Chromosome-scale assembly of the highly heterozygous genome of red clover (Trifolium pratense L.), an allogamous forage crop species

Red clover (Trifolium pratense L.) is used as a forage crop due to a variety of favorable traits relative to other crops. Improved varieties have been developed through conventional breeding approaches, but progress could be accelerated and gene discovery facilitated using modern genomic methods. Existing short-read based genome assemblies of the ~420 Megabase (Mb) genome are fragmented into >135,000 contigs with numerous errors in order and orientation within scaffolds, likely due to the biology of the plant which displays gametophytic self-incompatibility resulting in inherent high heterozygosity. A high-quality long-read based assembly of red clover is presented that reduces the number of contigs by more than 500-fold, improves the per-base quality, and increases the contig N50 statistic by three orders of magnitude. The 413.5 Mb assembly is nearly 20% longer than the 350 Mb short read assembly, closer to the predicted genome size. Quality measures are presented and full-length isoform sequence of RNA transcripts reported for use in assessing accuracy and for future annotation of the genome. The assembly accurately represents the seven main linkage groups present in the genome of an allogamous (outcrossing), highly heterozygous plant species.

Trifolium pratense ethanolic extract alters the gut microbiota composition and regulates serum lipid profile in the ovariectomized rats

Background Trifolium pratense (red clover) ethanolic extract (TPEE) has been used as a popular over-the-counter remedy for the management of menopausal symptoms. Prolonged consumption of herbal extract has been shown to regulate the composition of gut microbiota. This study was designed to elucidate the influence of TPEE on the gut microbiota composition in the ovariectomized (OVX) rats. Methods OVX rats were treated with TPEE at 125, 250, 500 mg/kg/day, or controls (pomegranate extract, 500 mg/kg/day; estradiol, 25 μg/kg/day) for 12 weeks. Gut microbiota analysis was conducted by extracting the microbial DNA from fecal samples and microbiome taxonomic profiling was carried out by using next-generation sequencing. The levels of serum biomarkers were analyzed using enzyme-linked immunosorbent assay (ELISA) kit. The prediction of functional biomarker of microbiota was performed using PICRUSt to investigate the potential pathways associated with gut health and serum lipid profile regulation. To study the correlation between gut microbiota composition and serum lipid levels, Spearman’s correlation coefficients were defined and analyzed. Additionally, gas chromatography–mass spectrometry analysis was conducted to uncover additional physiologically active ingredients. Results TPEE-treated OVX rats showed significant reduction in serum triglycerides (TG), total cholesterols (TCHOL), and LDL/VLDL levels but increase in HDL level. The alteration in the pathways involve in metabolism was the most common among the other KEGG categories. Particularly, TPEE also significantly reduced the relative abundance of sequences read associated with inflammatory bowel disease (IBD) and the peroxisome proliferator-activated receptor (PPAR) signalling pathway. TPEE intervention was seen to reduce the Firmicutes to Bacteroidetes (F/B) ratio in the OVX rats, denoting a reduction in microbial dysbiosis in the OVX rats. Correlation analysis at the phylum level revealed that Bacteriodetes and Proteobacteria were strongly correlated with serum TG, TCHOL and HDL levels. At the species level, Bifidobacterium pseudolongum group was seen to positively correlate with serum HDL level and negatively correlated with serum AST, ALT, LDL/VLDL, TCHOL, and TG levels. Conclusions TPEE treatment showed therapeutic benefits by improving the intestinal microbiota composition which strongly correlated with the serum lipid and cholesterol levels in the OVX rats.

Eco-friendly soil amendments improve growth, antioxidant activities, and root colonization in lingrain (Linum Usitatissimum L.) under drought conditions

This study’s primary purpose was to investigate the possible amelioration of limited irrigation conditions by mycorrhiza (AMF), vermicompost, and green manure for lingrain plants. This experiment was accomplished as a factorial based on the completely randomized design with three replications. The first factor was green manure (without green manure and with Trifolium pratense as green manure); the second factor consisted of Rhizophagus irregularis mycorrhiza, vermicompost, a combination of mycorrhiza and vermicompost and none of them, and also the third factor was irrigation regime (full irrigation and late-season water limitation). Green manure, vermicompost, and mycorrhiza single-use enhanced the plant’s underwater limitation conditions compared to the control. However, vermicompost and green manure or mycorrhiza developed a positive synergistic effect on most traits. Combining green manure with the dual fertilizer (mycorrhiza + vermicompost) resulted in the vermicompost and mycorrhiza synergistic effects, especially under limited irrigation. Consequently, the combination of green manure, mycorrhiza, and vermicompost experienced the highest amount of leaf relative water content, root colonization, leaf nitrogen, chlorophyll a, chlorophyll b, carotenoids, antioxidant enzymes activity, grain yield, and oil yield, which would lead to more resistance of plants to limited irrigation conditions.

Análisis de la sustentabilidad ecológica de distintas estrategias de manejo de malezas en el cultivo de lino oleaginoso (Linum usitatissimum L.) en Argentina

El lino se cultiva generalmente bajo un enfoque productivista, altamente dependiente de insumos externos, modelo que ha causado importantes problemas ecológicos. Entre ellos están los asociados con las malezas cuyo control se realiza principalmente con herbicidas. Ante esta realidad es necesario incorporar estrategias de manejo de malezas que mejoren la sustentabilidad de los agroecosistemas. Se comparó la sustentabilidad ecológica del control de malezas con herbicida respecto al manejo con alta densidad de lino y/o su intercultivo con Trifolium pratense L. En este trabajo se ajustaron y aplicaron algunos indicadores como: diversidad vegetal, % de cobertura de suelo, residuo remanente, materia seca restituida al suelo, % de residuo remanente, uso de herbicida, balance de nitrógeno, eficiencia energética y biomasa vegetal. La metodología usada fue adecuada y fácil de aplicar al objetivo del trabajo. Se observó que el control de malezas en el lino con herbicida fue la práctica de menor aporte a la sustentabilidad, por lo cual si se continúa en el largo plazo con dicha práctica se verá comprometida la misma. Mientras que el intercultivo fue ecológicamente más sustentable que el monocultivo, con puntos más favorables para sustentabilidad sin uso de herbicida, independientemente de la densidad de lino. Esto ocurrió tanto por una mejor conservación de la capacidad productiva del agroecosistema como por ejercer un menor impacto sobre el ambiente externo al mismo. Del presente trabajo surge que existen alternativas al control con herbicidas para el manejo de malezas en lino como es el caso del intercultivo con trébol rojo.

Primera aproximación de carga animal óptima en cuyes al pastoreo durante la época lluviosa en la sierra peruana

Se realizó una primera aproximación de la carga animal óptima en cuyes al pastoreo durante la época lluviosa en la sierra del Perú, utilizando una pastura perenne asociada conformada por Lolium multiflorum, Lolium perenne, Dactylis glomerata, Trifolium pratense, Trifolium repens y Medicago sativa en función a la ganancia de peso vivo individual y por unidad de área, consumo de alimento, conversión alimenticia, rendimiento de pastura, costos de producción y ratio beneficio costo. Se emplearon 132 cuyes machos G de 15 días de edad en un diseño de bloques completos al azar con tres ciclos de engorde de 60 días como bloque y cuatro tratamientos correspondientes a cargas de 400 (T1), 500 (T2), 600 (T3) y 700 (T4) cuyes/ha/año. Se trabajó en áreas de 200 m2 de pastura donde pastorearon grupos de 8, 10, 12 y 14 cuyes en T1, T2, T3 y T4, respectivamente. Los resultados demostraron que no hubo efecto de la carga animal sobre la ganancia de peso individual, consumo de alimento, conversión alimenticia y rendimiento de pastura (p>0.05). Los efectos sobre la ganancia por unidad de área y ratio beneficio costo (p<0.05) fueron consecuencia directa y esperada del diferente número de animales entre cargas animales. El rango de carga animal fue insuficiente para determinar una carga óptima, de manera que tentativamente se sugiere utilizar la carga de 700 cuyes/ha/año, por ser la carga que favorece lograr más cuyes con parámetros productivos similares a cargas menores y mejores parámetros económicos.

Seed Production of Red Clover (Trifolium pratense L.) under Danish Field Conditions

High and stable seed yield is critical for red clover (Trifolium pratense L.) seed production and the commercial exploitation of the crop. A three-year experiment was conducted from 2013 to 2015 under Danish field conditions to explore the influence of precipitation during peak flowering on the seed yield of three red clover cultivars. We investigated the flowering duration and intensity based on a visual scale assessment, seed yield, and thousand seed weight in all three experimental years. In 2014 and 2015 we measured the seed yield components of floret number per flower head, seed number per flower head, and seed set. During the experimental period, high seed yields of more than 1000 kg ha−1 were obtained for the diploid cultivars ‘Rajah’ and ‘Suez’. Although a relatively high seed yield of 500 kg ha−1 was obtained in the tetraploid cultivar ‘Amos’, this was only around half of the seed yield and seed set of the diploid cultivars. Precipitation during peak flowering positively influenced the seed yield and thousand seed weight for the three cultivars. We conclude that observations of flowering phenology are required to determine the impact of environmental conditions on seed yield in red clover cultivars. Further, adequate water supply during peak flowering is important to obtain the high seed yield of red clover.

An ethnobotanical survey of medicinal plants used by traditional healers for the treatment of cancer in Hammanskraal and Winterveld, Tshwane Metropolitan Municipality, South Africa

Background: Knowledge of medicinal plants used by the traditional healers are mostly confined among the locals and the adherents, hence, proper enquiry and documentation can help the ever dynamic scientific world to find permanent cure to the menace of such deadly diseases such as cancer. This study aimed at (1) specifically recording medicinal plants traditionally used for the treatment of cancer in Hammanskraal and Winterveld, South Africa, and (2) documenting the different methods of preparation and administration of those medicinal plants as recorded during the study. Method: An open-ended semi-structured questionnaire was administered to 90 willing traditional healers in Hammanskraal and Winterveld area, Tshwane Municipality, South Africa to document plants used for cancer treatments. The study was conducted over a period of six months (July – December, 2018). Descriptive statistics was used to present the obtained data. Results: The study recorded twenty-eight plant species belonging to 18 families for the treatment of different types of cancer. Plant species in the Fabaceae family particularly Lessertia frutescens (L.) Goldblatt and J.C. Manning, Senna italica Mill and Trifolium pratense L. were the most prominently mentioned (highest citation frequency) by the traditional healers for lung, and skin cancer treatment. Based on the citation frequency, the most treated cancer by the traditional healers is in the order: skin cancer > lung cancer > breast cancer > prostate cancer > cervical cancer. The method of preparation included decoction(32.3%), infusion (29%), paste (16.1%) and maceration (22.6%). Conclusion: In addition to the documentation of indigenous knowledge related to the use of medicinal plants in the traditional management of cancer in South Africa, this study opens a vista for investigations into the phytochemical and pharmacological properties of the documented plants. Keywords: Cancer; Indigenous plants; Medicinal plants; South Africa; Traditional practitioners.

The Influence of Ozone on Net Ecosystem Production of a Ryegrass–Clover Mixture under Field Conditions

In order to understand the effect of phytotoxic tropospheric ozone (O3) on terrestrial vegetation, we quantified the impact of current O3 concentration ([O3]) on net ecosystem production (NEP) when compared to the conditions of the pre-industrial era. We compared and tested linear mixed-effects models based on [O3] and stomatal O3 flux (Fsto). The managed ryegrass–clover (Lolium perenne and Trifolium pratense) mixture was grown on arable land in the Czech Republic, Central Europe. Values of [O3] and Fsto were measured and calculated based on resistance analogy, respectively, while NEP was calculated from eddy covariance CO2 fluxes. We found the Fsto-based model more precise when compared to measured NEP. High Fsto was found even at low [O3], while broad summer maximum of [O3] was not necessarily followed by significant NEP decline, due to low soil water content leading to a low stomatal conductivity and Fsto. Comparing to low pre-industrial O3 conditions, current levels of O3 resulted in the reduction of cumulative NEP over the entire growing season, up to 29.7 and 13.5% when the [O3]-based and Fsto-based model was applied, respectively. During the growing season, an O3-induced reduction of NEP ranged between 13.1% in May and 26.2% in July when compared to pre-industrial Fsto levels. Looking to the future, high [O3] and Fsto may lead to the reduction of current NEP by approximately 13.3% on average during the growing season, but may increase by up to 61–86.6% in autumn, indicating further O3-induced acceleration of the senescence. These findings indicate the importance of Fsto and its inclusion into the models estimating O3 effects on terrestrial vegetation. The interaction between environmental factors and stomatal conductance is therefore discussed in detail.

Herbicide Potential of Selected Essential Oils From Plants of Lamiaceae and Asteraceae Families

Essential oils from Monarda fistulosa L., Satureja pilosa Vel., Origanum vulgare subsp. hirtum Ietswaart. (Greek oregano), Micromeria dalmatica Benth., Thymus longedentatus (Degen & Urum.) Ronniger, and Artemisa campestris L. were evaluated as inhibitors of seed germination in target plants Lolium perenne L. and Trifolium pratense L. using in vitro assays. The essential oils were applied on the seeds as aqueous solutions at concentrations ranging from 0.5–3.0 µL/mL. Complete inhibition was established at a concentration of 1.5 µL/mL with the most effective essential oils. Oregano oil was evaluated for its inhibitory activity on seed germination under field conditions; the essential oil was applied as an aqueous solution at concentrations of 3, 5, and 10 µL/mL on superabsorbent Terawet. The mass obtained was mixed with the seeds of the target plants and planted in a field. After 1 month, the results were evaluated based on the weight of the aerial parts of the plants from control and experimental areas. At the highest concentration studied, the weight of the plants was 77% lower in the experimental areas than in the controls. The chemical composition of the essential oils was analyzed using gas chromatography–mass spectrometry. The results showed that carvacrol-rich essential oils had a strong inhibitory effect on seed germination. The inclusion of the essential oil on a superabsorbent was a good way to preserve its herbicidal activity under field conditions as this prevented its rapid evaporation.

GBS and a newly developed mRNA-GBS approach to link population genetic and transcriptome analyses reveal pattern differences between sites and treatments in red clover (Trifolium pratense L.)

The important worldwide forage crop red clover (Trifolium pratense L.) is widely cultivated as cattle feed and for soil improvement. Wild populations and landraces have great natural diversity that could be used to improve cultivated red clover. However, to date, there is still insufficient knowledge about the natural genetic and phenotypic diversity of the species. Here, we developed a low-cost transcriptome analysis (mRNA-GBS) with reduced complexity and compared the results with population genetic (GBS) and previously published mRNA-Seq data, to assess whether analysis of intraspecific variation within and between populations and transcriptome responses is possible simultaneously. The mRNA-GBS approach was successful. SNP analyses from the mRNA-GBS approach revealed comparable patterns to the GBS results, but it was not possible to link transcriptome analyses with reduced complexity and sequencing depth to previously published greenhouse and field expression studies. The use of short sequences upstream of the poly(A) tail of mRNA to reduce complexity are promising approaches that combine population genetics and expression profiling to analyze many individuals with trait differences simultaneously and cost-effectively, even in non-model species. Our mRNA-GBS approach revealed too many additional short mRNA sequences, hampering sequence alignment depth and SNP recovery. Optimizations are being discussed. Nevertheless, our study design across different regions in Germany was also challenging as the use of differential expression analyses with reduced complexity, in which mRNA is fragmented at specific sites rather than randomly, is most likely counteracted under natural conditions by highly complex plant reactions at low sequencing depth.