A simple and efficient CRISPR/Cas9 platform for induction of single and multiple,heritable mutations in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

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.

     

The possibility of application of agglomerate elastomers (EPP) as media for biological bed in aquaculture

The aim of the study was to determine the possibility of experimental media (agglomerate elastomers EPP) application as biological media bed, which serves the purpose of water purification in recirculating aquaculture systems (RAS). RAS enables mass‐production of fish in small volume of water in a limited area. This involves the possibility of multiple usage of water during culture. However, for that purpose of maintaining proper physico‐chemical parameters, water purification from products of metabolism, especially toxic nitrogen compounds, is required. One of the simplest and most effective ways to achieve it is combining application of two types of water filtration: mechanical and biological. It is needed to study new media for biological bed with proper filling is able to purify water from toxic nitrogen compounds.     

Effect of temperature on growth and survival of maraena whitefish Coregonus maraena (Bloch 1779) larvae in controlled conditions

This 28‐day study investigated the effect of three rearing temperatures, 11, 15 and 19°C, on survival and growth of maraena whitefish fry in a recirculating aquaculture system. Three groups of larvae in three repetitions were reared in recirculating system. Each group comprised 200 larvae. Feeding level was fixed at 500–700 Artemia sp. metanauplii per fish per day. Larvae were fed fresh live brine shrimp at 10 ml/tank every 3 hr. Significantly higher body weight (p = 0.00), total length (p = 0.00), larval yield (p = 0.00) and condition factor (p = 0.00) were obtained at 19°C compared to 15 and 11°C, as well as at 15°C compared to 11°C. Significantly higher survival (p = 0.00) was observed in larvae reared at 11 and 15°C compare to 19°C and no significant differences were observed between 11°C compared to 15°C. No significant differences in size heterogeneity among treatments were found (p = 0.46). In larviculture, the optimal assessed temperature for growth of maraena whitefish was 19°C, with highest survival observed at 11°C, at the end of this 28 days trial. The findings in this study apply to the particular study location and may not be applicable more broadly.     

耕地经营规模及其质量禀赋对农户生产环节外包行为的影响——基于中国广西水稻种植农户的调研数据

为了解耕地经营规模及耕地质量禀赋对农户生产环节外包行为的影响,以广西2014年典型地区的水稻种植为例,利用Poisson模型和Logit模型,以耕地经营规模及其质量禀赋作为核心解释变量,分析其对农户水稻生产环节外包决策的影响。结果表明:耕地经营规模扩大会使农户更倾向于生产环节中采用外包,尤其是在整地、收获这些劳动密集型环节;耕地肥沃程度会显著提高农户在生产环节采用外包服务的可能性和频率;出于外包成本的考虑,随着耕地经营规模的扩大,农户会减少在诸如灌溉等作业频繁的生产环节的外包可能性,也会减少整个生产环节外包的次数。     

Morphological and molecular identification and PCR amplification to determine the toxigenic potential of Fusarium spp. isolated from maize ears in southern Poland

The average amount of precipitation in spring and summer 2010 and 2011 coupled with relatively high temperatures caused massive Fusarium spp. infection of maize and yield losses in southern Poland. In order to examine the cause of this disease outbreak, Fusarium spp. were isolated and fungal strains were identified based on morphological characters and species-specific PCR assays. A total of 200 maize samples were processed, resulting in the obtention of 71 strains, which belonged to five Fusarium species, F. poae being the predominant one (74.56%). Other isolates were identified as F. graminearum, F. oxysporum, F. verticillioides and F. proliferatum. PCR-based detection of mycotoxin-synthesis-pathway genes was also used to determine the potential of the analyzed strains to produce trichothecenes (DON and NIV) and fumonisins (FUM). Only 14 isolates revealed the potential to produce DON (11 strains) and FUM (3 strains). HPLC analyses of grain samples revealed the presence of DON only – other mycotoxins were not detected. Moreover, 57.1% of potentially mycotoxin-producing isolates indicated the toxicity in a biological test.     

Dirofilaria repens—An etiological factor or an incidental finding in cytologic and histopathologic biopsies from dogs

Dirofilaria repens is an endemic, zoonotic parasite of carnivores, causing subcutaneous dirofilariasis, which is mostly asymptomatic. The aim of this study was to describe 22 cases of canine subcutaneous dirofilariasis. The cytologic and histopathologic samples were collected from dogs, which presented with various clinical signs such as cutaneous/subcutaneous nodules, hydropericardium, ascites, and lymphadenomegaly. The cytologic or histopathologic examination revealed purulent, pyogranulomatous, granulomatous or eosinophilic dermatitis/panniculitis, and the presence of D repens microfilariae or adults. The microfilariae or adults were also found incidentally in neoplastic cutaneous or subcutaneous tumors and in a sialocele. For the first time, microfilariae were also detected and described in pericardial and abdominal effusions and in enlarged reactive lymph nodes. Although it is well known that D repens can cause dermatitis and panniculitis in dogs, no previous reports of D repens microfilariae in body cavity fluids were found. The role of this parasite in the accumulation of body cavity fluid or in reactive lymphadenomegaly requires further investigation. Due to its zoonotic potential, and apparently underestimated pathogenicity, each case of canine subcutaneous dirofilariasis should be treated.     

Anticoagulant effect of polyphenols-rich extracts from black chokeberry and grape seeds

Blood coagulation consists of a series of zymogens that can be converted by limited proteolysis to active enzymes leading to the generation of thrombin. Fresh plasma and human thrombin was incubated with extracts from berries of Aronia melanocarpa or seeds of Vitis vinifera (0.5; 5; 50 μg/ml). The in vitro experiments showed that both extracts prolonged clotting time and decreased the maximal velocity of fibrin polymerization in human plasma. Moreover thrombin incubation with both extracts results in the inhibition of amidolytic activity of this enzyme. It gives hopes for development of diet supplements, which may be preventing thrombosis in pathological states.     

Variability of Selected Biochemical Parameters in Young Stallions During the 100-day Performance Test

The aim of the study was to evaluate variations in biochemical blood parameters of stallions during a 100-day performance test. The study was carried out with 20 clinically healthy stallions aged 3-4 years. The degree of adaptation to exercise loads during a 100-day performance test was assessed three times: (I) on the first day of training, (II) on the 46th day of training, and (III) on the 97^th day of training. Sample collection and basic clinical tests were performed before training (1), directly after training (2), and after a 30-minute rest (3). Venous blood samples were analyzed for the concentration of total protein, glucose (GLU), and lactate, the activity of lactate dehydrogenase (LDH), and creatine kinase (CK), and total antioxidant status (TAS). Postexercise changes in the analyzed parameters showed that the loads were of moderate intensity. As a result of the 100-day training, postexercise variation in GLU concentration and CK and LDH activity was lower than at the beginning, which indicates that the body was stimulated to adapt to performing exercise tasks. The resting level of TAS was also found to increase. The results obtained suggest that the stallions are well prepared for increasing exercise loads.     

A site index model for lodgepole pine (Pinus contorta Dougl. var. latifolia) in northern Sweden

A model was developed for predicting site index (SI) values and top height growth of lodgepole pine in northern Sweden. Data on 169 experimental plots from 61 experiments were used for model construction. Six dynamic site equations derived using the generalized algebraic difference approach (GADA) were tested. All of the equations estimate height and SI values on the basis of a known height and total age, and are base-age invariant. Unbiased parameter estimates were obtained by non-linear modelling without accounting for autocorrelation of residuals. The special formulation of the Hossfeld model and the GADA-transformed logistic function produced the most reliable SI curves. The Hossfeld model is recommended as it did not produce extreme outlying estimates for young stands. The developed model is polymorphic with variable asymptotes and can predict SI and dominant height growth for lodgepole pine between 20 and 50 years old.