Approcci molecolari e bioinformatici volti alla caratterizzazione di Vgt1, QTL coinvolto nella regolazione dell'epoca di fioritura in Zea Mays

The genetic control of flowering time has been addressed by many quantitative trait locus (QTL) studies. A survey of the results from 29 independent studies reporting information on 441 QTLs led to the production of a QTL consensus map, which enabled the identification of 59 chromosome regions distributed on all chromosomes and shown to be frequently involved in the genetic control of flowering time and related traits. One of the major QTLs for flowering time, the Vegetative to generative transition 1 (Vgt1) locus , corresponds to an upstream (70 kb) non-coding regulatory element of ZmRap2.7, a repressor of flowering. A transposon (MITE) insertion was identified as a major allelic difference within Vgt1. One of the hypotheses is that Vgt1 might function by modifying ZmRap2.7 chromatin through an epigenetic mechanism. Therefore, the methylation state at Vgt1 was investigated using an approach that combines digestion with McrBc, an endonuclease that acts upon methylated DNA, and quantitative PCR. The analyses were performed on genomic DNA from leaves of six different maize lines at four stages of development. The results showed a trend of reduction of methylation from the first to the last stage with the exception of a short genomic region flanking the MITE insertion, which showed a constant and very dense methylation throughout leaf development and for both alleles. Preliminary results from bisulfite sequencing of a small portion of Vgt1 revealed differential methylation of a single cytosine residue between the two alleles. ZmRap2.7 expression was assayed in the four developmental stages afore mentioned for the six genotypes, in order to establish a link between methylation at Vgt1 and ZmRap2.7 transcription. To assess the role of Vgt1 as a transcriptional enhancer, two reporter vectors for stable transformation of plants have been developed.

Mappaggio fine di un QTL principale per la resa in granella sul cromosoma 3B di frumento duro

In durum wheat, two major QTL for grain yield (Qyld.idw-2B and Qyld.idw-3B) and related traits were identified in a recombinant population derived from Kofa and Svevo (Maccaferri et al. 2008). To further investigate the genetic and physiological basis of allelic variation for this important trait, the fine mapping of Qyld.idw-2B e Qyld.idw-3B was done during the PhD. In this regard, new molecular markers were added to increase the map resolution in the target interval. For Qyld.idw-2B region COS markers derived from the synteny between wheat and rice/ sorghum /brachypodiu genomes were screened. While for Qyld.idw-3B region SSR, ISBP and COS markers obtained from BAC end-sequences and BAC sequences generated during the construction of the 3B physical map (Paux et al., 2008) were screened. In the RIL population a final map resolution of 2,8 markers/cM for Qyld.idw-2B and 0,6 markers/cM for Qyld.idw-3B were obtained. Eighteen pairs of near-isogenic lines (NILs) for Qyld.idw-3B were obtained from F4:5 heterogeneous inbred families. In order to confirm the phenotypic effect of the QTL all pairs were evaluated in field trials (2010 and 2011) for all traits. Three pairs of NILs, with contrasted haplotypes at the target region, were crossed to produce a large F2 population (ca. 7,500 plants in total) that was screened for the identification of recombinants. A total of 233 homozygous F4:5 segmental isolines were obtained and the phenotypic and genotypic characterization of these materials were done. A fine mapping for Qyld.idw-3B was obtained and the QTL peak was identified in a interval of 0,4 cM. All markers were anchored to the Chinese Spring physical map of chr. 3B, which allowed us to identify the BAC Contigs spanning the QTL region and to assign the QTL peak to Contig 954. Sequencing of this contig has revealed the presence of 42 genes.

Identifizierung und Quantifizierung der Ketocarotinoide in Dauerstadien von Grünalgen und Ketocarotinoidbiosynthese im Modellorganismus Chlamydomonas reinhardtii

Grünalgen bilden zur Überdauerung schlechter Umweltbedingungen Ruhestadien, die sich durch Ausbildung einer festen Zellwand, die Reduktion des Plastiden und die starke Akkumulation von Speicherfetten und Ketocarotinoiden im Zytosol auszeichnen. Obwohl Ketocarotinoide in Grünalgen seit über vierzig Jahren beforscht werden, gab es hierzu noch wenige molekularbiologische Untersuchungen. Im Vorfeld meiner Promotion wurde durch unsere Arbeitsgruppe entdeckt, dass auch der molekular gut zugängliche Modellorganismus Chlamydomonas reinhardtii im Zygotenstadium große Mengen an Ketocarotinoiden bildet. Neben dem zu erwartenden Ketocarotinoid Astaxanthin fanden wir große Mengen des bisher nur in einer Grünalge beschriebenen 4-Ketoluteins. Vorversuche ließen die Vermutung aufkommen, dass dieses Pigment bei der Untersuchung der Pigmentausstattung in Dauerstadien von vielen Grünalgen bisher übersehen wurde. rnIn der vorliegenden Arbeit wurde daher zunächst die Pigmentzusammensetzung von Dauerstadien der bereits gut untersuchten Grünalgen Muriella zofingiensis und Scenedesmus rubescens durch Vergleich mit dem Ketocarotinoidmuster aus Dauerstadien von C. reinhardtii und Fritschiella tuberosa reevaluiert und dabei erstmals das Vorkommen signifikanter Mengen an 4-Ketolutein nachgewiesen. Außerdem zeigte sich, dass die als bisheriger Modellorganismus der Ketocarotinoidbiosynthese in Grünalgen sehr gut untersuchte Alge Haematococcus pluvialis eher eine Ausnahme darstellt, da ihre Dauerstadien als einzige der hier untersuchten Algen nur minimale Mengen von 4 Ketolutein aufwiesen. Diese Beobachtungen machen es sehr wahrscheinlich, dass die Fähigkeit zur Bildung von 4-Ketolutein unter den Grünalgen wesentlich weiter verbreitet ist als bisher angenommen. Das sekundäre Carotinoid 4-Ketolutein kam in den Dauerstadien der Grünalgen neben seiner freien Form ausschließlich als Monoacylester vor, im Gegensatz zu Astaxanthin, das als mono- und diacylierte Form auftrat. rnÜber die Analyse der Pigmentausstattung hinaus konnten die entscheidenden Schritte des Synthesewegs der Ketocarotinoide in C. reinhardtii durch funktionelle Charakterisierung der beteiligten Enzyme in Bakterien aufgeklärt werden. Als Basis für die Charakterisierungen wurde ein umfangreiches Portfolio von carotinogenen E. coli-Bakterien etabliert, darunter α Carotin und Lutein produzierende Stämme, die bisher nicht zur Verfügung standen. Das wurde durch die Klonierung der Lycopinzyklase (OluLCY) aus der Grünalge Ostreococcus lucimarinus möglich, die eine Sonderolle unter den Zyklasen einnimmt, da sie die Lycopin-β-Zyklase und Lycopin-ε-Zyklase in einem Fusionsenzym vereint. Vorteile dieses Fusionsenzyms sind die Expressionskontrolle durch nur einen Promotor und die weitgehend konstante Stöchiometrie seiner Produkte α-Carotin und β-Carotin, was die OluLCY für die biotechnologische Anwendung prädestiniert.rnDie funktionelle Charakterisierung der Carotinoidbiosyntheseenzyme aus C. reinhardtii umfasste das Schlüsselenzym der Ketocarotinoidbiosynthese, die β-Carotin-Ketolase (BKT), sowie die Carotinoid-Hydroxylasen CHYB, CYP97A5 und CYP97C3. Dabei wurde für das BKT-Enzym aus C. reinhardtii nachgewiesen, dass es nicht nur die Ketolierung von β Carotin zu Canthaxanthin und von Zeaxanthin zu Astaxanthin, sondern auch die Bildung der von α-Carotin abgeleiteten Ketocarotinoide wie 4-Keto-α-Carotin und 4 Ketolutein katalysieren kann.rn

Fine Mapping of qroot-yield-1.06, a QTL for Root, Plant Vigor and Yield in Maize

Root-yield-1.06 is a major QTL affecting root system architecture (RSA) and other agronomic traits in maize. The effect of this QTL has been evaluated with the development of near isogenic lines (NILs) differing at the QTL position. The objective of this study was to fine map qroot-yield-1.06 by marker-assisted searching for chromosome recombinants in the QTL interval and concurrent root phenotyping in both controlled and field conditions, through successive generations. Complementary approaches such as QTL meta-analysis and RNA-seq were deployed in order to help prioritizing candidate genes within the QTL target region. Using a selected group of genotypes, field based root analysis by ‘shovelomics’ enabled to accurately collect RSA information of adult maize plants. Shovelomics combined with software-assisted root imaging analysis proved to be an informative and relatively highly automated phenotyping protocol. A QTL interval mapping was conducted using a segregating population at the seedling stage grown in controlled environment. Results enabled to narrow down the QTL interval and to identify new polymorphic markers for MAS in field experiments. A collection of homozygous recombinant NILs was developed by screening segregating populations with markers flanking qroot-yield-1.06. A first set of lines from this collection was phenotyped based on the adapted shovelomics protocol. QTL analysis based on these data highlighted an interval of 1.3 Mb as completely linked with the target QTL but, a larger safer interval of 4.1 Mb was selected for further investigations. QTL meta-analysis allows to synthetize information on root QTLs and two mQTLs were identified in the qroot-yield-1.06 interval. Trascriptomics analysis based on RNA-seq data of the two contrasting QTL-NILs, confirmed alternative haplotypes at chromosome bin 1.06. qroot-yield-1.06 has now been delimited to a 4.1-Mb interval, and thanks to the availability of additional untested homozygous recombinant NILs, the potentially achievable mapping resolution at qroot-yield-1.06 is c. 50 kb.

Calcolo strutturale ed ottimizzazione sedile in materiale composito per elicottero ultraleggero

Il lavoro svolto consiste nel processo di ottimizzazione strutturale di un sedile in materiale composito per un elicottero ultraleggero, attraverso un processo iterativo di simulazione FEM e modifiche al progetto, interpretando i dati ricavati dalle simulazioni, nel rispetto della normativa FAR 27. I risultati ottenuti hanno permesso di dimezzare il peso iniziale e resistere meglio agli sforzi, con implicazioni benefiche anche sul costo finale del prodotto che presenta minori costi del materiale e tempi di lavorazione ridotti sia in fase di laminazione che in fase di ciclo di cura in autoclave.

Dinoflagellate cyst counts and age model from sediment samples of ODP Hole 175-1085A

Sediment samples from ODP Site 1085 were investigated in order to obtain more information on the initiation and development of the Benguela upwelling system during the middle and upper Miocene. In particular, our intent was to establish the causes of the upwelling as well as the response of the upwelling regime to the development of the Antarctic Circumpolar Current. Based on changes in the calcareous dinoflagellate cyst association, we found an initial increase of the dinoflagellate cyst productivity, probably related to the initiation of upwelling about 11.8 Ma ago. Two distinct increases in cyst productivity in conjunction with temperature decreases of the upper water masses reflect upwelling pulses off Namibia and occur at the end of the Miocene cooling events Mi5 (about 11.5 Ma) and Mi6 (about 10.5 Ma). Both cooling events are associated with an ice volume increase in Antarctica and are thought to have led to an increase in southeasterly winds, possibly causing these two upwelling pulses. We demonstrate a decrease in dinoflagellate cyst productivity and enhanced terrigenous input via the Orange River after the Mi5 event. At about 11.1 Ma, the dinoflagellate cyst productivity increases again. The polar cyst species Caracomia arctica occurs here for the first time. This implies an influence of subantarctic mode water and therefore a change in the quality of the upwelling water which allowed the Benguela upwelling to develop into modern conditions. From about 10.4 Ma, C. arctica forms a permanent part of the association, pointing to an establishment of the upwelling regime.

(Table 2) Radiolaria species in nodule samples from the Clarion-Clipperton province, Pacific Ocean

Radiolaria were studied in 19 manganese nodules raised from the bottom. The nodules occurred mainly on the surface of thin Quaternary sediments covering Tertiary deposits of various ages (Middle Eocene to Early Miocene). Radiolaria in nodule cores and in inner and surface layers were studied. We found 85 radiolaria species and groups of species. Usually 1-4 to 6-19 radiolaria species were detected in each of the samples. Species belonging to Middle Eocene, Late Miocene to Early Oligocene, and Oligocene to Early Miocene were found. Rare Neogene species were revealed only in fractured surface layers. Age of the nodules is mainly Oligocene. Seismic waves cause sediment vibration, loosening disintegration, and removal of suspension by bottom currents. The vibration effect causes ancient nodules to float up to the surface of Quaternary sediment. This hypothesis suggests the reason for characteristics of the Clarion-Clipperton zone: regional stratigraphic hiatus, accumulation of residual fields of nodules, and the ''floating up'' of nodules to the surface of the Quaternary sediments.

Radiolarian biostratigraphy in the central Indian Ocean

Identifiable radiolarians of stratigraphic importance were recovered at eight of the sites drilled on Leg 115. The assemblages range in age from Holocene to middle Eocene (Dictyoprora mongolfieri Zone, about 48 Ma). Faunal preservation is particularly good in two stratigraphic intervals: the Holocene through upper Miocene (0-9 Ma), and the lowermost Oligocene to middle Eocene (35-48 Ma). Fluctuating rates of silica accumulation at these drill sites during the Cenozoic reflect changing tectonic and paleoceanographic conditions. In particular, the gradual closure of the Indonesian and Tethyan seaways and the northward migration of the Indian subcontinent severely restricted zonal circulation and silica accumulation in tropical latitudes during the late Oligocene through middle Miocene. By the late Miocene the Indian subcontinent had moved sufficiently north of the equator to allow trans-Indian zonal circulation patterns to become reestablished, and biosiliceous sedimentation resumed. The composition of the radiolarian assemblages in the tropical Indian Ocean is closely comparable with that of the 'stratotype' sequences in the equatorial Pacific. However, there are some notable exceptions in Indian Ocean assemblages: (1) the scarcity of the genera Pterocanium and Spongaster in the Neogene; (2) the absence of the stratigraphically important Podocyrtis lineage, P. diamesa -> P. phyxis -> P. ampla, in the middle Eocene; and (3) the scarcity of taxa of the genus Dorcadospyris, with the exception of D. ateuchus. The succession of radiolarian events was tabulated for those stratigraphic intervals where the assemblages were well preserved. We identified 55 events in the middle Eocene to earliest Oligocene, and 31 events in the late Miocene to Holocene. The succession of events is closely comparable with that of the tropical Pacific. However, there are exceptions that appear to be real, rather than artifacts of sample preservation, mixing, and core disturbance.