Generation of drought-resistant transgenic cereals using transcription factors isolated from wheat grain

Tese de doutoramento em Ciências da Saúde, ramo de Medicina, na especialidade de Medicina Interna (Cardiologia), apresentada à Faculdade de Medicina da Universidade de Coimbr

Application of next-generation sequencing technology to study genetic diversity and identify unique SNP markers in bread wheat from Kazakhstan

BACKGROUND: New SNP marker platforms offer the opportunity to investigate the relationships between wheat cultivars from different regions and assess the mechanism and processes that have led to adaptation to particular production environments. Wheat breeding has a long history in Kazakhstan and the aim of this study was to explore the relationship between key varieties from Kazakhstan and germplasm from breeding programs for other regions. RESULTS: The study revealed 5,898 polymorphic markers amongst ten cultivars, of which 2,730 were mapped in the consensus genetic map. Mapped SNP markers were distributed almost equally across the A and B genomes, with between 279 and 484 markers assigned to each chromosome. Marker coverage was approximately 10-fold lower in the D genome. There were 863 SNP markers identified as unique to specific cultivars, and clusters of these markers (regions containing more than three closely mapped unique SNPs) showed specific patterns on the consensus genetic map for each cultivar. Significant intra-varietal genetic polymorphism was identified in three cultivars (Tzelinnaya 3C, Kazakhstanskaya rannespelaya and Kazakhstanskaya 15). Phylogenetic analysis based on inter-varietal polymorphism showed that the very old cultivar Erythrospermum 841 was the most genetically distinct from the other nine cultivars from Kazakhstan, falling in a clade together with the American cultivar Sonora and genotypes from Central and South Asia. The modern cultivar Kazakhstanskaya 19 also fell into a separate clade, together with the American cultivar Thatcher. The remaining eight cultivars shared a single sub-clade but were categorised into four clusters. CONCLUSION: The accumulated data for SNP marker polymorphisms amongst bread wheat genotypes from Kazakhstan may be used for studying genetic diversity in bread wheat, with potential application for marker-assisted selection and the preparation of a set of genotype-specific markers.Yuri Shavrukov, Radoslaw Suchecki, Serik Eliby, Aigul Abugalieva, Serik Kenebayev and Peter Langridg

Complex regulation by Apetala2 domain-containing transcription factors revealed through analysis of the stress-responsive TdCor410b promoter from durum wheat

Expression of the wheat dehydrin gene Cor410b is induced several fold above its non-stressed levels upon exposure to stresses such as cold, drought and wounding. Deletion analysis of the TdCor410b promoter revealed a single functional C-repeat (CRT) element. Seven transcription factors (TFs) were shown to bind to this CRT element using yeast one-hybrid screens of wheat and barley cDNA libraries, of which only one belonged to the DREB class of TFs. The remaining six encoded ethylene response factors (ERFs) belong to three separate subfamilies. Analysis of binding selectivity of these TFs indicated that all seven could bind to the CRT element (GCCGAC), and that three of the six ERFs could bind both to the CRT element and the ethylene-responsive GCC-box (GCCGCC). The TaERF4 subfamily members specifically bound the CRT element, and did not bind either the GCC-box or DRE element (ACCGAC). Molecular modeling and site-directed mutagenesis identified a single residue Pro42 in the Apetala2 (AP2) domain of TaERF4-like proteins that is conserved in monocotyledonous plants and is responsible for the recognition selectivity of this subfamily. We suggest that both DREB and ERF proteins regulate expression of the Cor410b gene through a single, critical CRT element. Members of the TaERF4 subfamily are specific, positive regulators of Cor410b gene expression.Omid Eini, Nannan Yang, Tatiana Pyvovarenko, Katherine Pillman, Natalia Bazanova, Natalia Tikhomirov, Serik Eliby, Neil Shirley, Shoba Sivasankar, Scott Tingey, Peter Langridge, Maria Hrmova, Sergiy Lopat

Tissue specific promoters from rice and wheat for modifying grain characteristics

Trabalho final do 6º Ano Médico com vista à atribuição do grau de mestre no âmbito do ciclo de estudos de Mestrado Integrado em Medicina, apresentada à Faculdade de Medicina da Universidade de CoimbraA tiroidite, inflamação da glândula tiroideia, é, juntamente com o bócio, a afecção endócrina mais frequente, sendo comummente encontrada em medicina ambulatória. Associada a uma função tiroideia normal, aumentada ou diminuída (frequentemente com evolução de uma condição para outra), as circunstâncias da sua descoberta podem ser variadas e a distinção dos seus vários tipos baseia-se, essencialmente, no panorama clínico, rapidez de instalação sintomática, história familiar e presença ou ausência de sintomas prodrómicos e dor no pescoço. Vários critérios podem ser utilizados para a sua classificação, nomeadamente histológicos e clínicos (acompanhada ou não de dor tiroideia), sendo, contudo, a evolução da doença o critério classicamente utilizado. Assim, de acordo com este último, os diferentes subtipos de tiroidite podem ser agrupados em: tiroidite aguda, tiroidite subaguda ou tiroidite crónica. A primeira é uma forma dolorosa de tiroidite extremamente rara causada por uma infecção bacteriana, fúngica ou parasitária da tiróide e surgindo, sobretudo, na criança/adolescente e adulto jovem. Nas tiroidites subagudas, podemos encontrar a tiroidite subaguda granulomatosa ou de De Quervain – causa mais frequente de dor tiroideia, eventualmente, de origem viral – e as tiroidites subagudas linfocíticas – tiroidites esporádica indolor, do pós-parto, iatrogénica (interferão, interleucina-2, lítio), tóxica (amiodarona), por irradiação (iodo 131, irradiação externa) ou traumática (cirurgia, punção, traumatismo externo). Finalmente, nas tiroidites crónicas, caracterizadas, portanto, por um maior tempo de evolução, podemos destacar a tiroidite de Riedel, de natureza fibrótica e fisiopatologia desconhecida, e a tiroidite de Hashimoto, a tiroidite mais frequente e causa mais comum de hipotiroidismo nas regiões com aporte suficiente de iodo e da qual as tiroidites esporádica indolor e do pós-parto se aproximam pelo seu carácter autoimune. O diagnóstico destas afecções é feito pelo contexto e achados clínicos, incluindo a presença ou ausência de dor, textura e autoanticorpos. Adicionalmente, o grau de absorção de iodo radioactivo pela iii glândula é reduzido na maioria dos pacientes com inflamação viral, induzida por radiação, traumática, autoimune ou induzida por drogas. O tratamento é, primeiramente, dirigido ao alívio sintomático da dor tiroideia e restabelecimento do eutiroidismo.Thyroiditis, inflammation of the thyroid gland, is, along with goiter, the most frequent endocrine pathology, being commonly found in ambulatory medicine. Associated with normal, elevated or depressed thyroid function (often with evolution from one condition to another), the circumstances of its discovery are varied and the distinction of its several types is based primarily on the clinical setting, rapidity of symptom onset, family history and presence or absence of prodromal symptoms and neck pain. Different criteria can be used to its classification, namely histological and clinical (with thyroid pain or not). Nevertheless, the evolution of the disease is the classically used criterion. Thus, accordingly with this one, the several subtypes of thyroiditis can be divided into: acute thyroiditis, subacute thyroiditis and chronic thyroiditis. The first one is an extremely rare painful form of thyroiditis cause by a bacterial, fungic or parasitic infection of the thyroid and it in children/adolescents and young adults. In subacute thyroiditis, we can have the subacute granulomatous or de De Quervain thyroiditis – most common cause of thyroid pain, eventually of viral original – and the subacute lymphocytic thyroiditis – sporadic painless, postpartum, iatrogenic (interferon, interleukin-2, lithium), toxic (amiodarone), by irradiation (iodine 131 and external irradiation) or traumatic (surgery, puncture or external traumatism) thyroiditis. Finally, in chronic thyroiditis, characterized, therefore, by a longer time of evolution, we can mention Riedel’s thyroiditis, of fibrotic nature and unknown physiopathology, and Hashimoto’s thyroiditis, the most frequent thyroiditis and the most common cause of hypothyroidism in the regions with an adequate iodine supply, from which the sporadic painful and postpartum thyroiditis are close by their autoimune character. Diagnosis is by clinical context and findings, including the presence or absence of pain, tenderness and autoantibodies. In addition, the degree of radioactive iodine uptake by the gland is reduced in most patients with viral, radiation-induced, traumatic, autoimmune, or drug-induced inflammation of the thyroid. Treatment primarily is directed at symptomatic relief of the thyroid pain and restoration of euthyroidism

A biolistic method for high-throughput production of transgenic wheat plants with single gene insertions

Published: 26 June 2018Background: The relatively low efficiency of biolistic transformation and subsequent integration of multiple copies of the introduced gene/s significantly complicate the genetic modification of wheat (Triticum aestivum) and other plant species. One of the key factors contributing to the reproducibility of this method is the uniformity of the DNA/gold suspension, which is dependent on the coating procedure employed. It was also shown recently that the relative frequency of single copy transgene inserts could be increased through the use of nanogram quantities of the DNA during coating. Results: A simplified DNA/gold coating method was developed to produce fertile transgenic plants, via microprojectile bombardment of callus cultures induced from immature embryos. In this method, polyethyleneglycol (PEG) and magnesium salt solutions were utilized in place of the spermidine and calcium chloride of the standard coating method, to precipitate the DNA onto gold microparticles. The prepared microparticles were used to generate transgenics from callus cultures of commercial bread wheat cv. Gladius resulting in an average transformation frequency of 9.9%. To increase the occurrence of low transgene copy number events, nanogram amounts of the minimal expression cassettes containing the gene of interest and the hpt gene were used for co-transformation. A total of 1538 transgenic wheat events were generated from 15,496 embryos across 19 independent experiments. The variation of single copy insert frequencies ranged from 16.1 to 73.5% in the transgenic wheat plants, which compares favourably to published results. Conclusions: The DNA/gold coating procedure presented here allows efficient, large scale transformation of wheat. The use of nanogram amounts of vector DNA improves the frequency of single copy transgene inserts in transgenic wheat plants.Ainur Ismagul, Nannan Yang, Elina Maltseva, Gulnur Iskakova, Inna Mazonka, Yuri Skiba, Huihui Bi, Serik Eliby, Satyvaldy Jatayev, Yuri Shavrukov, Nikolai Borisjuk and Peter Langridg

Processes and vectors for producing transgenic plants

Publication Number: WO/2002/046440 International Application No.: PCT/EP2001/014421 Publication Date: 13.06.2002 International Filing Date: 07.12.2001 Chapter 2 Demand Filed: 01.07.2002This invention describes a process for gene expression in plants utilizing translational vectors. Said translational vectors cause a gene of interest to be stably integrated into a transriptionally active host genomic DNA such that the transcription the gene of interest is controlled by a promoter of the host organism. Said translational vectors are preferably based on internal ribosome entry site (IRES) elements that are of plant origin, plant viral origin, synthetic origin or are isolated from other organisms

Process of controlled shuffling of chromosome fragments for plant breeding

Publication Number: WO/2003/001900 International Application No.: PCT/EP2002/007134 Publication Date: 09.01.2003 International Filing Date: 27.06.2002 Chapter 2 Demand Filed: 03.01.2003Disclosed is a method of constructing a wild-species genomic library of chromosome fragments incorporated in a crop-species genome. First, a number of transformants for donor and recipient plant species is produced, carrying the DNA constructs necessary for the exchange of chromosomal fragments mediated by site-specific recombination. The donor and recipient are chosen such that, upon sexual cross or somatic cell fusion, they produce unstable progeny or demonstrate preferential segregation or sorting out. Second, the crossing between donor and recipient species and formation of chromosomal recombinants of donor and recipient plant species is induced. Third, taking advantage of the instability of hybrids between donor and recipient, recombinant cells and plants of the recipient are selected which contain specific chromosome fragments of the donor species. Also disclosed are transgenic plants, libraries and breeding material produced by the methods