A catalogue of recombination coldspots in interspecific tomato hybrids.

Increasing natural resistance and resilience in plants is key for ensuring food security within a changing climate. Breeders improve these traits by crossing cultivars with their wild relatives and introgressing specific alleles through meiotic recombination. However, some genomic regions are devoid of recombination especially in crosses between divergent genomes, limiting the combinations of desirable alleles. Here, we used pooled-pollen sequencing to build a map of recombinant and non-recombinant regions between tomato and five wild relatives commonly used for introgressive tomato breeding. We detected hybrid-specific recombination coldspots that underscore the role of structural variations in modifying recombination patterns and maintaining genetic linkage in interspecific crosses. Crossover regions and coldspots show strong association with specific TE superfamilies exhibiting differentially accessible chromatin between somatic and meiotic cells. About two-thirds of the genome are conserved coldspots, located mostly in the pericentromeres and enriched with retrotransposons. The coldspots also harbor genes associated with agronomic traits and stress resistance, revealing undesired consequences of linkage drag and possible barriers to breeding. We presented examples of linkage drag that can potentially be resolved by pairing tomato with other wild species. Overall, this catalogue will help breeders better understand crossover localization and make informed decisions on generating new tomato varieties.

Genome architecture and genetic diversity of allopolyploid okra (Abelmoschus esculentus).

The allopolyploid okra (Abelmoschus esculentus) unveiled telomeric repeats flanking distal gene-rich regions and short interstitial TTTAGGG telomeric repeats, possibly representing hallmarks of chromosomal speciation. Ribosomal RNA (rRNA) genes organize into 5S clusters, distinct from the 18S-5.8S-28S units, indicating an S-type rRNA gene arrangement. The assembly, in line with cytogenetic and cytometry observations, identifies 65 chromosomes and a 1.45 Gb genome size estimate in a haploid sibling. The lack of aberrant meiotic configurations implies limited to no recombination among sub-genomes. k-mer distribution analysis reveals 75% has a diploid nature and 15% heterozygosity. The configurations of Benchmarking Universal Single-Copy Ortholog (BUSCO), k-mer, and repeat clustering point to the presence of at least two sub-genomes one with 30 and the other with 35 chromosomes, indicating the allopolyploid nature of the okra genome. Over 130 000 putative genes, derived from mapped IsoSeq data and transcriptome data from public okra accessions, exhibit a low genetic diversity of one single nucleotide polymorphisms per 2.1 kbp. The genes are predominantly located at the distal chromosome ends, declining toward central scaffold domains. Long terminal repeat retrotransposons prevail in central domains, consistent with the observed pericentromeric heterochromatin and distal euchromatin. Disparities in paralogous gene counts suggest potential sub-genome differentiation implying possible sub-genome dominance. Amino acid query sequences of putative genes facilitated phenol biosynthesis pathway annotation. Comparison with manually curated reference KEGG pathways from related Malvaceae species reveals the genetic basis for putative enzyme coding genes that likely enable metabolic reactions involved in the biosynthesis of dietary and therapeutic compounds in okra.

Genome assembly and analysis of Lactuca virosa: implications for lettuce breeding.

Lettuce (Lactuca sativa L.) is a leafy vegetable crop with ongoing breeding efforts related to quality, resilience, and innovative production systems. To breed resilient and resistant lettuce in the future, valuable genetic variation found in close relatives could be further exploited. Lactuca virosa (2x = 2n = 18), a wild relative assigned to the tertiary lettuce gene pool, has a much larger genome (3.7 Gbp) than Lactuca sativa (2.5 Gbp). It has been used in interspecific crosses and is a donor to modern crisphead lettuce cultivars. Here, we present a de novo reference assembly of L. virosa with high continuity and complete gene space. This assembly facilitated comparisons to the genome of L. sativa and to that of the wild species L. saligna, a representative of the secondary lettuce gene pool. To assess the diversity in gene content, we classified the genes of the 3 Lactuca species as core, accessory, and unique. In addition, we identified 3 interspecific chromosomal inversions compared to L. sativa, which each may cause recombination suppression and thus hamper future introgression breeding. Using 3-way comparisons in both reference-based and reference-free manners, we show that the proliferation of long-terminal repeat elements has driven the genome expansion of L. virosa. Further, we performed a genome-wide comparison of immune genes, nucleotide-binding leucine-rich repeat, and receptor-like kinases among Lactuca spp. and indicated the evolutionary patterns and mechanisms behind their expansions. These genome analyses greatly facilitate the understanding of genetic variation in L. virosa, which is beneficial for the breeding of improved lettuce varieties.

The genome of Lactuca saligna, a wild relative of lettuce, provides insight into non-host resistance to the downy mildew Bremia lactucae.

Lactuca saligna L. is a wild relative of cultivated lettuce (Lactuca sativa L.), with which it is partially interfertile. Hybrid progeny suffer from hybrid incompatibility (HI), resulting in reduced fertility and distorted transmission ratios. Lactuca saligna displays broad-spectrum resistance against lettuce downy mildew caused by Bremia lactucae Regel and is considered a non-host species. This phenomenon of resistance in L. saligna is called non-host resistance (NHR). One possible mechanism behind this NHR is through the plant-pathogen interaction triggered by pathogen recognition receptors, including nucleotide-binding leucine-rich repeat (NLR) proteins and receptor-like kinases (RLKs). We report a chromosome-level genome assembly of L. saligna (accession CGN05327), leading to the identification of two large paracentric inversions (>50 Mb) between L. saligna and L. sativa. Genome-wide searches delineated the major resistance clusters as regions enriched in NLRs and RLKs. Three of the enriched regions co-locate with previously identified NHR intervals. RNA-seq analysis of Bremia-infected lettuce identified several differentially expressed RLKs in NHR regions. Three tandem wall-associated kinase-encoding genes (WAKs) in the NHR8 interval display particularly high expression changes at an early stage of infection. We propose RLKs as strong candidates for determinants of the NHR phenotype of L. saligna.

Technology-driven approaches for meiosis research in tomato and wild relatives.

Meiosis is a specialized cell division during reproduction where one round of chromosomal replication is followed by genetic recombination and two rounds of segregation to generate recombined, ploidy-reduced spores. Meiosis is crucial to the generation of new allelic combinations in natural populations and artificial breeding programs. Several plant species are used in meiosis research including the cultivated tomato (Solanum lycopersicum) which is a globally important crop species. Here we outline the unique combination of attributes that make tomato a powerful model system for meiosis research. These include the well-characterized behavior of chromosomes during tomato meiosis, readily available genomics resources, capacity for genome editing, clonal propagation techniques, lack of recent polyploidy and the possibility to generate hybrids with twelve related wild species. We propose that further exploitation of genome bioinformatics, genome editing and artificial intelligence in tomato will help advance the field of plant meiosis research. Ultimately this will help address emerging themes including the evolution of meiosis, how recombination landscapes are determined, and the effect of temperature on meiosis.

Domestication Shapes Recombination Patterns in Tomato.

Meiotic recombination is a biological process of key importance in breeding, to generate genetic diversity and develop novel or agronomically relevant haplotypes. In crop tomato, recombination is curtailed as manifested by linkage disequilibrium decay over a longer distance and reduced diversity compared with wild relatives. Here, we compared domesticated and wild populations of tomato and found an overall conserved recombination landscape, with local changes in effective recombination rate in specific genomic regions. We also studied the dynamics of recombination hotspots resulting from domestication and found that loss of such hotspots is associated with selective sweeps, most notably in the pericentromeric heterochromatin. We detected footprints of genetic changes and structural variants, among them associated with transposable elements, linked with hotspot divergence during domestication, likely causing fine-scale alterations to recombination patterns and resulting in linkage drag.

Chasing breeding footprints through structural variations in Cucumis melo and wild relatives.

Cucumis melo (melon or muskmelon) is an important crop in the family of the Cucurbitaceae. Melon is cross pollinated and domesticated at several locations throughout the breeding history, resulting in highly diverse genetic structure in the germplasm. Yet, the relations among the groups and cultivars are still incomplete. We shed light on the melonbreeding history, analyzing structural variations ranging from 50 bp up to 100 kb, identified from whole genome sequences of 100 selected melon accessions and wild relatives. Phylogenetic trees based on SV types completely resolve cultivars and wild accessions into two monophyletic groups and clustering of cultivars largely correlates with their geographic origin. Taking into account morphology, we found six mis-categorized cultivars. Unique inversions are more often shared between cultivars, carrying advantageous genes and do not directly originate from wild species. Approximately 60% of the inversion breaks carry a long poly A/T motif, and following observations in other plant species, suggest that inversions in melon likely resulted from meiotic recombination events. We show that resistance genes in the linkage V region are expanded in the cultivar genomes compared to wild relatives. Furthermore, particular agronomic traits such as fruit ripening, fragrance, and stress response are specifically selected for in the melon subspecies. These results represent distinctive footprints of selective breeding that shaped today's melon. The sequences and genomic relations between land races, wild relatives, and cultivars will serve the community to identify genetic diversity, optimize experimental designs, and enhance crop development.

Meiotic recombination profiling of interspecific hybrid F1 tomato pollen by linked read sequencing.

Genome wide screening of pooled pollen samples from a single interspecific F1 hybrid obtained from a cross between tomato, Solanum lycopersicum and its wild relative, Solanum pimpinellifolium using linked read sequencing of the haploid nuclei, allowed profiling of the crossover (CO) and gene conversion (GC) landscape. We observed a striking overlap between cold regions of CO in the male gametes and our previously established F6 recombinant inbred lines (RILs) population. COs were overrepresented in non-coding regions in the gene promoter and 5'UTR regions of genes. Poly-A/T and AT rich motifs were found enriched in 1 kb promoter regions flanking the CO sites. Non-crossover associated allelic and ectopic GCs were detected in most chromosomes, confirming that besides CO, GC represents also a source for genetic diversity and genome plasticity in tomato. Furthermore, we identified processed break junctions pointing at the involvement of both homology directed and non-homology directed repair pathways, suggesting a recombination machinery in tomato that is more complex than currently anticipated.

Comparative analysis of repetitive sequences among species from the potato and the tomato clades.

BACKGROUND AND AIMS: The genus Solanum includes important vegetable crops and their wild relatives. Introgression of their useful traits into elite cultivars requires effective recombination between hom(e)ologues, which is partially determined by genome sequence differentiation. In this study we compared the repetitive genome fractions of wild and cultivated species of the potato and tomato clades in a phylogenetic context. METHODS: Genome skimming followed by a clustering approach was used as implemented in the RepeatExplorer pipeline. Repeat classes were annotated and the sequences of their main domains were compared. KEY RESULTS: Repeat abundance and genome size were correlated and the larger genomes of species in the tomato clade were found to contain a higher proportion of unclassified elements. Families and lineages of repetitive elements were largely conserved between the clades, but their relative proportions differed. The most abundant repeats were Ty3/Gypsy elements. Striking differences in abundance were found in the highly dynamic Ty3/Gypsy Chromoviruses and Ty1/Copia Tork elements. Within the potato clade, early branching Solanum cardiophyllum showed a divergent repeat profile. There were also contrasts between cultivated and wild potatoes, mostly due to satellite amplification in the cultivated species. Interspersed repeat profiles were very similar among potatoes. The repeat profile of Solanum etuberosum was more similar to that of the potato clade. CONCLUSIONS: The repeat profiles in Solanum seem to be very similar despite genome differentiation at the level of collinearity. Removal of transposable elements by unequal recombination may have been responsible for structural rearrangements across the tomato clade. Sequence variability in the tomato clade is congruent with clade-specific amplification of repeats after its divergence from S. etuberosum and potatoes. The low differentiation among potato and its wild relatives at the level of interspersed repeats may explain the difficulty in discriminating their genomes by genomic in situ hybridization techniques.

Intact DNA purified from flow-sorted nuclei unlocks the potential of next-generation genome mapping and assembly in Solanum species.

Next-generation genome mapping through nanochannels (Bionano optical mapping) of plant genomes brings genome assemblies to the 'nearly-finished' level for reliable and detailed gene annotations and assessment of structural variations. Despite the recent progress in its development, researchers face the technical challenges of obtaining sufficient high molecular weight (HMW) nuclear DNA due to cell walls which are difficult to disrupt and to the presence of cytoplasmic polyphenols and polysaccharides that co-precipitate or are covalently bound to DNA and might cause oxidation and/or affect the access of nicking enzymes to DNA, preventing downstream applications. Here we describe important improvements for obtaining HMW DNA that we tested on Solanum crops and wild relatives. The methods that we further elaborated and refined focus on •Improving flexibility of using different tissues as source materials, like fast-growing root tips and young leaves from seedlings or in vitro plantlets.•Obtaining nuclei suspensions through either lab homogenizers or by chopping.•Increasing flow sorting efficiency using DAPI (4',6-diamidino-2-phenylindole) and PI (propidium iodide) DNA stains, with different lasers (UV or 488 nm) and sorting platforms such as the FACSAria and FACSVantage flow sorters, thus making it appropriate for more laboratories working on plant genomics. The obtained nuclei are embedded into agarose plugs for processing and isolating uncontaminated HMW DNA, which is a prerequisite for nanochannel-based next-generation optical mapping strategies.

DNA sequence and shape are predictive for meiotic crossovers throughout the plant kingdom.

A better understanding of genomic features influencing the location of meiotic crossovers (COs) in plant species is both of fundamental importance and of practical relevance for plant breeding. Using CO positions with sufficiently high resolution from four plant species [Arabidopsis thaliana, Solanum lycopersicum (tomato), Zea mays (maize) and Oryza sativa (rice)] we have trained machine-learning models to predict the susceptibility to CO formation. Our results show that CO occurrence within various plant genomes can be predicted by DNA sequence and shape features. Several features related to genome content and to genomic accessibility were consistently either positively or negatively related to COs in all four species. Other features were found as predictive only in specific species. Gene annotation-related features were especially predictive for maize, whereas in tomato and Arabidopsis propeller twist and helical twist (DNA shape features) and AT/TA dinucleotides were found to be the most important. In rice, high roll (another DNA shape feature) and low CA dinucleotide frequency in particular were found to be associated with CO occurrence. The accuracy of our models was sufficient for Arabidopsis and rice (area under receiver operating characteristic curve, AUROC > 0.5), and was high for tomato and maize (AUROC â‰« 0.5), demonstrating that DNA sequence and shape are predictive for meiotic COs throughout the plant kingdom.

Identification of methylated GnTI-dependent N-glycans in Botryococcus brauni.

In contrast to mammals and vascular plants, microalgae show a high diversity in the N-glycan structures of complex N-glycoproteins. Although homologues for ß1,2-N-acetylglucosaminyltransferase I (GnTI), a key enzyme in the formation of complex N-glycans, have been identified in several algal species, GnTI-dependent N-glycans have not been detected so far. We have performed an N-glycoproteomic analysis of the hydrocarbon oils accumulating green microalgae Botryococcus braunii. Thereby, the analysis of intact N-glycopeptides allowed the determination of N-glycan compositions. Furthermore, insights into the role of N-glycosylation in B. braunii were gained from functional annotation of the identified N-glycoproteins. In total, 517 unique N-glycosylated peptides have been identified, including intact N-glycopeptides that harbored N-acetylhexosamine (HexNAc) at the nonreducing end. Surprisingly, these GnTI-dependent N-glycans were also found to be modified with (di)methylated hexose. The identification of GnTI-dependent N-glycans in combination with N-glycan methylation in B. braunii revealed an uncommon type of N-glycan processing in this microalgae.

Botryococcus braunii strains compared for biomass productivity, hydrocarbon and carbohydrate content.

Botryococcus braunii can produce both long-chain hydrocarbons as well as carbohydrates in large quantities, and is therefore a promising industrial organism for the production of biopolymer building blocks. Many studies describe the use of different strains of Botryococcus braunii but differences in handling and cultivation conditions make the comparison between strains difficult. In this study, 16 B. braunii strains obtained from six culture collections were compared for their biomass productivity and hydrocarbon and carbohydrate content. Biomass productivity was highest for AC768 strain with 1.8gL-1day-1, while hydrocarbon production ranged from none to up to 42% per gram biomass dry weight, with Showa showing the highest hydrocarbon content followed by AC761. The total carbohydrate content varied from 20% to 76% per gram of the biomass dry weight, with CCALA777 as the highest producer. Glucose and galactose are the main monosaccharides in most strains and fucose content reached 463mgL-1 in CCALA778.

Distribution, position and genomic characteristics of crossovers in tomato recombinant inbred lines derived from an interspecific cross between Solanum lycopersicum and Solanum pimpinellifolium.

We determined the crossover (CO) distribution, frequency and genomic sequences involved in interspecies meiotic recombination by using parent-assigned variants of 52 F6 recombinant inbred lines obtained from a cross between tomato, Solanum lycopersicum, and its wild relative, Solanum pimpinellifolium. The interspecific CO frequency was 80% lower than reported for intraspecific tomato crosses. We detected regions showing a relatively high and low CO frequency, so-called hot and cold regions. Cold regions coincide to a large extent with the heterochromatin, although we found a limited number of smaller cold regions in the euchromatin. The CO frequency was higher at the distal ends of chromosomes than in pericentromeric regions and higher in short arm euchromatin. Hot regions of CO were detected in euchromatin, and COs were more often located in non-coding regions near the 5' untranslated region of genes than expected by chance. Besides overrepresented CCN repeats, we detected poly-A/T and AT-rich motifs enriched in 1-kb promoter regions flanking the CO sites. The most abundant sequence motifs at CO sites share weak similarity to transcription factor-binding sites, such as for the C2H2 zinc finger factors class and MADS box factors, while InterPro scans detected enrichment for genes possibly involved in the repair of DNA breaks.

Cnidaria: fast, reference-free clustering of raw and assembled genome and transcriptome NGS data.

BACKGROUND: Identification of biological specimens is a requirement for a range of applications. Reference-free methods analyse unprocessed sequencing data without relying on prior knowledge, but generally do not scale to arbitrarily large genomes and arbitrarily large phylogenetic distances. RESULTS: We present Cnidaria, a practical tool for clustering genomic and transcriptomic data with no limitation on genome size or phylogenetic distances. We successfully simultaneously clustered 169 genomic and transcriptomic datasets from 4 kingdoms, achieving 100% identification accuracy at supra-species level and 78% accuracy at the species level. CONCLUSION: CNIDARIA allows for fast, resource-efficient comparison and identification of both raw and assembled genome and transcriptome data. This can help answer both fundamental (e.g. in phylogeny, ecological diversity analysis) and practical questions (e.g. sequencing quality control, primer design).

Introgression browser: high-throughput whole-genome SNP visualization.

Breeding by introgressive hybridization is a pivotal strategy to broaden the genetic basis of crops. Usually, the desired traits are monitored in consecutive crossing generations by marker-assisted selection, but their analyses fail in chromosome regions where crossover recombinants are rare or not viable. Here, we present the Introgression Browser (iBrowser), a bioinformatics tool aimed at visualizing introgressions at nucleotide or SNP (Single Nucleotide Polymorphisms) accuracy. The software selects homozygous SNPs from Variant Call Format (VCF) information and filters out heterozygous SNPs, multi-nucleotide polymorphisms (MNPs) and insertion-deletions (InDels). For data analysis iBrowser makes use of sliding windows, but if needed it can generate any desired fragmentation pattern through General Feature Format (GFF) information. In an example of tomato (Solanum lycopersicum) accessions we visualize SNP patterns and elucidate both position and boundaries of the introgressions. We also show that our tool is capable of identifying alien DNA in a panel of the closely related S. pimpinellifolium by examining phylogenetic relationships of the introgressed segments in tomato. In a third example, we demonstrate the power of the iBrowser in a panel of 597 Arabidopsis accessions, detecting the boundaries of a SNP-free region around a polymorphic 1.17 Mbp inverted segment on the short arm of chromosome 4. The architecture and functionality of iBrowser makes the software appropriate for a broad set of analyses including SNP mining, genome structure analysis, and pedigree analysis. Its functionality, together with the capability to process large data sets and efficient visualization of sequence variation, makes iBrowser a valuable breeding tool.

Structural homology in the Solanaceae: analysis of genomic regions in support of synteny studies in tomato, potato and pepper.

We have analysed the structural homology in euchromatin regions of tomato, potato and pepper with special attention for the long arm of chromosome 2 (2L). Molecular organization and colinear junctions were delineated using multi-color BAC FISH analysis and comparative sequence alignment. We found large-scale rearrangements including inversions and segmental translocations that were not reported in previous comparative studies. Some of the structural rearrangements are specific for the tomato clade, and differentiate tomato from potato, pepper and other Solanaceous species. Although local gene vicinity is largely preserved, there are many small-scale synteny perturbations. Gene adjacency in the aligned segments was frequently disrupted for 47% of the ortholog pairs as a result of gene and LTR retrotransposon insertions, and occasionally by single gene inversions and translocations. Our data also suggests that long distance intra-chromosomal rearrangements and local gene rearrangements have evolved frequently during speciation in the Solanum genus, and that small changes are more prevalent than large-scale differences. The occurrence of sonata and harbinger transposable elements and other repeats near or at junction breaks is considered in the light of repeat-mediated rearrangements and a reconstruction scenario for an ancestral 2L topology is discussed.

Genomic organisation of the Mal d 1 gene cluster on linkage group 16 in apple.

European populations exhibit progressive sensitisation to food allergens, and apples are one of the foods for which sensitisation is observed most frequently. Apple cultivars vary greatly in their allergenic characteristics, and a better understanding of the genetic basis of low allergenicity may therefore allow allergic individuals to increase their fruit intake. Mal d 1 is considered to be a major apple allergen, and this protein is encoded by the most complex allergen gene family. Not all Mal d 1 members are likely to be involved in allergenicity. Therefore, additional knowledge about the existence and characteristics of the different Mal d 1 genes is required. In the present study, we investigated the genomic organisation of the Mal d 1 gene cluster in linkage group 16 of apple through the sequencing of two bacterial artificial chromosome clones. The results provided new information on the composition of this family with respect to the number and orientation of functional and pseudogenes and their physical distances. The results were compared with the apple and peach genome sequences that have recently been made available. A broad analysis of the whole apple genome revealed the presence of new genes in this family, and a complete list of the observed Mal d 1 genes is supplied. Thus, this study provides an important contribution towards a better understanding of the genetics of the Mal d 1 family and establishes the basis for further research on allelic diversity among cultivars in relation to variation in allergenicity. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9588-4) contains supplementary material, which is available to authorized users.

SNP markers retrieval for a non-model species: a practical approach.

BACKGROUND: SNP (Single Nucleotide Polymorphism) markers are rapidly becoming the markers of choice for applications in breeding because of next generation sequencing technology developments. For SNP development by NGS technologies, correct assembly of the huge amounts of sequence data generated is essential. Little is known about assembler's performance, especially when dealing with highly heterogeneous species that show a high genome complexity and what the possible consequences are of differences in assemblies on SNP retrieval. This study tested two assemblers (CAP3 and CLC) on 454 data from four lily genotypes and compared results with respect to SNP retrieval. RESULTS: CAP3 assembly resulted in higher numbers of contigs, lower numbers of reads per contig, and shorter average read lengths compared to CLC. Blast comparisons showed that CAP3 contigs were highly redundant. Contrastingly, CLC in rare cases combined paralogs in one contig. Redundant and chimeric contigs may lead to erroneous SNPs. Filtering for redundancy can be done by blasting selected SNP markers to the contigs and discarding all the SNP markers that show more than one blast hit. Results on chimeric contigs showed that only four out of 2,421 SNP markers were selected from chimeric contigs. CONCLUSION: In practice, CLC performs better in assembling highly heterogeneous genome sequences compared to CAP3, and consequently SNP retrieval is more efficient. Additionally a simple flow scheme is suggested for SNP marker retrieval that can be valid for all non-model species.

Evidence for RNA recombination between distinct isolates of Pepino mosaic virus.

Genetic recombination plays an important role in the evolution of virus genomes. In this study we analyzed publicly available genomic sequences of Pepino mosaic virus (PepMV) for recombination events using several bioinformatics tools. The genome-wide analyses not only confirm the presence of previously found recombination events in PepMV but also provide the first evidence for double recombinant origin of the US2 isolate.