressione dei geni della via biosintetica delle antocianine durante la fase di maturazione dei frutti, sono stati fatti 4 campionamenti, il primo a 45gg dalla piena fioritura, poi a 60, 90, 120 giorni. Foglie e bucce sono state prelevate in campo e poste immediatamente in azoto liquido. Dai risultati con Real Time �� emerso che vi �� una maggiore espressione nelle prime fasi di sviluppo in Max Red Bartlett per poi calare enormemente in giugno. Si potrebbe ipotizzare che ci sia una reazione di feed back da parte della piante considerando che in questa fase il frutto non si accresce. I livelli di espressione poi aumentano verso la fase finale della maturazione del frutto. In agosto, con l���ultimo campionamento vi �� una espressione assai maggiore in Max Red Bartlett per quei geni posti a valle della via biosintetica per la sintesi delle antocianine. Questo risultato �� confermato anche dal livello di espressione che si riscontra nelle foglie. In cui i geni F3H, LDOX e UFGT hanno un livello di espressione nettamente maggiore in Max Red Bartlett rispetto a William. Recentemente Takos et al (2006) hanno pubblicato uno studio su un gene regolatore della famiglia Myb e ci�� ha permesso di ampliare i nostri studi anche su questo gene. L���altissima omologia di sequenza, anche a livello di introni, non ha permesso di individuare polimorfismi tra le variet�� Abate Fetel e Max Red Bartlett, per nessun gene ad eccezione proprio del gene regolatore Myb. I risultati ottenuti in questa tesi dimostrano che in pero l���espressione relativa del gene Myb codificante per una proteina regolatrice mostra una netta sovra-espressione nel primo stadio di maturazione del frutto, in Max Red Bartlett 25 volte maggiore che in William. All���interno della sequenza del gene un polimorfismo prodotto da un microsatellite ha permesso il mappaggio del gene nel linkage group 9 in Max Red Bartlett e in Abate Fetel. Confrontando questo dato di mappa con quello del carattere morfologico rosso, mappato nel linkage group 4, si deduce che la mutazione non agisce direttamente sulla sequenza di questo gene regolatore, bench�� sia espresso maggiormente in Max Red Bartlett rispetto a William ma agisca in un altro modo ancora da scoprire. Infine per entrambe le variet�� (William e Max Red Bartlett) sono state effettuate analisi fenotipiche in diversi step. Innanzi tutto si �� proceduto con una analisi preliminare in HPLC per osservare se vi fossero differenze nella produzione di composti con assorbenza specifica delle antocianine e dei flavonoidi in generale. Si �� potuto quindi osservare la presenza di due picchi in Max Red Bartlett ma non in William. La mancanza di standard che coincidessero con i picchi rilevati dallo spettro non ha permesso in questa fase di fare alcuna ipotesi riguardo alla loro natura. Partendo da questo risultato l���investigazione �� proceduta attraverso analisi di spettrometria di massa associate ad una cromatografia liquida identificando con una certa precisione due composti: la cianidina-3-0-glucoside e la quercitina-3-o-glucoside. In particolare la cianidina sembra essere la molecola responsabile della colorazione della buccia nei frutti di pero. Successive analisi sono state fatte sempre con lo spettrometro di massa ma collegato ad un gas cromatografo per verificare se vi fossero delle differenze anche nella produzione di zuccheri e pi�� in generale di molecole volatili. L���assenza di variazioni significative ha dimostrato che la mutazione coinvolge solo il colore della buccia e non le caratteristiche gustative e organolettiche di William che restano inalterate nel mutante.

Studio genetico ed epidemiologico su Erysiphe necator agente eziologico dell'Oidio della vite

The objective was to analyse population structure and to determine genetic diversity of Erysiphe necator (syn. Uncinula necator) populations obtained from some vineyards located in the South-East Po valley (Italy). Powdery mildew is one of the most important fungal diseases of grapes (Vitis vinifera L.) throughout the world. The causal agent is the haploid, heterothallic ascomycete E. necator. It is an obligate biotrophic fungus and it can be found only on green organs of plants belonging to the family Vitaceae. For this pathogen, two sympatric populations (groups A and B) have been described in Europe and Australia. The two genetic groups differ at multiple genetic loci and previous studies reported a lack of interfertility among isolates of the two groups. There are now several well documented examples of plant pathogen species, such as Leptosphaeria maculans, Gaeumannomyces graminis var. tritici, Botrytis cinerea and Erysiphe syringae, which are indeed composed of genetically differentiated clades, that have led to the description of new groups or even new species. Several studies have suggested that genetic E. necator group A and B correlated with ecological features of the pathogen; some researchers proposed that group A isolates over-winter as resting mycelium within dormant buds, and in spring originate infected shoots, known as Flag shoots, while group B isolates would survive as ascospores in overwintering cleistothecia. However, the association between genetic groups and mode of over-wintering has been challenged by recent studies reporting that flag-shoot may be originated indifferently by group A or group B isolate. Previous studies observed a strong association between the levels of disease severity at the end of the growing season and the initial compositions of E. necator populations in commercial vineyards. The frequencies of E. necator genetic groups vary considerably among vineyards, and the two groups may coexist in the same vineyard. This finding suggests that we need more information on the genetics and epidemiology of E. necator for optimize the crop management In this study we monitored E. necator populations in different vineyards in Emilia ��� Romagna region (Italy), where the pathogen overwinters both as flagshoots and as cleistothecia. During the grape growing season, symptomatic leaves were sampled early in the growing season and both leaves and berries later during the epidemic growth of the disease. From each sample, single-conidial isolate was obtained. Each isolates was grown on V. vinifera leaf cv. Primitivo and after harvesting the mycelium, the DNA was purified and used as template for PCR amplification with SCAR primers (Sequences Characterised Amplified Region ), ���-tubulin, IGS sequences and Microsatellite markers (SSR). Amplified DNA from b-tubulin and IGS loci was digested with AciI and XhoI restriction enzymes, respectively, to show single-nucleotide polymorphisms specific for the two genetic groups. The results obtained indicated that SCAR primers are not useful to study the epidemiology. of E. necator conversely the b-tubulin IGS sequences and SSR. Summarize the results obtained with b-tubulin, IGS sequences, in treated vineyards we have found individuals of group B along all grape growing season, whereas in the untreated vineyard individuals of the two genetic groups A and B coexisted throughout the season, with no significant change of their frequency. DNA amplified from ascospores of single cleistothecia showed the presence of markers diagnostic for either groups A and B and were seldom observed also the coexistence of both groups within a claistothecium. These results indicate that individuals of the two groups mated in nature and were able to produced ascospores. With SSR we showed the possibility of recombination between A and B groups in field isolates. During winter, cleistothecia were collected repeatedly in the same vineyards sampling leaves fallen on ground, exfoliating bark from trunks, and from soil. From each substrate, was assess the percentage of cleistothecia containing viable ascospores. Our results confirmed that cleisthotecia contained viable ascospores, therefore they have the potential to be an additional and important source of primary inoculum in Emilia-Romagna vineyards.

Deep space orbit determination and guidance of the LICIACube microsatellite mission

Recently, the JPL's MarCO mission demonstrated that these probes are also mature enough to be employed in the deep space, even though with the limitations related to the employed commercial components. Currently, other deep space CubeSats are planned either as stand-alone missions or as companions of a traditional large probe. Therefore, developing a dedicated navigation suite is crucial to reaching the mission's goals, considering the limitations of the onboard components compared to typical deep space missions. In this framework, the LICIACube mission represents an ideal candidate test-bench, as it performs a flyby of the Didymos asteroid system subject to a strong position, epochs, and pointing requirements. This mission will also allow us to infer the capabilities of such microsatellites and highlight their limitations compared with the benefits of a lighter design and tailoring efforts. In this work, the OD and guidance methods and tools adopted for classical deep space missions have been tailored for the CubeSat applications and validated through extensive analyses. In addition, navigation procedures and interfaces have been designed in view of the operations foreseen in late 2022. The pre-launch covariance analysis has been performed to assess the mission's feasibility for the nominal trajectory and its associated uncertainties, based on conservative assumptions on the main parameters. Extensive sensitivity analyses have been carried out to understand the main mission parameters affecting the performance and to demonstrate the robustness of the designed trajectory and operation schedule in fulfilling the mission requirements. The developed system was also stressed by tuning the models to access different reconstruction methods for the maneuvers. The analysis demonstrated the feasibility of the LICIACube mission navigation in compliance with the mission requirements, compatible with the limited resources available, both in space and on the ground.

Characterization and cloning of chemically-induced mutants in barley (Hordeum vulgare L.)

Induced mutagenesis has been exploited for crop improvement and for investigating gene function and regulation. To unravel molecular mechanisms of stress resilience, we applied state-of-the-art genomics-based gene cloning methods to barley mutant lines showing altered root and shoot architecture and disease lesion mimic phenotypes. With a novel method that we named complementation by sequencing, we cloned NEC3, the causal gene for an orange-spotted disease lesion mimic phenotype. NEC3 belongs to the CYP71P1 gene family and it is involved in serotonin biosynthesis. By comparative phylogenetic analysis we showed that CYP71P1 emerged early in angiosperm evolution but was lost in some lineages including Arabidopsis thaliana. By BSA-Seq, we cloned the gene whose mutation increased leaf width, and we showed that the gene corresponded to the previously cloned BROADLEAF1. By BSA coupled to WGS sequencing, we cloned EGT1 and EGT2, two genes that regulate root gravitropic set point angle. EGT1 encodes a Tubby-like F-box protein and EGT2 encodes a Sterile Alpha Motive protein; EGT2 is phylogenetically related to AtSAM5 in Arabidopsis and to WEEP in peach where it regulates branch angle. Both EGT1 and EGT2 are conserved in wheat. We hypothesized that both participate to an anti-gravitropic offset mechanism since their disruption causes mutant roots to grow along the gravity vector. By the MutMap+ method, we cloned the causal gene of a short and semi-rigid root mutant and found that it encodes for an endoglucanase and is the ortholog of OsGLU3 in rice whose mutant has the same phenotype, suggesting that the gene is conserved in barley and rice. The mutants and the corresponding genes which were cloned in this work are involved in the response to stress and can potentially contribute to crop adaptation.

Characterization of an international tetraploid wheat germplasm including landraces and primitive wheat towards improved resilience to abiotic and biotic stresses and quality

This thesis aimed to characterise two large tetraploid germplasm collections. The Global Durum Panel, involving modern cultivars and landrances and the Tetraploid Global Collection which comprises all the tetraploid wheat subgroups. Two distinct parallel studies were carried out. The first is focused on the characterisation of both collection for yield and quality related traits. The panel were phenotyped for two consecutive years each. In this phase the following traits were collected: the number of fertile spikelets per spike, the number of fertile florets of central spikelet for the spike-related traits. The following grain related traits were also phenotyped: the thousand kernel weight, the average grain area, average grain length, average grain width, grain brightness, grain redness, grain yellowness. GWAS analysis were performed for each collected trait and major QTLs were subjected to candidate gene analysis. Major QTLs emerging from GWA study were located on chromosome 2A with a strong bibliographic evidence for grain number-related traits such as the fertile spikelet number, the number of fertile florets per central spikelet. On the other hand two evident peaks were detected on chromosomes 6A and 7B for grain size and weight related traits. The second work was focused on the characterisation of the Global Durum Panel for root system architecture components, namely the root growth angle. GWAS analysis was perfomed and three major QTLs were detected on chromosome 2A, 6A and 7A. These three QTLs all have a bibliographic evidence.

Red flesh fruit in european pear (Pyrus communis): genetic sources, QTLs, candidate genes and tools for breeding

Red flesh fruit is a character which interest is increasing in several commercial species. Following a review of the research on the biosynthesis and accumulation of anthocyanin in pears (Chapter 1) the general aim of the project is reported in Chapter 2. Chapter 3 reports the results of a molecular analysis of 33 red-fleshed pear accessions, genotyped with 18 SSR markers with the aim of improving germplasm conservation strategies to support ongoing breeding programs. The molecular profiles revealed both cases of synonymy and homonymy and 6 unique genotypes were identified. The S-allele were established to highlight the genetic relationships among these landraces. Four of the unique genotypes have been clustered based on pomological data. In the Chapter 4, the work was directed to identify the putative genomic regions involved in the appearance of this character in pear fruit. A crossing population (���Carmen��� x ���Cocomerina Precoce���) segregating for the trait was phenotyped for 2 consecutive years and used for QTL analysis. A strong QTL was identified in a small genomic region related to the red flesh fruit trait at 27 Mb from the start of LG5. Two candidate genes were detected in this genomic region: ���PcMYB114��� and ���PcABCC2���. SSR marker SSR114 was found able to detect the red flesh phenotype segregation in all the red-fleshed pear accessions and segregating progenies tested. Chapter 5 focuses on examining the trend of anthocyanin synthesis and accumulation during the fruit development, from fruit set to ripening time. Three different trials were planned: qPCR and HPLC methods were performed to correlate the genes expression with the anthocyanin accumulation in ���Cocomerina Precoce��� and six progenies. Total transcriptome sequencing was used to compare the differential genes expression between red and white-fleshed fruit. Chapter 6 reviews and analyses all the earlier study findings while providing new potential future perspectives.