Cenh3: An Emerging Player in Haploid Induction Technology.

True-breeding lines are required for the development and production of crop varieties. In a classical breeding approach these lines are obtained through inbreeding, and often 7-9 generations of inbreeding is performed to achieve the desired level of homozygosity, over a period of several years. In contrast, the chromosomes of haploids can be doubled to produce true-breeding lines in a single generation. Over the last century, scientists have developed a variety of techniques to induce haploids and doubled haploids, though these techniques apply only to particular crop varieties. Ravi and Chan (2010) discovered that haploids could be obtained in Arabidopsis through the manipulation of the centromere-specific histone 3 variant, CENH3. Their approach, which involved extensive modifications to a transgenic CENH3, held promise of being translated to crop species, and has been successfully employed in maize (see Kelliher et al., 2016). Refinements of this technology have since been developed which indicate that non-transgenic modifications to CENH3 will also induce haploids. The complementation of a cenh3 null by CENH3 from closely related plant species can result in plants that are fertile but haploid-inducing on crossing by CENH3 wt plants- suggesting that introgression of alien CENH3 may produce non-transgenic haploid inducers. Similarly, a remarkably wide variety of point mutations in CENH3, inducible by chemical agents, have recently been shown to result in haploid induction on crossing by wild-type CENH3 plants. These CENH3-variant plants grow normally, are fully fertile on self-pollination, and may be present in existing mutagenized collections

Thinking about Drinking: Explaining drinking behaviour by drinker prototypes

__Abstract__ Worldwide, about 11.5% of drinkers engage in weekly heavy episodic drinking. Heavy or excessive drinking is defined as consuming 60 or more grams of pure alcohol; equal to 6 or more standard glasses. In the Netherlands4 and many other countries, excessive drinking is especially prevalent among adolescents and young adults (18–25 years old, 20%). In the general population, the prevalence and consumption is higher among males (15.4%) than females (10.5%). Furthermore, higher educated people (90%) report a higher prevalence of alcohol use after the age of 34 than lower educated people (66%), irrespective of gender. However, excessive drinking is generally more prevalent among lower educate

EPR spectroscopy of iron- and nickel-doped [ZnAl]-layered double hydroxides: modeling active sites in heterogeneous water oxidation catalysts

Iron-doped nickel layered double hydroxides (LDHs) are among the most active heterogeneous water oxidation catalysts. Due to inter-spin interactions, however, the high density of magnetic centers results in line-broadening in magnetic resonance spectra. As a result, gaining atomic-level insight into the catalytic mechanism via electron paramagnetic resonance (EPR) is not generally possible. To circumvent spin-spin broadening, iron and nickel atoms were doped into non-magnetic [ZnAl]-LDH materials and the coordination environments of the isolated Fe(III) and Ni(II) sites were characterized. Multifrequency EPR spectroscopy identified two distinct Fe(III) sites (S = 5/2) in [Fe:ZnAl]-LDH. Changes in zero field splitting (ZFS) were induced by dehydration of the material, revealing that one of the Fe(III) sites is solvent-exposed (i.e. at an edge, corner, or defect site). These solvent-exposed sites feature an axial ZFS of 0.21 cm⁻¹ when hydrated. The ZFS increases dramatically upon dehydration (to -1.5 cm⁻¹), owing to lower symmetry and a decrease in the coordination number of iron. The ZFS of the other (“inert”) Fe(III) site maintains an axial ZFS of 0.19-0.20 cm⁻¹ under both hydrated and dehydrated conditions. We observed a similar effect in [Ni:ZnAl]-LDH materials; notably, Ni(II) (S = 1) atoms displayed a single, small ZFS (±0.30 cm⁻¹) in hydrated material, whereas two distinct Ni(II) ZFS values (±0.30 and ±1.1 cm⁻¹) were observed in the dehydrated samples. Although the magnetically-dilute materials were not active catalysts, the identification of model sites in which the coordination environments of iron and nickel were particularly labile (e.g. by simple vacuum drying) is an important step towards identifying sites in which the coordination number may drop spontaneously in water, a probable mechanism of water oxidation in functional materials

High atomic weight, high-energy radiation (HZE) induces transcriptional responses shared with conventional stresses in addition to a core "DSB" response specific to clastogenic treatments.

Plants exhibit a robust transcriptional response to gamma radiation which includes the induction of transcripts required for homologous recombination and the suppression of transcripts that promote cell cycle progression. Various DNA damaging agents induce different spectra of DNA damage as well as "collateral" damage to other cellular components and therefore are not expected to provoke identical responses by the cell. Here we study the effects of two different types of ionizing radiation (IR) treatment, HZE (1 GeV Fe(26+) high mass, high charge, and high energy relativistic particles) and gamma photons, on the transcriptome of Arabidopsis thaliana seedlings. Both types of IR induce small clusters of radicals that can result in the formation of double strand breaks (DSBs), but HZE also produces linear arrays of extremely clustered damage. We performed these experiments across a range of time points (1.5-24 h after irradiation) in both wild-type plants and in mutants defective in the DSB-sensing protein kinase ATM. The two types of IR exhibit a shared double strand break-repair-related damage response, although they differ slightly in the timing, degree, and ATM-dependence of the response. The ATM-dependent, DNA metabolism-related transcripts of the "DSB response" were also induced by other DNA damaging agents, but were not induced by conventional stresses. Both Gamma and HZE irradiation induced, at 24 h post-irradiation, ATM-dependent transcripts associated with a variety of conventional stresses; these were overrepresented for pathogen response, rather than DNA metabolism. In contrast, only HZE-irradiated plants, at 1.5 h after irradiation, exhibited an additional and very extensive transcriptional response, shared with plants experiencing "extended night." This response was not apparent in gamma-irradiated plants

Genomic stability in response to high versus low linear energy transfer radiation in Arabidopsis thaliana.

Low linear energy transfer (LET) gamma rays and high LET HZE (high atomic weight, high energy) particles act as powerful mutagens in both plants and animals. DNA damage generated by HZE particles is more densely clustered than that generated by gamma rays. To understand the genetic requirements for resistance to high versus low LET radiation, a series of Arabidopsis thaliana mutants were exposed to either 1GeV Fe nuclei or gamma radiation. A comparison of effects on the germination and subsequent growth of seedlings led us to conclude that the relative biological effectiveness (RBE) of the two types of radiation (HZE versus gamma) are roughly 3:1. Similarly, in wild-type lines, loss of somatic heterozygosity was induced at an RBE of about a 2:1 (HZE versus gamma). Checkpoint and repair defects, as expected, enhanced sensitivity to both agents. The "replication fork" checkpoint, governed by ATR, played a slightly more important role in resistance to HZE-induced mutagenesis than in resistance to gamma induced mutagenesis

High-Resolution Analysis of the Efficiency, Heritability, and Editing Outcomes of CRISPR/Cas9-Induced Modifications of NCED4 in Lettuce (Lactuca sativa).

CRISPR/Cas9 is a transformative tool for making targeted genetic alterations. In plants, high mutation efficiencies have been reported in primary transformants. However, many of the mutations analyzed were somatic and therefore not heritable. To provide more insights into the efficiency of creating stable homozygous mutants using CRISPR/Cas9, we targeted LsNCED4 (9-cis-EPOXYCAROTENOID DIOXYGENASE4), a gene conditioning thermoinhibition of seed germination in lettuce. Three constructs, each capable of expressing Cas9 and a single gRNA targeting different sites in LsNCED4, were stably transformed into lettuce (Lactuca sativa) cvs. Salinas and Cobham Green. Analysis of 47 primary transformants (T1) and 368 T2 plants by deep amplicon sequencing revealed that 57% of T1 plants contained events at the target site: 28% of plants had germline mutations in one allele indicative of an early editing event (mono-allelic), 8% of plants had germline mutations in both alleles indicative of two early editing events (bi-allelic), and the remaining 21% of plants had multiple low frequency mutations indicative of late events (chimeric plants). Editing efficiency was similar in both genotypes, while the different gRNAs varied in efficiency. Amplicon sequencing of 20 T1 and more than 100 T2 plants for each of the three gRNAs showed that repair outcomes were not random, but reproducible and characteristic for each gRNA. Knockouts of NCED4 resulted in large increases in the maximum temperature for seed germination, with seeds of both cultivars capable of germinating >70% at 37°. Knockouts of NCED4 provide a whole-plant selectable phenotype that has minimal pleiotropic consequences. Targeting NCED4 in a co-editing strategy could therefore be used to enrich for germline-edited events simply by germinating seeds at high temperature

Cultural Implications of Late Quaternary Environmental Change in Northeastern Texas

Northeastern Texas is one of the most intensely studied archaeological regions of the state, principally for the two reasons that (1) archaeologically-rich Caddoan manifestations have long attracted interest and (2) many large land-modifying projects, such as reservoirs and strip mines, have occasioned environmental studies which include investigations of cultural resources. This greater amount of activity relative to other regions in the state has generally prevailed for more than a century (Guy 1990) and prospects are good that archaeologists will continue to intensely research the area in the foreseeable future. Unfortunately, however, they will be hard-pressed to keep pace with the destruction of archaeological sites. The region is growing in population and developing economically which inevitably results in land modifications destructive of archaeological evidence. Also, many sites are being willfully destroyed by commercial dealers in antiquities and by relic collectors. This contribution to historic preservation planning is concerned with gaining a better understanding of the past environmental factors that stimulated responses by past inhabitants of Northeastern Texas, and the natural environmental context of the archaeological record in Northeastern Texas. The area has some overall environmental similarities--such as being in a single physiographic section, and falling within a single climatic region--that might be misconstrued to imply a uniformity of conditions critical in pre-industrial human adaptations. In fact, the conditions important in human ecology vary significantly across space and have varied equally significantly over time

Transient error approximation in a Lévy queue

Motivated by a capacity allocation problem within a finite planning period, we conduct a transient analysis of a single-server queue with Lévy input. From a cost minimization perspective, we investigate the error induced by using stationary congestion measures as opposed to time-dependent measures. Invoking recent results from fluctuation theory of Lévy processes, we derive a refined cost function, that accounts for transient effects. This leads to a corrected capacity allocation rule for the transient single-server queue. Extensive numerical experiments indicate that the cost reductions achieved by this correction can be significant

Point Mutations in Centromeric Histone Induce Post-zygotic Incompatibility and Uniparental Inheritance.

The centromeric histone 3 variant (CENH3, aka CENP-A) is essential for the segregation of sister chromatids during mitosis and meiosis. To better define CENH3 functional constraints, we complemented a null allele in Arabidopsis with a variety of mutant alleles, each inducing a single amino acid change in conserved residues of the histone fold domain. Many of these transgenic missense lines displayed wild-type growth and fertility on self-pollination, but exhibited frequent post-zygotic death and uniparental inheritance when crossed with wild-type plants. The failure of centromeres marked by these missense mutation in the histone fold domain of CENH3 reproduces the genome elimination syndromes described with chimeric CENH3 and CENH3 from diverged species. Additionally, evidence that a single point mutation is sufficient to generate a haploid inducer provide a simple one-step method for the identification of non-transgenic haploid inducers in existing mutagenized collections of crop species. As proof of the extreme simplicity of this approach to create haploid-inducing lines, we performed an in silico search for previously identified point mutations in CENH3 and identified an Arabidopsis line carrying the A86V substitution within the histone fold domain. This A87V non-transgenic line, while fully fertile on self-pollination, produced postzygotic death and uniparental haploids when crossed to wild type