CAPS markers in plant biology

Cleaved Amplified Polymorphic Sequences (CAPS) markers are applicable in a wide range of tasksin plant biology. They have been developed for plant genetics and breeding and become especially useful. This mini-review analyzes information about the application of CAPS markers within the past3–5 years. In the presented study, special attention is focused on CAPS markers linked with genes controlling important agricultural traits in different crops. The main principles of the developmentand analysis of CAPS markers, as well as advantages and disadvantages of this type of molecular markers, are briefly outlined in the beginning of this review. CAPS markers are based on PCR amplification of DNA fragments with specific primers followed by digestion with restriction enzymes and separation of the products in agarose gel. Functional CAPS markers can be developed on the known sequence of a gene of interest for the analyses of its structure, function, expression, and regulation. CAPS closely linked to the gene of interest are especially helpful for Marker-Assisted Selection, and they are widely used in the breeding of wheat, barley, soybean, potato, tomato, and other crops for tolerance to various pathogens. CAPS markers are often used for the preparation of genetic maps and fine mapping of studied genes. For some plants, first moleculargenetic maps were prepared using CAPS. This method was also successfully used for the mapping of both individual genes and QTLs controlling such important traits as plant growth habit, grain quality, and tolerance to pathogens in cereals, as well as the shape of tomato fruit. CAPS have important applications in the analyses of genetic polymorphism and phylogeny, particularly, in closely related species. Thus, CAPS are an effective tool for molecular-genetic research and plant breeding

Identification of a QTL on chromosome 7AS for sodium exclusion in bread wheat

Tese de doutoramento em Engenharia Física (Instrumentação), apresentada à Faculdade de Ciências e Tecnologia da Universidade de CoimbraO número de ciclotrões com capacidade para acelerar protões até cerca de 20 MeV tem vindo a aumentar em todo o mundo. Apesar de o objectivo principal das instalaçõoes que contêm estas máquinas ser a produção de radionuclídeos para tomografia por emissão de positrões (PET, do inglês positron emission tomography), algumas dessas instalações estão equipadas com várias linhas de feixe que podem ser adaptadas para investigação científica. Por exemplo, radiobiologia, radiofisiolgia e outros estudos de dosimetria podem ser realizados utilizando uma destas linhas de feixe devidamente adaptada para o efeito. Neste trabalho, uma das linhas de feixe do ciclotrão PET da Universidade de Coimbra foi complementada e instrumentada por forma a possibilitar a irradiação de um arranjo experimental com um feixe de protões de elevada qualidade. Esta nova linha de feixe foi construída de raíz e sem causar qualquer interferência com as demais linhas do ciclotrão, dedicadas à produção de radionuclídeos. São apresentados resultados tanto experimentais como de simulação, estes últimos obtidos através dos pacotes de simulação SRIM/TRIM e Geant4, tendo como objectivo a medição do pico de Bragg depositado pelo feixe de protões do ciclotrão PET, com uma energia nominal de 18 MeV. Utilizando um bloco de plástico cintilador e uma câmara fotográfica com ligação à internet da marca D-link foi possível medir tanto o alcance como a largura do feixe de protões com uma resolução espacial inferior a 0,1 mm. Os alcances do feixe medidos após este passar pelo meio de um tubo de alumínio com vácuo com 40 cm de comprimento e um segundo tubo com 2,4 m de comprimento permitiram confirmar que a energia efectiva do feixe é de 18 MeV. Mediu-se também o pico de Bragg utilizando um alvo constituído por várias folhas de alumínio intercaladas com lâminas de polietileno. O sinal de corrente foi recolhido das várias folhas de alumínio através de amplificadores de transimpedância fabricados no âmbito desta tese. Verificou-se que o pico de Bragg assim medido é consistente com simulações realizadas utilizando o pacote SRIM/TRIM. Após a instalação da linha de feixe no perímetro exterior do ciclotrão, esta foi caracterizada, calibrada e validada. Para tal, o sinal induzido pela passagem do feixe por uma folha de alumínio com 20 μm de espessura é lido através do amplificador de transimpedância mencionado. Este sinal amplificado providencia informação de dose em tempo real através de um programa desenvolvido em C/C++. Para além da dose, as principais variáveis de monitorização que este programa providencia incluem a corrente do feixe, a carga integrada em conjunto com a taxa de dose. Deste modo a dose e a corrente integrada (carga total) entregue até um dado instante na montagem experimental pode ser controlada por meio de um obturador controlado por computador. Feixes de protões com correntes tão baixas como 10 pA podem deste modo ser aferidas. A folha de alumínio foi escolhida por ser resistente à radiação, possuir baixa densidade e baixa probabilidade de radioactividade induzida pelo feixe e, finalmente, por representar um custo negligenciável. Junta-se a estas vantagens o facto de o método potenciar o cálculo da dose entregue a um alvo durante uma irradiação, com uma perda mínima da energia do feixe de protões, e com dispersão igualmente mínima. Resultados experimentais e simulações com o Geant4 são apresentados que revelam a aplicação, pela primeira vez, de um feixe de 18 MeV proveniente de um ciclotrão para irradiação de uma região seleccionada de um alvo. Fazendo uso do sistema de dosimetria apresentado no parágrafo anterior foi possível irradiar de modo homogéneo uma região circular com 18 mm de diâmetro. Torna-se assim possível irradiar culturas celulares localizadas em placas multi-poços com um diâmetro por poço de 16 mm, como é usual em experiências de radiobiologia. Verificou-se que o controlo do campo magnético aplicado dentro do ciclotrão é crucial para se obter uma irradiação uniforme em todo o campo do alvo. Para tal, efetua-se antes de cada irradiação e com o obturador fechado, um varrimento à corrente que gera o campo magnético dentro do ciclotrão, medindo-se um perfil quase gaussiano e tomando-se o seu valor central para se obter uma irradiação homogénea. As taxas de dose no alvo (entre 500 mGy/s e 5 mGy/s) são obtidas através de um disco em rotação posicionado na trajectória do feixe. O disco, com 150 mm de raio e uma fenda de 0,5 mm na sua extermidade, permite reduzir a taxa de dose por um factor de 5×10−4. Finalmente, vários filmes do tipo Gafchromic EBT2 foram expostos a diferente valores de dose por forma a validar toda a instalação para irradiação de um alvo com feixes de protões. Para tal validação fez-se uso do sistema de dosimetria em filme 2D do Serviço de Radioterapia do Centro Hospitalar Universitário de Coimbra. A dose absoluta nos filmes irradiados com protões foi verificada neste sistema e apresentou uma precisão melhor que 2%.The number of cyclotrons capable of accelerating protons to about 20MeV is increasing throughout the world. Originally aiming at the production of positron emission tomography (PET) radionuclides, some of these facilities are equipped with several beamlines suitable for scientific research. Radiobiology, radiophysiology, and other dosimetric studies can be performed using these beamlines. In this work, a PET cyclotron was fitted with a long beam transport line to irradiate with a good quality proton beam experimental setups. The beamline was configured as a natural extension of one of the cyclotron beam ports, while keeping available the other beam ports for PET radionuclides production. Experimental results are reported, together with SRIM/TRIM and Geant4 simulations, which aim at measuring the Bragg peak of the 18 -MeV proton beam from the PET cyclotron. By using a piece of plastic scintillator and a D-link Ethernet-based camera, the proton beam range and width were measured with a spatial resolution of 0.1mm. The ranges of the proton beam in the plastic scintillator were used to assess its energy after trespassing one or two Havar R windows and either a 40-cm-long or a 2.4-m-long aluminum pipe. The initial energy of the proton beam from the PET cyclotron was found to be 18 MeV. Additionally, the Bragg peak of the protons from the PET cyclotron was assessed using a stacked target consisting of several aluminum foils interleaved with polyethylene sheets, readout by in-house made transimpedance electronics. The measured Bragg peak is consistent with simulations performed using the SRIM/TRIM simulation toolkit. An out-of-yoke irradiation setup using the accelerated proton beam coming from the PET cyclotron was developed, characterized, calibrated, and validated. A 20-μm-thick aluminum transmission foil is readout by in-house made transimpedance electronics, providing online dose information via a C/C++ program. xvii Overview The main monitoring variables include beam current, integrated charge together with dose rate. The beam monitor is able to readout and deliver these variables in real-time. Hence the dose and integrated current (total charge) delivered upto a given instant to an experimental setup may be controlled via a computercontrolled shutter that was installed in the beam path. Proton beam currents down to 10 pA can be assessed using the thin aluminum foil. The aluminum was chosen for this task because it is radiation hard, it has low density and low radiation activity, and finally because it is easily available at negligible cost. In addition, this method allows for calculating the dose delivered to a target during an irradiation with high efficiency, and with minimal proton energy loss and scattering. Experimental results and Geant4 simulations are reported, which aim at using for the first time the 18 -MeV proton beam from a PET cyclotron to irradiate a selected region of a target using the developed dosimetry system. By using this system, a homogeneous beam spot on target with a diameter of 18mm can be controlled. This allows controlled irradiation of cell cultures located in typical biological multi-well dishes with diameters of 16mm each. It was found that the control of the magnetic field applied inside the cyclotron plays a major role for achieving said homogeneity. For that, scanning the magnet current and measuring the corresponding dose rate reveals a quasi-Gaussian shaped curve that must be known before any irradiation procedure (the final shutter is closed during such measurements). The optimum magnet current is taken from the center of the Gaussian-shaped curve, hence producing a homogenous dose on target. The measured dose rate on target ranges from 500mGy/s down to 5mGy/s. This is achieved with a 150mm radius rotating disk with a slit of 0.5mm width, that decreases target dose rates by a factor of 5 × 10−4. Several Gafchromic R EBT2 films were exposed to different values of dose for validating the developed irradiation setup using the 2D film dosimetry system of the Department of Radiotherapy of Coimbra University Hospital Center. The absolute dose in the irradiated films were assessed with a precision better than 2%. It is planned, in the near future, to irradiate small animals, cell cultures, or other materials or samples

Genetics of Na(+) exclusion and salinity tolerance in Afghani durum wheat landraces

Bakground: Selecting for low concentration of Na⁺ in the shoot provides one approach for tackling salinity stress that adversely affects crop production. Novel alleles for Na⁺ exclusion can be identified and then introduced into elite crop cultivars. Results: We have identified loci associated with lower Na⁺ concentration in leaves of durum wheat landraces originating from Afghanistan. Seedlings of two F₂ populations derived from crossings between Australian durum wheat (Jandaroi) and two Afghani landraces (AUS-14740 and AUS-14752) were grown hydroponically and evaluated for Na⁺ and K⁺ concentration in the third leaf. High heritability was found for both third leaf Na⁺ concentration and the K⁺/Na⁺ ratio in both populations. Further work focussed on line AUS-14740. Bulk segregant analysis using 9 K SNP markers identified two loci significantly associated with third leaf Na⁺ concentration. Marker regression analysis showed a strong association between all traits studied and a favourable allele originating from AUS-14740 located on the long arm of chromosome 4B. Conclusions: The candidate gene in the relevant region of chromosome 4B is likely to be the high affinity K⁺ transporter B1 (HKT1;5-B1). A second locus associated with third leaf Na⁺ concentration was located on chromosome 3BL, with the favourable allele originating from Jandaroi; however, no candidate gene can be identified.Nawar Jalal Shamaya, Yuri Shavrukov, Peter Langridge, Stuart John Roy and Mark Teste

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

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

Constitutive overexpression of the TaNF-YB4 gene in transgenic wheat significantly improves grain yield

First published online: July 27, 2015Heterotrimeric nuclear factors Y (NF-Ys) are involved in regulation of various vital functions in all eukaryotic organisms. Although a number of NF-Y subunits have been characterized in model plants, only a few have been functionally evaluated in crops. In this work, a number of genes encoding NF-YB and NF-YC subunits were isolated from drought-tolerant wheat (Triticum aestivum L. cv. RAC875), and the impact of the overexpression of TaNF-YB4 in the Australian wheat cultivar Gladius was investigated. TaNF-YB4 was isolated as a result of two consecutive yeast two-hybrid (Y2H) screens, where ZmNF-YB2a was used as a starting bait. A new NF-YC subunit, designated TaNF-YC15, was isolated in the first Y2H screen and used as bait in a second screen, which identified two wheat NF-YB subunits, TaNF-YB2 and TaNF-YB4. Three-dimensional modelling of a TaNF-YB2/TaNF-YC15 dimer revealed structural determinants that may underlie interaction selectivity. The TaNF-YB4 gene was placed under the control of the strong constitutive polyubiquitin promoter from maize and introduced into wheat by biolistic bombardment. The growth and yield components of several independent transgenic lines with up-regulated levels of TaNF-YB4 were evaluated under well-watered conditions (T1-T3 generations) and under mild drought (T2 generation). Analysis of T2 plants was performed in large deep containers in conditions close to field trials. Under optimal watering conditions, transgenic wheat plants produced significantly more spikes but other yield components did not change. This resulted in a 20-30% increased grain yield compared with untransformed control plants. Under water-limited conditions transgenic lines maintained parity in yield performance.Dinesh Yadav, Yuri Shavrukov, Natalia Bazanova, Larissa Chirkova, Nikolai Borisjuk, Nataliya Kovalchuk, Ainur Ismagul, Boris Parent, Peter Langridge, Maria Hrmova and Sergiy Lopat

Salt-induced expression of intracellular vesicle trafficking genes, CaRab-GTP, and their association with Na(+) accumulation in leaves of chickpea (Cicer arietinum L.)

Background: Chickpea is an important legume and is moderately tolerant to salinity stress during the growing season. However, the level and mechanisms for salinity tolerance can vary among accessions and cultivars. A large family of CaRab-GTP genes, previously identified in chickpea, is homologous to intracellular vesicle trafficking superfamily genes that play essential roles in response to salinity stress in plants. Results: To determine which of the gene family members are involved in the chickpea salt response, plants from six selected chickpea accessions (Genesis 836, Hattrick, ICC12726, Rupali, Slasher and Yubileiny) were exposed to salinity stress and expression profiles resolved for the major CaRab-GTP gene clades after 5, 9 and 15 days of salt exposure. Gene clade expression profiles (using degenerate primers targeting all members of each clade) were tested for their relationship to salinity tolerance measures, namely plant biomass and Na+ accumulation. Transcripts representing 11 out of the 13 CaRab clades could be detected by RT-PCR, but only six (CaRabA2, -B, -C, -D, -E and -H) could be quantified using qRT-PCR due to low expression levels or poor amplification efficiency of the degenerate primers for clades containing several gene members. Expression profiles of three gene clades, CaRabB, -D and -E, were very similar across all six chickpea accessions, showing a strongly coordinated network. Salt-induced enhancement of CaRabA2 expression at 15 days showed a very strong positive correlation (R2 = 0.905) with Na+ accumulation in leaves. However, salinity tolerance estimated as relative plant biomass production compared to controls, did not correlate with Na+ accumulation in leaves, nor with expression profiles of any of the investigated CaRab-GTP genes. Conclusion: A coordinated network of CaRab-GTP genes, which are likely involved in intracellular trafficking, are important for the salinity stress response of chickpea plants.Crystal Sweetman, Gulmira Khassanova, Troy K. Miller, Nicholas J. Booth, Akhylbek Kurishbayev, Satyvaldy Jatayev, Narendra K. Gupta, Peter Langridge, Colin L.D. Jenkins, Kathleen L. Soole, David A. Day and Yuri Shavruko

Genes encoding transcription factors TaDREB5 and TaNFYC-A7 are differentially expressed in leaves of bread wheat in response to drought, dehydration and ABA

Two groups of six spring bread wheat varieties with either high or low grain yield under the dry conditions of Central and Northern Kazakhstan were selected for analysis. Experiments were set up with the selected wheat varieties in controlled environments as follows: (1) slowly progressing drought imposed on plants in soil, (2) rapid dehydration of whole plants grown in hydroponics, (3) dehydration of detached leaves, and (4) ABA treatment of whole plants grown in hydroponics. Representatives of two different families of transcription factors (TFs), TaDREB5 and TaNFYC-A7, were found to be linked to yield-under-drought using polymorphic Amplifluor-like SNP marker assays. qRT-PCR revealed differing patterns of expression of these genes in the leaves of plants subjected to the above treatments. Under drought, TaDREB5 was significantly up-regulated in leaves of all high-yielding varieties tested and down-regulated in all low-yielding varieties, and the level of expression was independent of treatment type. In contrast, TaNFYC-A7 expression levels showed different responses in the high- and low-yield groups of wheat varieties. TaNFYC-A7 expression under dehydration (treatments 2 and 3) was higher than under drought (treatment 1) in all high-yielding varieties tested, while in all low-yielding varieties the opposite pattern was observed: the expression levels of this gene under drought were higher than under dehydration. Rapid dehydration of detached leaves and intact wheat plants grown in hydroponics produced similar changes in gene expression. ABA treatment of whole plants caused rapid stomatal closure and a rise in the transcript level of both genes during the first 30 min, which decreased 6 h after treatment. At this time-point, expression of TaNFYC-A7 was again significantly up-regulated compared to untreated controls, while TaDREB5 returned to its initial level of expression. These findings reveal significant differences in the transcriptional regulation of two drought-responsive and ABA-dependent TFs under slowly developing drought and rapid dehydration of wheat plants. The results obtained suggest that correlation between grain yield in dry conditions and TaNFYC-A7 expression levels in the examined wheat varieties is dependent on the length of drought development and/or strength of drought; while in the case of TaDREB5, no such dependence is observed.Lyudmila Zotova, Akhylbek Kurishbayev, Satyvaldy Jatayev, Gulmira Khassanova, Askar Zhubatkanov, Dauren Serikbay, Sergey Sereda, Tatiana Sereda, Vladimir Shvidchenko, Sergiy Lopato, Colin Jenkins, Kathleen Soole, Peter Langridge, and Yuri Shavruko

Adaptive mechanisms of plants against salt stress and salt shock

Salinization process occurs when soil is contaminated with salt, which consequently influences plant growth and development leading to reduction in yield of many food crops. Responding to a higher salt concentration than the normal range can result in plant developing complex physiological traits and activation of stress-related genes and metabolic pathways. Many studies have been carried out by different research groups to understand adaptive mechanism in many plant species towards salinity stress. However, different methods of sodium chloride (NaCl) applications definitely give different responses and adaptive mechanisms towards the increase in salinity. Gradual increase in NaCl application causes the plant to have salt stress or osmotic stress, while single step and high concentration of NaCl may result in salt shock or osmotic shock. Osmotic shock can cause cell plasmolysis and leakage of osmolytes in plant. Also, the gene expression pattern is influenced by the type of methods used in increasing the salinity. Therefore, this chapter discusses the adaptive mechanism in plant responding to both types of salinity increment, which include the morphological changes of plant roots and aerial parts, involvement of signalling molecules in stress perception and regulatory networks and production of osmolyte and osmoprotective proteins