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.

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.

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.

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.