A reference genome for Nicotiana tabacum enables map-based cloning of homeologous loci implicated in nitrogen utilization efficiency.

BACKGROUND: Tobacco (Nicotiana tabacum) is an important plant model system that has played a key role in the early development of molecular plant biology. The tobacco genome is large and its characterisation challenging because it is an allotetraploid, likely arising from hybridisation between diploid N. sylvestris and N. tomentosiformis ancestors. A draft assembly was recently published for N. tabacum, but because of the aforementioned genome complexities it was of limited utility due to a high level of fragmentation. RESULTS: Here we report an improved tobacco genome assembly, which, aided by the application of optical mapping, achieves an N50 size of 2.17 Mb and enables anchoring of 64% of the genome to pseudomolecules; a significant increase from the previous value of 19%. We use this assembly to identify two homeologous genes that explain the differentiation of the burley tobacco market class, with potential for greater understanding of Nitrogen Utilization Efficiency and Nitrogen Use Efficiency in plants; an important trait for future sustainability of agricultural production. CONCLUSIONS: Development of an improved genome assembly for N. tabacum enables what we believe to be the first successful map-based gene discovery for the species, and demonstrates the value of an improved assembly for future research in this model and commercially-important species.

Phase calibration target for quantitative phase imaging with ptychography.

Quantitative phase imaging (QPI) utilizes refractive index and thickness variations that lead to optical phase shifts. This gives contrast to images of transparent objects. In quantitative biology, phase images are used to accurately segment cells and calculate properties such as dry mass, volume and proliferation rate. The fidelity of the measured phase shifts is of critical importance in this field. However to date, there has been no standardized method for characterizing the performance of phase imaging systems. Consequently, there is an increasing need for protocols to test the performance of phase imaging systems using well-defined phase calibration and resolution targets. In this work, we present a candidate for a standardized phase resolution target, and measurement protocol for the determination of the transfer of spatial frequencies, and sensitivity of a phase imaging system. The target has been carefully designed to contain well-defined depth variations over a broadband range of spatial frequencies. In order to demonstrate the utility of the target, we measure quantitative phase images on a ptychographic microscope, and compare the measured optical phase shifts with Atomic Force Microscopy (AFM) topography maps and surface profile measurements from coherence scanning interferometry. The results show that ptychography has fully quantitative nanometer sensitivity in optical path differences over a broadband range of spatial frequencies for feature sizes ranging from micrometers to hundreds of micrometers.

Quantitative electron phase imaging with high sensitivity and an unlimited field of view.

As it passes through a sample, an electron beam scatters, producing an exit wavefront rich in information. A range of material properties, from electric and magnetic field strengths to specimen thickness, strain maps and mean inner potentials, can be extrapolated from its phase and mapped at the nanoscale. Unfortunately, the phase signal is not straightforward to obtain. It is most commonly measured using off-axis electron holography, but this is experimentally challenging, places constraints on the sample and has a limited field of view. Here we report an alternative method that avoids these limitations and is easily implemented on an unmodified transmission electron microscope (TEM) operating in the familiar selected area diffraction mode. We use ptychography, an imaging technique popular amongst the X-ray microscopy community; recent advances in reconstruction algorithms now reveal its potential as a tool for highly sensitive, quantitative electron phase imaging.

Ptychographic microscope for three-dimensional imaging.

Ptychography is a coherent imaging technique that enables an image of a specimen to be generated from a set of diffraction patterns. One limitation of the technique is the assumption of a multiplicative interaction between the illuminating coherent beam and the specimen, which restricts ptychography to samples no thicker than a few tens of micrometers in the case of visible-light imaging at micron-scale resolution. By splitting a sample into axial sections, we demonstrated in recent work that this thickness restriction can be relaxed and whats-more, that coarse optical sectioning can be realized using a single ptychographic data set. Here we apply our technique to data collected from a modified optical microscope to realize a reduction in the optical sectioning depth to 2 µm in the axial direction for samples up to 150 µm thick. Furthermore, we increase the number of sections that are imaged from 5 in our previous work to 34 here. Our results compare well with sectioned images collected from a confocal microscope but have the added advantage of strong phase contrast, which removes the need for sample staining.

Ptychographic transmission microscopy in three dimensions using a multi-slice approach.

Generally, methods of three-dimensional imaging such as confocal microscopy and computed tomography rely on two essentials: multiple measurements (at a range of focus positions or rotations) and a weakly scattering specimen (to avoid distortion of the focal spot in the confocal microscope or to satisfy the projection approximation in tomography). Here we show that an alternative form of multi-measurement imaging, ptychography, can be extended to three dimensions and can successfully recover images in the presence of multiple scattering and when the projection approximation is not applicable. We demonstrate our technique experimentally using visible light, where it has applications in imaging thick samples such as biological tissues; however the results also have important implications for x ray and electron imaging.

An annealing algorithm to correct positioning errors in ptychography.

Ptychography offers the possibility of improving the resolution of atomic-scale (electron and X-ray) transmission microscopy without any of the demands of high quality lenses: its resolution is in theory only limited by the effective synthetic numerical aperture determined by the angular size of the detector. However, it has been realised experimentally that a major weakness of the approach is that the obtainable resolution is only as good as the accuracy to which the illuminating beam can be moved relative to the specimen. This can be catastrophic in the electron case because of thermal drift and hysteresis in the probe scan coils. We present here a computationally efficient extension of the 'ePIE' ptychographic reconstruction algorithm for correcting these errors retrospectively. We demonstrate its effectiveness using simulations and results from visible light and electron beam experiments that show it can correct positioning errors tens of times larger than the pixel size in the resulting image.

Ptychographic electron microscopy using high-angle dark-field scattering for sub-nanometre resolution imaging.

Diffractive imaging, in which image-forming optics are replaced by an inverse computation using scattered intensity data, could, in principle, realize wavelength-scale resolution in a transmission electron microscope. However, to date all implementations of this approach have suffered from various experimental restrictions. Here we demonstrate a form of diffractive imaging that unshackles the image formation process from the constraints of electron optics, improving resolution over that of the lens used by a factor of five and showing for the first time that it is possible to recover the complex exit wave (in modulus and phase) at atomic resolution, over an unlimited field of view, using low-energy (30 keV) electrons. Our method, called electron ptychography, has no fundamental experimental boundaries: further development of this proof-of-principle could revolutionize sub-atomic scale transmission imaging.

Relationship between bruchid resistance and seed mass in mungbean based on QTL analysis.

Bruchids (Coleoptera: Bruchidae) can cause serious damage to mungbean and several other leguminous crops and there is a strong association between small seed size and bruchid resistance. In investigating the feasibility of breeding large-seeded cultivars with high levels of bruchid resistance, we studied the relationship between these two traits by QTL analysis. A major locus conferring resistance to Callosobruchus chinensis was identified from a wild mungbean genotype, 'ACC41' (belonging to Vigna radiata var. sublobata), collected in Australia. The proportion of the C. chinensis resistance response that could be attributed to this single QTL varied among four different resistance assays. The highest value reached was 98.5%, suggesting that bruchid resistance in this genotype is likely conditioned by this single locus. The QTL was robust and its detection was not affected by the use of different sources of the insect, different lengths and conditions of seed storage, or different bruchid resistance assay methods. This bruchid resistance QTL was coincident with one of the loci conferring seed mass detected from the three seed sources produced in Australia. However, such a co-location was not detected for the seed source produced in China. Covariance analysis revealed a complex relationship between seed mass and bruchid resistance. Nevertheless, the effect of the bruchid resistance QTL remained highly significant for all four assays after the effect of seed mass was accounted for. These results, together with the relationship between the bruchid resistance QTL identified in this study and a second one detected previously in a wild mungbean genotype from Madagascar, are discussed.

Dianhydride-amine hydrogen bonded perylene tetracarboxylic dianhydride and tetraaminobenzene rows.

We have investigated the coadsorption of perylene tetracarboxylic dianhydride (PTCDA) and tetraaminobenzene (TAB) on the Ag/Si(111)-square root(3) x square root(3) R30 degree surface using scanning tunneling microscopy. At room temperature, PTCDA islands with square and herringbone ordering are formed which, on exposure to TAB, are converted into an intermixed phase in which PTCDA and TAB form alternating rows. From our images, we determine the relative placement of TAB and PTCDA molecules and conclude that the row structure is stabilized by hydrogen bonding between dianhydride and diamine groups. We confirm that this hydrogen bonding junction is stable using ab initio calculations and show that the proposed geometry is consistent with calculated intermolecular dimensions.

Bond breaking coupled with translation in rolling of covalently bound molecules.

The response of a C60 molecule to manipulation across a surface displays a long range periodicity which corresponds to a rolling motion. A period of three or four lattice constants is observed and is accompanied by complex subharmonic structure due to molecular hops through a regular, repeating sequence of adsorption states. Combining experimental data and ab initio calculations, we show that this response corresponds to a rolling motion in which two of the four Si-C60 covalent bonds act as a pivot over which the molecule rotates while moving through one lattice constant and identify a sequence of C60 bonding configurations that accounts for the periodic structure.

Construction of bacterial artificial chromosome libraries and their application in developing PCR-based markers closely linked to a major locus conditioning bruchid resistance in mungbean (Vigna radiata L. Wilczek).

Bacterial artificial chromosome (BAC) libraries have been widely used in different aspects of genome research. In this paper we report the construction of the first mungbean (Vigna radiata L. Wilczek) BAC libraries. These BAC clones were obtained from two ligations and represent an estimated 3.5 genome equivalents. This correlated well with the screening of nine random single-copy restriction fragment length polymorphism probes, which detected on average three BACs each. These mungbean clones were successfully used in the development of two PCR-based markers linked closely with a major locus conditioning bruchid (Callosobruchus chinesis) resistance. These markers will be invaluable in facilitating the introgression of bruchid resistance into breeding programmes as well as the further characterisation of the resistance locus.

Identification of a major locus conferring resistance to powdery mildew (Erysiphe polygoni DC) in mungbean (Vigna radiata L. Wilczek) by QTL analysis.

A major locus conferring resistance to the causal organism of powdery mildew, Erysiphe polygoni DC, in mungbean (Vigna radiata L. Wilczek) was identified using QTL analysis with a population of 147 recombinant inbred individuals. The population was derived from a cross between 'Berken', a highly susceptible variety, and ATF 3640, a highly resistant line. To test for response to powdery mildew, F7 and F8 lines were inoculated by dispersing decaying mungbean leaves with residual conidia of E. polygoni amongst the young plants to create an artificial epidemic and assayed in a glasshouse facility. To generate a linkage map, 322 RFLP clones were tested against the two parents and 51 of these were selected to screen the mapping population. The 51 probes generated 52 mapped loci, which were used to construct a linkage map spanning 350 cM of the mungbean genome over 10 linkage groups. Using these markers, a single locus was identified that explained up to a maximum of 86% of the total variation in the resistance response to the pathogen.