Background

Genome editing of monocot plants can be accomplished by using the components of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR associated Cas9) technology specifically optimized for these types of plants. Here, we present the development of RNA-guided Cas9 system for simplex and multiplex genome editing in barley.

Results

We developed a set of customizable RNA-guided Cas9 binary vectors and sgRNA modules for simplex and multiplex editing in barley. To facilitate the design of RNA-guided Cas9 constructs, the pBract derived binary vectors were adapted to Gateway cloning and only one restriction enzyme was required for construction of the sgRNA. We designed a synthetic, codon optimized Cas9 gene containing the N terminal SV40 nuclear localization signal and the UBQ10 Arabidopsis 1st intron. Two different sgRNAs were constructed for simplex editing and one polycistronic tRNA-gRNA construct (PTG) for multiplex editing using an endogenous tRNA processing system. The RNA-guided Cas9 constructs were validated in transgenic barley plants produced by Agrobacterium-mediated transformation. The highest mutation rate was observed in simplex editing of the cytokinin oxidase/dehydrogenase HvCKX1 gene, where mutations at the hvckx1 locus were detected in 88% of the screened T₀ plants. We also proved the efficacy of the PTG construct in the multiplex editing of two CKX genes by obtaining 9 plants (21% of all edited plants) with mutations induced in both HvCKX1 and HvCKX3. Analysis of the T₁ lines revealed that mutations in the HvCKX1 gene were transmitted to the next generation of plants. Among 220 screened T₁ plants we identified 85 heterozygous and 28 homozygous mutants, most of them bearing frameshift mutations in the HvCKX1 gene. We also observed independent segregation of mutations and the Cas9-sgRNA T-DNA insert in several T₁ plants. Moreover, the knockout mutations of the Nud gene generated phenotype mutants with naked grains, and the phenotypic changes were identifiable in T₀ plants.

Conclusions

We demonstrated the effectiveness of an optimized RNA-guided Cas9 system that can be used for generating homozygous knockout mutants in the progeny of transgenic barely plants. This is also the first report of successful multiplex editing in barley using a tRNA processing system.

Background

To investigate plant hybrid sterility, we studied interspecific hybrids of two cultivated rice species, Asian rice (Oryza sativa) and African rice (O. glaberrima). Male gametes of these hybrids display complete sterility owing to a dozen of hybrid sterility loci, termed HS loci, but this complicated genetic system remains poorly understood.

Results

Microspores from these interspecific hybrids form sterile pollen but are viable at the immature stage. Application of the anther culture (AC) method caused these immature microspores to induce callus. The segregation distortion of 11 among 13 known HS loci was assessed in the callus population. Using many individual calli, fine mapping of the HS loci was attempted based on heterozygotes produced from chromosome segment substitution lines (CSSLs). Transmission ratio distortion (TRD) from microspores was detected at 6 of 11 HS loci in the callus population. The fine mapping of S₁ and S₁₉ loci using CSSLs revealed precise distances of markers from the positions of HS loci exhibiting excessive TRD.

Conclusions

We demonstrated that AC to generate callus populations derived from immature microspores is a useful methodology for genetic study. The callus population facilitated detection of TRD at multiple HS loci and dramatically shortened the process for mapping hybrid sterility genes.

Context

Multiple ecological drivers generate spatial patterns in species’ distributions. Changes to natural disturbance regimes can place early successional habitat specialists at an increased risk of extinction by altering landscape patterns of habitat suitability.

Objectives

We developed a series of hypotheses to evaluate the effects of landscape structure, fire history, and site-level habitat quality on site occupancy by an early successional specialist, the eastern chestnut mouse (Pseudomys gracilicaudatus).

Methods

We obtained eight years of monitoring data from 26 sites in recently burned heathland in southeast Australia. We used generalised linear models to determine which explanatory variables were related to occupancy. We also explored predictability in patterns of small mammal species co-occurrence.

Results

Landscape structure (patch area, landscape heterogeneity) was strongly related to site occupancy. Site occupancy was associated with dead shrubs in the understory and rock cover on ground layer, but was not directly influenced by recent or historical fire. Contrary to contemporary ecological theory, we found no predictable species associations in our early successional community.

Conclusions

We recommend surveys take account of landscape configuration and proximity to suitable habitat for optimal results. Fire regimes expected to promote eastern chestnut mouse population growth should encourage the retention of critical habitat features rather than be based on temporal rates of successional stages. For management to adequately account for post-disturbance patterns in early successional communities, a species-by-species, multi-scaled approach to research is necessary.

Context

Policy decisions form a major driver of land use change, with important implications for socially and environmentally susceptible regions. It is well known that there can be major unintended consequences, especially where policies are not tailored to regionally specific contexts.

Objectives

In this paper we assess the implications of 60 years of agricultural policies on soil erosion prevention (SEP) by vegetation, an essential regulating ecosystem service in Mediterranean Europe.

Methods

To assess these implications we produced and analysed a time series of land cover/use and environmental conditions datasets (from 1951 to 2012) in relation to changing agricultural policies for a specific region in the southern Portugal. A set of indicators related to SEP allowed us to identify that land use intensification as increased soil erosion in the last 60 years.

Results

Particularly in the last 35 years, as a consequence of headage payments for cattle, the agricultural policy had a significant effect in the density and renewal of the tree cover, resulting in drastic effects for the provision of the SEP service. These are more significant after 1986, coinciding with the implementation of several Common Agricultural Policy instruments focused on increasing the modernization and productivity capacity of farm systems.

Conclusions

The results show some unintended effects of agricultural policy mechanisms on ecosystem service provision and highlight the need for context-based policies, tailored to the environmental constrains and potentials of each region.

Context

The role of agricultural landscapes in biodiversity conservation is an emerging topic in a world experiencing a worrying decrease of species richness. Farm systems may either decrease or increase biological diversity, depending on land-use intensities and management.

Objectives

We present an intermediate disturbance-complexity model (IDC) of cultural landscapes aimed at assessing how different levels of anthropogenic disturbance on ecosystems affect the capacity to host biodiversity depending on the land matrix heterogeneity. It is applied to the Mallorca Island, amidst the Mediterranean biodiversity hotspot.

Methods

The model uses the disturbance exerted when farmers alter the Net Primary Production through land-use change as well as when they remove a share of it (HANPP), together with Shannon–Wiener index (H′) of land-cover diversity. The model is tested with a twofold-scalar experimental design (1:50,000 and 1:5000) of a set of landscape units along three time points (1956, 1989, 2011). Species richness of breeding and wintering birds, taken as a biodiversity proxy, is used in an exploratory factor analysis.

Results

The results clearly show that when intermediate levels of HANPP are performed within intermediate levels of complexity (H′) in landscape patterns, like agro-forest mosaics, great bird species richness and high socio-ecological resilience can be maintained. Yet, these complex-heterogeneous landscapes are currently vanishing due to industrial farm intensification, rural abandonment and urban sprawl.

Conclusions

The results make apparent the usefulness of transferring the concept of intermediate disturbance-complexity interplay to cultural landscapes. Our spatial-explicit IDC model can be used as a tool for strategic environmental assessment of land-use planning.

Context

Seed dispersal is recognized as having profound effects on the distribution, dynamics and structure of plant populations and communities. However, knowledge of how landscape structure shapes carnivore-mediated seed dispersal patterns is still scarce, thereby limiting our understanding of large-scale plant population processes.

Objectives

We aim to determine how the amount and spatial configuration of forest cover impacted the relative abundance of carnivorous mammals, and how these effects cascaded through the seed dispersal kernels they generated.

Methods

Camera traps activated by animal movement were used for carnivore sampling. Colour-coded seed mimics embedded in common figs were used to know the exact origin of the dispersed seed mimics later found in carnivore scats. We applied this procedure in two sites differing in landscape structure.

Results

We did not find between-site differences in the relative abundance of the principal carnivore species contributing to seed dispersal patterns, Martes foina. Mean dispersal distance and the probability of long dispersal events were higher in the site with spatially continuous and abundant forest cover, compared to the site with spatially aggregated and scarcer forest cover. Seed deposition closely matched the spatial patterning of forest cover in both study sites, suggesting behaviour-based mechanisms underpinning seed dispersal patterns generated by individual frugivore species.

Conclusions

Our results provide the first empirical evidence of the impact of landscape structure on carnivore-mediated seed dispersal kernels. They also indicate that seed dispersal kernels generated strongly depend on the effect that landscape structure exerts on carnivore populations, particularly on habitat-use preferences.