Genes involved in auxin biosynthesis, transport and signalling underlie the extreme adventitious root phenotype of the tomato aer mutant.

The use of tomato rootstocks has helped to alleviate the soaring abiotic stresses provoked by the adverse effects of climate change. Lateral and adventitious roots can improve topsoil exploration and nutrient uptake, shoot biomass and resulting overall yield. It is essential to understand the genetic basis of root structure development and how lateral and adventitious roots are produced. Existing mutant lines with specific root phenotypes are an excellent resource to analyse and comprehend the molecular basis of root developmental traits. The tomato aerial roots (aer) mutant exhibits an extreme adventitious rooting phenotype on the primary stem. It is known that this phenotype is associated with restricted polar auxin transport from the juvenile to the more mature stem, but prior to this study, the genetic loci responsible for the aer phenotype were unknown. We used genomic approaches to define the polygenic nature of the aer phenotype and provide evidence that increased expression of specific auxin biosynthesis, transport and signalling genes in different loci causes the initiation of adventitious root primordia in tomato stems. Our results allow the selection of different levels of adventitious rooting using molecular markers, potentially contributing to rootstock breeding strategies in grafted vegetable crops, especially in tomato. In crops vegetatively propagated as cuttings, such as fruit trees and cane fruits, orthologous genes may be useful for the selection of cultivars more amenable to propagation.

A chromosome-level genome assembly of Solanum chilense, a tomato wild relative associated with resistance to salinity and drought.

Introduction: Solanum chilense is a wild relative of tomato reported to exhibit resistance to biotic and abiotic stresses. There is potential to improve tomato cultivars via breeding with wild relatives, a process greatly accelerated by suitable genomic and genetic resources. Methods: In this study we generated a high-quality, chromosome-level, de novo assembly for the S. chilense accession LA1972 using a hybrid assembly strategy with ~180 Gbp of Illumina short reads and ~50 Gbp long PacBio reads. Further scaffolding was performed using Bionano optical maps and 10x Chromium reads. Results: The resulting sequences were arranged into 12 pseudomolecules using Hi-C sequencing. This resulted in a 901 Mbp assembly, with a completeness of 95%, as determined by Benchmarking with Universal Single-Copy Orthologs (BUSCO). Sequencing of RNA from multiple tissues resulting in ~219 Gbp of reads was used to annotate the genome assembly with an RNA-Seq guided gene prediction, and for a de novo transcriptome assembly. This chromosome-level, high-quality reference genome for S. chilense accession LA1972 will support future breeding efforts for more sustainable tomato production. Discussion: Gene sequences related to drought and salt resistance were compared between S. chilense and S. lycopersicum to identify amino acid variations with high potential for functional impact. These variants were subsequently analysed in 84 resequenced tomato lines across 12 different related species to explore the variant distributions. We identified a set of 7 putative impactful amino acid variants some of which may also impact on fruit development for example the ethylene-responsive transcription factor WIN1 and ethylene-insensitive protein 2. These variants could be tested for their ability to confer functional phenotypes to cultivars that have lost these variants.

Missense mutation of a class B heat shock factor is responsible for the tomato bushy root-2 phenotype.

The bushy root-2 (brt-2) tomato mutant has twisting roots, and slower plant development. Here we used whole genome resequencing and genetic mapping to show that brt-2 is caused by a serine to cysteine (S75C) substitution in the DNA binding domain (DBD) of a heat shock factor class B (HsfB) encoded by SolycHsfB4a. This gene is orthologous to the Arabidopsis SCHIZORIZA gene, also known as AtHsfB4. The brt-2 phenotype is very similar to Arabidopsis lines in which the function of AtHsfB4 is altered: a proliferation of lateral root cap and root meristematic tissues, and a tendency for lateral root cap cells to easily separate. The brt-2 S75C mutation is unusual because all other reported amino acid substitutions in the highly conserved DBD of eukaryotic heat shock factors are dominant negative mutations, but brt-2 is recessive. We further show through reciprocal grafting that brt-2 exerts its effects predominantly through the root genotype even through BRT-2 is expressed at similar levels in both root and shoot meristems. Since AtHsfB4 is induced by root knot nematodes (RKN), and loss-of-function mutants of this gene are resistant to RKNs, BRT-2 could be a target gene for RKN resistance, an important trait in tomato rootstock breeding.Gene & accession numbersSolycHsfB4a - Solyc04g078770.

De Novo Genome Assembly Of Solanum Sitiens Reveals Structural Variation Associated With Drought And Salinity Tolerance.

MOTIVATION: Solanum sitiens is a self-incompatible wild relative of tomato, characterised by salt and drought resistance traits, with the potential to contribute through breeding programmes to crop improvement in cultivated tomato. This species has a distinct morphology, classification and ecotype compared to other stress resistant wild tomato relatives such as S. pennellii and S. chilense. Therefore, the availability of a reference genome for S. sitiens will facilitate the genetic and molecular understanding of salt and drought resistance. RESULTS: A high-quality de novo genome and transcriptome assembly for S. sitiens (Accession LA1974) has been developed. A hybrid assembly strategy was followed using Illumina short reads (∼159X coverage) and PacBio long reads (∼44X coverage), generating a total of ∼262 Gbp of DNA sequence. A reference genome of 1,245 Mbp, arranged in 1,483 scaffolds with a N50 of 1.826 Mbp was generated. Genome completeness was estimated at 95% using the Benchmarking Universal Single-Copy Orthologs (BUSCO) and the K-mer Analysis Tool (KAT). In addition, ∼63 Gbp of RNA-Seq were generated to support the prediction of 31,164 genes from the assembly, and to perform a de novo transcriptome. Lastly, we identified three large inversions compared to S. lycopersicum, containing several drought resistance related genes, such as beta-amylase 1 and YUCCA7. AVAILABILITY: S. sitiens (LA1974) raw sequencing, transcriptome and genome assembly have been deposited at the NCBI's Sequence Read Archive, under the BioProject number "PRJNA633104".All the commands and scripts necessary to generate the assembly are available at the following github repository: https://github.com/MCorentin/Solanum_sitiens_assembly. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Biostimulating effect of l-tryptophan on the yield and chemical and microbiological quality of perennial ryegrass (Lolium perenne) herbage and silage for ruminant.

BACKGROUND: This study aimed to evaluate the effect of l-tryptophan (l-TRP) used in the cultivation of Lolium perenne on the yield, and chemical and microbiological quality of its herbage and silage. l-Tryptophan was applied in doses of 5, 0.5, 0.05 kg ha-1 . The experiment was conducted with a control group (C) and a comparative control group (C+) with higher nitrogen fertilization. RESULTS: The dose of 5 kg ha-1 had a significant effect on herbage yield, which increased by 15% compared to group C and approximated the value achieved in group C+. The treatment with l-TRP caused a significant increase in water-soluble carbohydrate (WSC) content only in Tr5 (165 g kg-1 DM), which was reflected in a more beneficial course of fermentation, lower pH (4.59), and a higher sum of fermentation acids, including lactic acid (94.7 g kg-1 DM). Ryegrass treatment with a high l-TRP dose effectively reduced the loads of Clostridium spp. and fungi, and increased the count of Bacillus spp. The l-TRP significantly reduced N-NH3 content in Tr05 (98.6 g kg-1 TN) compared with C+ (123 g kg-1 TN) and butyric acid content in Tr05 (from 1.35 g kg-1 DM in the C to 0.38 g kg-1 DM). CONCLUSION: The most effective dose turned out to be a dose of 5 kg ha-1 , which allowed a higher yield and a better fermentation course to be achieved. This work presents the feasibility of using l-TRP to optimize nutrient consumption by Lolium perenne and ultimately to affect the quality of its silage as a feedstuff. © 2020 Society of Chemical Industry.

Mutagenesis of Puccinia graminis f. sp. tritici and Selection of Gain-of-Virulence Mutants.

Wheat stem rust caused by the fungus Puccinia graminis f. sp. tritici (Pgt), is regaining prominence due to the recent emergence of virulent isolates and epidemics in Africa, Europe and Central Asia. The development and deployment of wheat cultivars with multiple stem rust resistance (Sr) genes stacked together will provide durable resistance. However, certain disease resistance genes can suppress each other or fail in particular genetic backgrounds. Therefore, the function of each Sr gene must be confirmed after incorporation into an Sr-gene stack. This is difficult when using pathogen disease assays due to epistasis from recognition of multiple avirulence (Avr) effectors. Heterologous delivery of single Avr effectors can circumvent this limitation, but this strategy is currently limited by the paucity of cloned Pgt Avrs. To accelerate Avr gene cloning, we outline a procedure to develop a mutant population of Pgt spores and select for gain-of-virulence mutants. We used ethyl methanesulphonate (EMS) to mutagenize urediniospores and create a library of > 10,000 independent mutant isolates that were combined into 16 bulks of ~658 pustules each. We sequenced random mutants and determined the average mutation density to be 1 single nucleotide variant (SNV) per 258 kb. From this, we calculated that a minimum of three independently derived gain-of-virulence mutants is required to identify a given Avr gene. We inoculated the mutant library onto plants containing Sr43, Sr44, or Sr45 and obtained 9, 4, and 14 mutants with virulence toward Sr43, Sr44, or Sr45, respectively. However, only mutants identified on Sr43 and Sr45 maintained their virulence when reinolculated onto the lines from which they were identified. We further characterized 8 mutants with virulence toward Sr43. These also maintained their virulence profile on the stem rust international differential set containing 20 Sr genes, indicating that they were most likely not accidental contaminants. In conclusion, our method allows selecting for virulent mutants toward targeted resistance (R) genes. The development of a mutant library from as little as 320 mg spores creates a resource that enables screening against several R genes without the need for multiple rounds of spore multiplication and mutagenesis.

Tersect: a set theoretical utility for exploring sequence variant data.

SUMMARY: Comparing genomic features among a large panel of individuals across the same species is considered nowadays a core part of the bioinformatics analyses. This typically involves a series of complex theoretical expressions to compare, intersect, extract symmetric differences between individuals within a large set of genotypes. Several publically available tools are capable of performing such tasks; however, due to the sheer size of variants being queried, such tasks can be computationally expensive with a runtime ranging from few minutes up to several hours depending on the dataset size. This makes existing tools unsuitable for interactive data query or as part of genomic data visualization platforms such as genome browsers. Tersect is a lightweight, high-performance command-line utility which interprets and applies flexible set theoretical expressions to sets of sequence variant data. It can be used both for interactive data exploration and as part of a larger pipeline thanks to its highly optimized storage and indexing algorithms for variant data. AVAILABILITY AND IMPLEMENTATION: Tersect was implemented in C and released under the MIT license. Tersect is freely available at https://github.com/tomkurowski/tersect. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The effect of an additive containing three Lactobacillus species on the fermentation pattern and microbiological status of silage.

BACKGROUND: Appropriate combinations of lactic acid bacteria (LAB) strains should be selected to optimize the ensiling process, and the additives should be adjusted to the ensiled forage crops. The aim of this study was to determine the effect of inoculation with three Lactobacillus species on the chemical parameters and microbiological quality (beneficial and harmful microbiota) of grass silage. RESULTS: Three species: L. paracasei (LPa), L. brevis (LB) and L. plantarum (LPl), isolated from sugar beet silage and characterized based on 16S rDNA sequences and biochemical parameters, were analyzed in the study. Single strains and their combinations were used as silage inoculants. The basic chemical and microbiological (qPCR) parameters of silages were determined. Based on the results of agglomerative hierarchical clustering (AHC) and principal component analysis (PCA) it was determined that silages inoculated with single LAB strains and LPa + LB and commercial additive (0+) were the best quality ones, particularly with regard to microbiological parameters and they effectively lowered the pH value. A consortium of three Lactobacillus species had no influence on silage quality, whereas LPa + LB and LPl + LB combinations as well as a commercial additive exerted positive effects. Inoculation inhibited the growth of toxin-producing fungi. CONCLUSION: Only the appropriate LAB composition can improve the quality of the ensiled material (antagonistic relationship). Only the LPa + LB combination was able to improve the value of low dry silage; nevertheless, almost all combinations were able to reduced concentrations of toxin-producing fungi. © 2019 Society of Chemical Industry.

Early plant growth and bacterial community in rhizoplane of wheat and flax exposed to silver and titanium dioxide nanoparticles.

Silver and titanium dioxide nanoparticles (AgNPs and TiO2NPs) are highly useful, but they are also a significant reason for concern as they exert toxicity. The goal of research was to assess the role of three kinds of NPs in concentrations of 100 mg L-1 on early growth plants (wheat, flax) and bacterial community in rhizoplane. Titanium (IV) oxide anatase (TiO2NPs1) and titanium (IV) oxide nanopowder (TiO2NPs2) are commercial products. A suspension of AgNPs was prepared via a procedure of reduction with tannic acid. The response of Monocot and Dicot growth form plants to the tested NPs was different. Germination and seedling growth of wheat treated with TiO2NPs1 was better. The response of flax to NPs was noted as an increase of chlorophyll content. The bacterial community in wheat rhizoplane was not significantly modified, but there was a declining trend. In turn, a difference in the surface charge of NPs had an influence on the total bacterial community in Dicot rhizoplane. Positively charged TiO2NPs2 significantly decreased the quantity of total bacteria in contrast to negatively charged AgNPs and TiO2NPs1 which increased it. A qualitative analysis did not confirm the influence of the surface charge of NPs on an increase/decrease in the quantity of Pseudomonas and Bacillus bacteria, but did show that there was no toxicity of the tested NPs to the plant growth-promoting bacteria community. The rhizoplane microbiome was dependent on the species of plant, and the bacteria found in the communities are sensitive to NPs to a varying degree.

Parental Opinions and Attitudes about Children's Vaccination Safety in Silesian Voivodeship, Poland.

Despite mandatory vaccinations in Poland, the final decision on vaccination in children is taken by their parents or legal guardians. Understanding parents' attitudes and opinions regarding vaccinations is essential for planning and undertaking extensive and properly targeted educational actions aimed at preventing their hesitancy. In 2016, a cross-sectional study was conducted in the Silesian Voivodeship (Poland) in 11 randomly selected educational institutions. The authors' self-administered questionnaire contained 24 mixed-type questions. It was distributed among 3000 parents or legal guardians of children aged 6-13 years; prior consent of the relevant bioethics committee had been obtained. The response rate was 41.3% (N = 1239). Data were analysed using descriptive and analytical statistics, and focused on parental opinions regarding the safety of vaccines. Results of simple and multivariable analyses showed that perceived risk of adverse vaccine reaction (AVR), contraindications and perception of the qualification procedure for vaccination as substandard were significant factors associated with the rating of children's vaccination as unsafe (p < 0.001). Respondents with a lower level of education, compared with those with higher, more often declared vaccinations to be safe (p = 0.03); however, results of multivariable analysis did not confirm that effect. AVR occurrence, finding of contraindication to vaccinations and perception of qualification procedure for vaccination were found to be the most important factors responsible for influencing general public opinions in the field of vaccination safety.

BIFURCATE FLOWER TRUSS: a novel locus controlling inflorescence branching in tomato contains a defective MAP kinase gene.

A mutant line, bifurcate flower truss (bif), was recovered from a tomato genetics programme. Plants from the control line produced a mean of 0.16 branches per truss, whereas the value for bif plants was 4.1. This increase in branching was accompanied by a 3.3-fold increase in flower number and showed a significant interaction with exposure to low temperature during truss development. The control line and bif genomes were resequenced and the bif gene was mapped to a 2.01 Mbp interval on chromosome 12; all coding region polymorphisms in the interval were surveyed, and five candidate genes displaying altered protein sequences were detected. One of these genes, SlMAPK1, encoding a mitogen-activated protein (MAP) kinase, contained a leucine to stop codon mutation predicted to disrupt kinase function. SlMAPK1 is an excellent candidate for bif because knock-out mutations of an Arabidopsis orthologue MPK6 were reported to have increased flower number. An introgression browser was used to demonstrate that the origin of the bif genomic DNA at the BIF locus was Solanum galapagense and that the SlMAPK1 null mutant is a naturally occurring allele widespread only on the Galápagos Islands. This work strongly implicates SlMAPK1 as part of the network of genes controlling inflorescence branching in tomato.

The effect of fertiliser treatments on the severity of Fusarium head blight and mycotoxin biosynthesis in winter rye.

The fungi of the genus Fusarium cause Fusarium head blight (FHB), a devastating disease that reduces grain yield and quality. They also produce mycotoxins which may pose a serious threat to human and animal health. This study investigated the effects of NPK fertilisation, foliar application of Cu, Zn, and Mn, applied separately and in combination, and of the Nano-Gro® organic growth stimulator on the occurrence of FHB in cultivar Dankowskie Diament rye based on the mycological analysis of kernels and on the concentrations of Fusarium mycotoxins in grain. The severity of FHB caused by seven species of the genus Fusarium was influenced by weather conditions in the analysed growing seasons. The applied fertilisation and the Nano-Gro® organic growth stimulator exerted varied effects on FHB development and the biosynthesis of Fusarium mycotoxins (deoxynivalenol, nivalenol, zearalenone and fumonisins) in grain. The greatest reduction in deoxynivalenol and nivalenol concentrations was noted in 2013, and the levels of moniliformin were lower in treated samples than in absolute control (untreated) samples in both years of the study. The severity of FHB positively correlated with the concentrations of zearalenone, deoxynivalenol, nivalenol, and moniliformin in the grain samples. Greater accumulation of ergosterol was noted in the rye grain harvested in 2013 than in 2012, and fertiliser treatment led to higher ergosterol concentrations than did control treatment.