Genome Editing of the Octoploid Fragaria x ananassa Using the CRISPR/Cas9 System

Due to its octoploid nature, gene functional analyses in the cultivated strawberry (Fragaria × ananassa) are commonly carried out via gene silencing using self-complementary “hairpin” double-stranded RNA (RNAi) constructs. However, this system is not always as efficient as expected. First, an efficient silencing of the target gene is not always achieved, and second, its effect might not be stable after several clonal propagations of the transgenic lines. Recently, genome editing is becoming an important biotechnological tool for gene functional analysis and crop improvement, in particular since the development of the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR associated protein 9) system. To investigate the functionality of the CRISPR/Cas9 in strawberry, we designed two sgRNAs directed against two regions of the floral homeotic gene APETALA3 (AP3) in order to induce a deletion of around 200 nt. A vector containing both sgRNAs and Cas9 was used to transform leaf disks of F. × ananassa cv. Camarosa. Several independent stable transgenic lines displayed defects in stamen and fruit development, partially phenocopying that of the Arabidopsis ap3 mutants. Molecular analysis of the targeted AP3 locus indicated differences in gene editing among different transgenic lines and suggests mutations in all the possible AP3 alleles. Phenotypic analyses indicate that impaired fruit development might be caused by the lack of proper development of the anthers due to the CRISPR/Cas9 induced mutation in AP3. In summary, we show that the CRISPR/Cas9 system is a functional tool to perform genome editing in the octoploid F. × ananassa. We propose this system as an alternative to the traditional RNAi strategy to stably mutagenize a particular gene of interest for functional analyses in this species.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Targeted gene modification in Fragaria vesca mediated by CRISPR/Cas9 system

Genome editing is becoming an important biotechnological tool for gene function analysis and crop improvement, being the CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR associated protein 9) system the most widely used. The natural CRISPR/Cas9 system has been reduced to two components: a single-guide RNA (sgRNA) for target recognition via RNA-DNA base pairing, which is commonly expressed using a promoter for small-RNAs (U6 promoter), and the Cas9 endonuclease for DNA cleavage (1). To validate the CRISPR/Cas9 system in strawberry plants, we designed two sgRNAs directed against the floral homeotic gene APETALA3 (sgRNA-AP3#1 and sgRNA-AP3#2). This gene was selected because ap3 mutations induce clear developmental phenotypes in which petals and stamens are missing or partially converted to sepals and carpels respectively (2). In this work, we used two different U6 promoters to drive the sgRNA-AP3s expression: AtU6-26 from Arabidopsis (4), and a U6 promoter from Fragaria vesca (FvU6) (this work). We also tested two different coding sequences of Cas9: a human- (hSpCas9) (3) and a plant-codon optimized (pSpCas9) (this work). Transient expression experiments using both CRISPR/Cas9 systems (AtU6-26:sgRNA-AP3#1_35S:hSpCas9_AtU6-26:sgRNA-AP3#2 and FvU6:sgRNA-AP3#1_35S:pSpCas9_FvU6:sgRNA-AP3#2) were performed infiltrating Agrobacterium tumefaciens into F. vesca fruits. PCR amplification and sequencing analyses across the target sites showed a deletion of 188-189 bp corresponding to the region comprised between the two cutting sites of Cas9, confirming that the CRISPR/Cas9 system is functional in F. vesca. Remarkably, the two systems showed different mutagenic efficiency that could be related to differences in expression of the U6 promoters as well as differences in the Cas9 transcripts stability and translation. Stable transformants for both F. vesca (2n) and Fragaria X anannassa (8n) are currently being established to test whether is possible to obtain heritable homozygous mutants derived from CRISPR/Cas9 strategies in strawberry. Thus, our work offers a promising tool for genome editing and gene functional analysis in strawberry. This tool might represent a more efficient alternative to the sometimes inefficient RNAi silencing methods commonly used in this species.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Fruit over sunbed : carotenoid skin coloration is found more attractive than melanin coloration

Skin coloration appears to play a pivotal part in facial attractiveness. Skin yellowness contributes to an attractive appearance and is influenced both by dietary carotenoids and by melanin. While both increased carotenoid coloration and increased melanin coloration enhance apparent health in Caucasian faces by increasing skin yellowness, it remains unclear firstly, whether both pigments contribute to attractiveness judgements, secondly, whether one pigment is clearly preferred over the other, and thirdly, whether these effects depend on the sex of the face. Here, in three studies, we examine these questions using controlled facial stimuli transformed to be either high or low in (a) carotenoid coloration, or (b) melanin coloration. We show, firstly, that both increased carotenoid coloration and increased melanin coloration are found attractive compared to lower levels of these pigments. Secondly, we show that carotenoid coloration is consistently preferred over melanin coloration when levels of coloration are matched. In addition, we find an effect of the sex of stimuli with stronger preferences for carotenoids over melanin in female compared to male faces, irrespective of the sex of the observer. These results are interpreted as reflecting preferences for sex-typical skin coloration: men have darker skin than women and high melanisation in male faces may further enhance this masculine trait, thus carotenoid coloration is not less desirable, but melanin coloration is relatively more desirable in males compared to females. Taken together, our findings provide further support for a carotenoid-linked health-signalling system that is highly important in mate choice.PostprintPeer reviewe

Large Scale Features of Rotating Forced Turbulence

Large scale features of a randomly isotropically forced incompressible and unbounded rotating fluid are examined in perturbation theory. At first order in both the random force amplitude and the angular velocity we find two types of modifications to the fluid equation of motion. The first correction transforms the molecular shear viscosity into a (rotation independent) effective viscosity. The second perturbative correction leads to a new large scale non-dissipative force proportional to the fluid angular velocity in the slow rotation regime. This effective force does no net work and alters the dispersion relation of inertial waves propagating in the fluid. Both dynamically generated corrections can be identified with certain components of the most general axisymmetric ``viscosity tensor'' for a Newtonian fluid.Comment: 12 pages, 2 figures, RevTeX, and accepted for publication in Phys. Rev.

Exploring the links between HIV/AIDS, social capital, and development

This paper attempts to quantify the impact of the HIV/AIDS epidemic on social capital with cross-country data. Using data from the World Values Survey, the authors estimate reduced-form regressions of the main determinants of social capital controlling for HIV prevalence, institutional quality, social distance, and economic indicators. The results obtained indicate that HIV prevalence affects social capital negatively. The empirical estimates suggest that a one standard deviation increase in HIV prevalence will lead to a decline of at least 1 percent in trust, controlling for other determinants of social capital. Moving from a country with a relatively low level of HIV prevalence, such as Estonia, to a country with a relatively high level, such as Uganda, there is a more than 11 percentpoint decline in social capital. These results are robust in a number of dimensions and highlight the empirical importance of an additional mechanism through which HIV/AIDS hinders the development process.Population Policies,Social Capital,HIV AIDS,Disease Control&Prevention,Inequality

First report of Diaphorina citri (Hemiptera: Psyllidae) in Costa Rica

In November 2003, the Asian citrus psyllid (Diaphorina citri) was found for the first time in Costa Rica, in Alajuela, Heredia and San Jose provinces. Figure 1 shows an adult female and nymphal instars of Asiatic citrus psyllid

Targeted mutagenesis of FaTM6 in the Octoploid Strawberry (Fragaria x ananassa) using the CRISPR/Cas9 System

The B-class of MADS-box transcription factors has been studied in many plant species, but remain functionally uncharacterized in the Rosaceae family. APETALA3 (AP3), a member of this class, controls the identity of petals and stamens in Arabidopsis thaliana. In this work, we identified two members of the AP3 lineage in the cultivated strawberry (Fragaria × ananassa): FaAP3 and FaTM6. Interestingly, FaTM6, and not FaAP3, shows an expression pattern equivalent to that of AP3 in Arabidopsis. Genome editing using Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system is becoming a robust tool for targeted and stable mutagenesis of DNA. However, whether it can be efficiently used in an octoploid species such as F. × ananassa is not known. In our study, we report the application of the CRISPR/Cas9 in F. × ananassa to characterize the function of FaTM6 in flower development. An exhaustive analysis by high-throughput sequencing of the FaTM6 locus spanning the target sites showed a high efficiency genome editing already in the T0 generation. The phenotypic characterization of the mutant lines indicates that FaTM6 plays a key role in petal and especially in anther development in strawberry. Our results validate the CRISPR/Cas9 strategy for gene functional analysis in an octoploid species such as F. × ananassa, and offer new opportunities for engineering strawberry to improve traits of interest in breeding programs.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

An assessment of bottom current controlled sedimentation in Pacific Ocean abyssal environments

The effect of the circulation of polar-sourced dense waters on the deep seafloor is still largely unknown. This paper presents multibeam bathymetric evidence for the existence of present-day contourite drifts on the central Pacific ocean at depths ranging between 4000 and 5000 m, based on the identification of their diagnostic erosive (moats, linear moats, furrows and abyssal sinuous channels) and depositional (plastered, elongated separated and abyssal contourite-fan drifts) contourite morphological features. These contourite drifts display a patchy distribution, sculpted by the secondary flows of the Lower Circumpolar Water (LCPW) and the North Pacific Deep Waters (NPDW), as well as local dynamics of the flow in the form of internal waves and bottom currents modulated by tides, which are capable of accelerating the typically sluggish bottom currents and contribute to the erosion, transport and deposition of sediments. The formation of contourites is also predominantly controlled by the presence of large-scale tectonic and volcanic features (ridges, seamounts and deep elongated valleys), which affect the morphology and distribution of the basins and induce the acceleration of bottom currents. Overall, this study allows to re-assess the effects, distribution and significance of bottom-current controlled sedimentation in abyssal environments, where examples of contourite features are still rather scarce in part because of the technical challenges and their remote location, but also because the effect of bottom currents is not well known in the central oceanic basins and only capable of marginally affecting their sedimentological record