Pangenome analyses reveal impact of transposable elements and ploidy on the evolution of potato species.

Potato (Solanum sp., family Solanaceae) is the most important noncereal food crop globally. It has over 100 wild relatives in the Solanum section Petota, which features species with both sexual and asexual reproduction and varying ploidy levels. A pangenome of Solanum section Petota composed of 296 accessions was constructed including diploids and polyploids compared via presence/absence variation (PAV). The Petota core (genes shared by at least 97% of the accessions) and shell genomes (shared by 3 to 97%) are enriched in basic molecular and cellular functions, while the cloud genome (genes present in less than 3% of the member accessions) showed enrichment in transposable elements (TEs). Comparison of PAV in domesticated vs. wild accessions was made, and a phylogenetic tree was constructed based on PAVs, grouping accessions into different clades, similar to previous phylogenies produced using DNA markers. A cladewise pangenome approach identified abiotic stress response among the core genes in clade 1+2 and clade 3, and flowering/tuberization among the core genes in clade 4. The TE content differed between the clades, with clade 1+2, which is composed of species from North and Central America with reproductive isolation from species in other clades, having much lower TE content compared to other clades. In contrast, accessions with in vitro propagation history were identified and found to have high levels of TEs. Results indicate a role for TEs in adaptation to new environments, both natural and artificial, for Solanum section Petota.

Development of a Quick-Install Rapid Phenotyping System.

In recent years, there has been a growing need for accessible High-Throughput Plant Phenotyping (HTPP) platforms that can take measurements of plant traits in open fields. This paper presents a phenotyping system designed to address this issue by combining ultrasonic and multispectral sensing of the crop canopy with other diverse measurements under varying environmental conditions. The system demonstrates a throughput increase by a factor of 50 when compared to a manual setup, allowing for efficient mapping of crop status across a field with crops grown in rows of any spacing. Tests presented in this paper illustrate the type of experimentation that can be performed with the platform, emphasizing the output from each sensor. The system integration, versatility, and ergonomics are the most significant contributions. The presented system can be used for studying plant responses to different treatments and/or stresses under diverse farming practices in virtually any field environment. It was shown that crop height and several vegetation indices, most of them common indicators of plant physiological status, can be easily paired with corresponding environmental conditions to facilitate data analysis at the fine spatial scale.

The plastome of Arctic Oxytropis arctobia (Fabaceae) is significantly different from that of O. splendens and other related species.

Anatomical and physiological specializations for plant adaptation to harsh climates result from molecular mechanisms that can be encoded in the nucleus or organelle. In this study, the complete plastomes of an arctic species, Oxytropis arctobia Bunge (Fabaceae), and a closely related temperate species, O. splendens Douglas ex Hook., were assembled, annotated, and analyzed to identify differences that might help explain their adaptation to different environments. This is consistent with the previously sequenced O. bicolor DC. and O. glabra plastomes, O. arctobia and O. splendens plastomes both have the common features of the inverted repeat-lacking clade (IRLC), as well as atpF intron loss, which is unique to the genus. However, significant differences were observed between the plastomes of O. arctobia and O. splendens and other closely related species (Oxytropis spp. and Astragalus spp.), including a 3 kb inversion, two large insertions (>1 kb), significant modifications of the accD gene, and an overall larger size.

Structural genome analysis in cultivated potato taxa.

KEY MESSAGE: Twelve potato accessions were selected to represent two principal views on potato taxonomy. The genomes were sequenced and analyzed for structural variation (copy number variation) against three published potato genomes. The common potato (Solanum tuberosum L.) is an important staple crop with a highly heterozygous and complex tetraploid genome. The other taxa of cultivated potato contain varying ploidy levels (2X-5X), and structural variations are common in the genomes of these species, likely contributing to the diversification or agronomic traits during domestication. Increased understanding of the genomes and genomic variation will aid in the exploration of novel agronomic traits. Thus, sequencing data from twelve potato landraces, representing the four ploidy levels, were used to identify structural genomic variation compared to the two currently available reference genomes, a double monoploid potato genome and a diploid inbred clone of S. chacoense. The results of a copy number variation analysis showed that in the majority of the genomes, while the number of deletions is greater than the number of duplications, the number of duplicated genes is greater than the number of deleted ones. Specific regions in the twelve potato genomes have a high density of CNV events. Further, the auxin-induced SAUR genes (involved in abiotic stress), disease resistance genes and the 2-oxoglutarate/Fe(II)-dependent oxygenase superfamily proteins, among others, had increased copy numbers in these sequenced genomes relative to the references.

Promzea: a pipeline for discovery of co-regulatory motifs in maize and other plant species and its application to the anthocyanin and phlobaphene biosynthetic pathways and the Maize Development Atlas.

BACKGROUND: The discovery of genetic networks and cis-acting DNA motifs underlying their regulation is a major objective of transcriptome studies. The recent release of the maize genome (Zea mays L.) has facilitated in silico searches for regulatory motifs. Several algorithms exist to predict cis-acting elements, but none have been adapted for maize. RESULTS: A benchmark data set was used to evaluate the accuracy of three motif discovery programs: BioProspector, Weeder and MEME. Analysis showed that each motif discovery tool had limited accuracy and appeared to retrieve a distinct set of motifs. Therefore, using the benchmark, statistical filters were optimized to reduce the false discovery ratio, and then remaining motifs from all programs were combined to improve motif prediction. These principles were integrated into a user-friendly pipeline for motif discovery in maize called Promzea, available at http://www.promzea.org and on the Discovery Environment of the iPlant Collaborative website. Promzea was subsequently expanded to include rice and Arabidopsis. Within Promzea, a user enters cDNA sequences or gene IDs; corresponding upstream sequences are retrieved from the maize genome. Predicted motifs are filtered, combined and ranked. Promzea searches the chosen plant genome for genes containing each candidate motif, providing the user with the gene list and corresponding gene annotations. Promzea was validated in silico using a benchmark data set: the Promzea pipeline showed a 22% increase in nucleotide sensitivity compared to the best standalone program tool, Weeder, with equivalent nucleotide specificity. Promzea was also validated by its ability to retrieve the experimentally defined binding sites of transcription factors that regulate the maize anthocyanin and phlobaphene biosynthetic pathways. Promzea predicted additional promoter motifs, and genome-wide motif searches by Promzea identified 127 non-anthocyanin/phlobaphene genes that each contained all five predicted promoter motifs in their promoters, perhaps uncovering a broader co-regulated gene network. Promzea was also tested against tissue-specific microarray data from maize. CONCLUSIONS: An online tool customized for promoter motif discovery in plants has been generated called Promzea. Promzea was validated in silico by its ability to retrieve benchmark motifs and experimentally defined motifs and was tested using tissue-specific microarray data. Promzea predicted broader networks of gene regulation associated with the historic anthocyanin and phlobaphene biosynthetic pathways. Promzea is a new bioinformatics tool for understanding transcriptional gene regulation in maize and has been expanded to include rice and Arabidopsis.

The Y-segment of novel cold dehydrin genes is conserved and codons in the PR-10 genes are under positive selection in Oxytropis (Fabaceae) from contrasting climates.

While the arctic flora is particularly threatened by climate changes, the molecular aspects allowing colonization of this harsh environment remain largely enigmatic. Genes with a likely functional or evolutive role for arctic Oxytropis (Fabaceae) were previously discovered given a sharp differential expression between arctic and temperate species, but the evolutionary forces in action were unknown within the respective species. Here, we analyze gene duplication patterns and positive and negative selection between genes from species of contrasting environments, which can reveal potential gene functions. Genes were amplified and sequenced from two arctic (Oxytropis arctobia and O. maydelliana) and two temperate (O. campestris subsp. johannensis and O. splendens) species. Detection of codons under positive or negative selection and phylogenetic analyses were used to further elucidate pathogenesis-related class 10 (PR-10), ripening-related proteins, cold dehydrins gene families and light-harvesting complex (lhcaIII and lhcbI) genes from Oxytropis. Overall, results showed that the three gene families duplicated in tandem prior to the Oxytropis genus diversification; that genes overexpressed in arctic species evolve under higher constraints at the sequence level in these species; that evolving novel protein variants in PR-10 genes were required for initial adaptation to the Arctic, and that Oxytropis cold dehydrins are of a novel (K-like-Y(4)-K-S) structure, where the Y-segment is under stringent evolutive constraints in the arctic species. This suggests a scenario not previously described for arctic plants, where tandem duplications precede gene recruitment that later become both highly expressed and under stringent constraints in the arctic species.

Plastaumatic: Automating plastome assembly and annotation.

Plastome sequence data is most often extracted from plant whole genome sequencing data and need to be assembled and annotated separately from the nuclear genome sequence. In projects comprising multiple genomes, it is labour intense to individually process the plastomes as it requires many steps and software. This study developed Plastaumatic - an automated pipeline for both assembly and annotation of plastomes, with the scope of the researcher being able to load whole genome sequence data with minimal manual input, and therefore a faster runtime. The main structure of the current automated pipeline includes trimming of adaptor and low-quality sequences using fastp, de novo plastome assembly using NOVOPlasty, standardization and quality checking of the assembled genomes through a custom script utilizing BLAST+ and SAMtools, annotation of the assembled genomes using AnnoPlast, and finally generating the required files for NCBI GenBank submissions. The pipeline is demonstrated with 12 potato accessions and three soybean accessions.

Genome sequencing of adapted diploid potato clones.

Cultivated potato is a vegetatively propagated crop, and most varieties are autotetraploid with high levels of heterozygosity. Reducing the ploidy and breeding potato at the diploid level can increase efficiency for genetic improvement including greater ease of introgression of diploid wild relatives and more efficient use of genomics and markers in selection. More recently, selfing of diploids for generation of inbred lines for F1 hybrid breeding has had a lot of attention in potato. The current study provides genomics resources for nine legacy non-inbred adapted diploid potato clones developed at Agriculture and Agri-Food Canada. De novo genome sequence assembly using 10× Genomics and Illumina sequencing technologies show the genome sizes ranged from 712 to 948 Mbp. Structural variation was identified by comparison to two references, the potato DMv6.1 genome and the phased RHv3 genome, and a k-mer based analysis of sequence reads showed the genome heterozygosity range of 1 to 9.04% between clones. A genome-wide approach was taken to scan 5 Mb bins to visualize patterns of heterozygous deleterious alleles. These were found dispersed throughout the genome including regions overlapping segregation distortions. Novel variants of the StCDF1 gene conferring earliness of tuberization were found among these clones, which all produce tubers under long days. The genomes will be useful tools for genome design for potato breeding.

Phased, chromosome-scale genome assemblies of tetraploid potato reveal a complex genome, transcriptome, and predicted proteome landscape underpinning genetic diversity.

Cultivated potato is a clonally propagated autotetraploid species with a highly heterogeneous genome. Phased assemblies of six cultivars including two chromosome-scale phased genome assemblies revealed extensive allelic diversity, including altered coding and transcript sequences, preferential allele expression, and structural variation that collectively result in a highly complex transcriptome and predicted proteome, which are distributed across the homologous chromosomes. Wild species contribute to the extensive allelic diversity in tetraploid cultivars, demonstrating ancestral introgressions predating modern breeding efforts. As a clonally propagated autotetraploid that undergoes limited meiosis, dysfunctional and deleterious alleles are not purged in tetraploid potato. Nearly a quarter of the loci bore mutations are predicted to have a high negative impact on protein function, complicating breeder's efforts to reduce genetic load. The StCDF1 locus controls maturity, and analysis of six tetraploid genomes revealed that 12 allelic variants of StCDF1 are correlated with maturity in a dosage-dependent manner. Knowledge of the complexity of the tetraploid potato genome with its rampant structural variation and embedded deleterious and dysfunctional alleles will be key not only to implementing precision breeding of tetraploid cultivars but also to the construction of homozygous, diploid potato germplasm containing favorable alleles to capitalize on heterosis in F1 hybrids.

PR-10, defensin and cold dehydrin genes are among those over expressed in Oxytropis (Fabaceae) species adapted to the arctic.

In many studied plants, typical responses to cold treatment include up-regulating the hydrophilic COR/LEA genes and down-regulating photosynthesis-related genes, carbohydrate metabolism, GDSL-motif lipase, hormone metabolism and oxidative regulation genes. However, next to nothing is known about gene expression in arctic plants, which are actually adapted to a harsh, cold environment. The molecular mechanisms behind the many specific adaptations of arctic plants, such as slow growth, well-developed root systems and short stature, are not well understood. In this study, we examine whole plantlet transcriptome differences between two arctic and two temperate Oxytropis (Fabaceae) species, grown under their respective controlled environmental conditions. Gene expression differences are analyzed using cDNA library subtraction followed by expressed sequence tags sequencing and annotation. Sequences from a total of nearly 2,000 clones cluster into 121 and 368 unique genes from the arctic and from the temperate plants, respectively. The predominant biological process for genes from the arctic-enriched library is "response to stimulus". A concurrent overexpression of pathogenesis-related class 10 proteins (PR-10), plant defensin and cold dehydrin genes is a novel feature for species adapted to stressful growth environment. The temperate-enriched genes are involved in photosynthesis, translation and nucleosome assembly. Interestingly, both arctic and temperate-enriched libraries also contain genes involved in ribosome biogenesis and assembly, however of different types. Real-time reverse transcription PCR of cold dehydrin and two PR-10 genes, as well as the light harvesting complex b1 genes demonstrates that the gene expression is dependent on species and growth conditions.

Transcript profiling reveals expression differences in wild-type and glabrous soybean lines.

BACKGROUND: Trichome hairs affect diverse agronomic characters such as seed weight and yield, prevent insect damage and reduce loss of water but their molecular control has not been extensively studied in soybean. Several detailed models for trichome development have been proposed for Arabidopsis thaliana, but their applicability to important crops such as cotton and soybean is not fully known. RESULTS: Two high throughput transcript sequencing methods, Digital Gene Expression (DGE) Tag Profiling and RNA-Seq, were used to compare the transcriptional profiles in wild-type (cv. Clark standard, CS) and a mutant (cv. Clark glabrous, i.e., trichomeless or hairless, CG) soybean isoline that carries the dominant P1 allele. DGE data and RNA-Seq data were mapped to the cDNAs (Glyma models) predicted from the reference soybean genome, Williams 82. Extending the model length by 250 bp at both ends resulted in significantly more matches of authentic DGE tags indicating that many of the predicted gene models are prematurely truncated at the 5' and 3' UTRs. The genome-wide comparative study of the transcript profiles of the wild-type versus mutant line revealed a number of differentially expressed genes. One highly-expressed gene, Glyma04g35130, in wild-type soybean was of interest as it has high homology to the cotton gene GhRDL1 gene that has been identified as being involved in cotton fiber initiation and is a member of the BURP protein family. Sequence comparison of Glyma04g35130 among Williams 82 with our sequences derived from CS and CG isolines revealed various SNPs and indels including addition of one nucleotide C in the CG and insertion of ~60 bp in the third exon of CS that causes a frameshift mutation and premature truncation of peptides in both lines as compared to Williams 82. CONCLUSION: Although not a candidate for the P1 locus, a BURP family member (Glyma04g35130) from soybean has been shown to be abundantly expressed in the CS line and very weakly expressed in the glabrous CG line. RNA-Seq and DGE data are compared and provide experimental data on the expression of predicted soybean gene models as well as an overview of the genes expressed in young shoot tips of two closely related isolines.

The complete plastome sequences of nine diploid potato clones.

Potato (Solanum tuberosum L.) is the world's fourth most important food crop and essential for global food security. The potato chloroplast genomes, the plastomes, are highly conserved and are largely studied for their maternal lineages. In this study, we assembled the complete circular plastome sequences of nine diploid potato clones, with sizes ranging between 155,296 bp and 155,564 bp. Annotation of these plastomes reveals that they each have 141 genes in a similar order. The computational chloroplast DNA typing reveals three plastid DNA types among the nine plastomes and they are grouped according to these types in the phylogeny.

The complete mitogenome assemblies of 10 diploid potato clones reveal recombination and overlapping variants.

The potato mitogenome is complex and to understand various biological functions and nuclear-cytoplasmic interactions, it is important to characterize its gene content and structure. In this study, the complete mitogenome sequences of nine diploid potato clones along with a diploid Solanum okadae clone were characterized. Each mitogenome was assembled and annotated from Pacific Biosciences (PacBio) long reads and 10X genomics short reads. The results show that each mitogenome consists of multiple circular molecules with similar structure and gene organization, though two groups (clones 07506-01, DW84-1457, 08675-21 and H412-1 in one group, and clones W5281-2, 12625-02, 12120-03 and 11379-03 in another group) could be distinguished, and two mitogenomes (clone 10908-06 and OKA15) were not consistent with those or with each other. Significant differences in the repeat structure of the 10 mitogenomes were found, as was recombination events leading to multiple sub-genomic circles. Comparison between individual molecules revealed a translocation of ∼774 bp region located between a short repeat of 40 bp in molecule 3 of each mitogenome, and an insertion of the same in molecule 2 of the 10908-06 mitogenome. Finally, phylogenetic analyses revealed a close relationship between the mitogenomes of these clones and previously published potato mitogenomes.

Complete mitogenome assemblies from a panel of 13 diverse potato taxa.

Mitochondrial DNA is maternally inherited and is shown to affect nuclear-cytoplasmic interactions in potato. Analyzing the mitogenome helps understand the evolutionary relationships and improve breeding programs in potato. We report complete mitogenome sequences from a panel of 13 potato accessions of various taxa. Each mitogenome has three independent circular molecules, except one of the S. bukasovii sample BUK2, which has a single circular molecule. Each mitogenome code for 37 non-redundant protein-coding genes, three rRNAs, 20 tRNAs, and 19 hypothetical open reading frames. Phylogenetic analysis reveals congruency between plastome and mitogenome phylogeny.

Genome assembly of six polyploid potato genomes.

Genome assembly of polyploid plant genomes is a laborious task as they contain more than two copies of the genome, are often highly heterozygous with a high level of repetitive DNA. Next Generation genome sequencing data representing one Chilean and five Peruvian polyploid potato (Solanum spp.) landrace genomes was used to construct genome assemblies comprising five taxa. Third Generation sequencing data (Linked and Long-read data) was used to improve the assembly for one of the genomes. Native landraces are valuable genetic resources for traits such as disease and pest resistance, environmental tolerance and other qualities of interest such as nutrition and fiber for breeding programs. The need for conservation and enhanced understanding of genetic diversity of cultivated potato from South America is also crucial to North American and European cultivars. Here, we report draft genomes from six polyploid potato landraces representing five taxa, illustrating how Third Generation Sequencing can aid in assembling polyploid genomes.

Complete plastome assemblies from a panel of 13 diverse potato taxa.

The chloroplasts are a crucial part of photosynthesizing plant cells and are extensively utilized in phylogenetic studies mainly due to their maternal inheritance. Characterization and analysis of complete plastome sequences is necessary to understand their diversity and evolutionary relationships. Here, a panel of thirteen plastomes from various potato taxa are presented. Though they are highly similar with respect to gene order and content, there is also a great extent of SNPs and InDels between them, with one of the Solanum bukasovii plastomes (BUK2) having the highest number of SNPs and InDels. Five different potato plastome types (C, S, A, W, W2) are present in the panel. Interestingly, the S. tuberosum subsp. tuberosum (TBR) accession has a W-type plastome, which is not commonly found in this species. The S-type plastome has a conserved 48 bp deletion not found in other types, which is responsible for the divergence of the S-type from the C-type plastome. Finally, a phylogenetic analysis shows that these plastomes cluster according to their types. Congruence between the nuclear genome and the plastome phylogeny of these accessions was seen, however with considerable differences, supporting the hypothesis of introgression and hybridization between potato species.

Application of iTRAQ to catalogue the skeletal muscle proteome in pigs and assessment of effects of gender and diet dephytinization.

In this study iTRAQ was used to produce a highly confident catalogue of 542 proteins identified in porcine muscle (false positive<5%). To our knowledge this is the largest reported set of skeletal muscle proteins in livestock. Comparison with human muscle proteome demonstrated a low level of false positives with 83% of the proteins common to both proteomes. In addition, for the first time we assess variations in the muscle proteome caused by sexually dimorphic gene expression and diet dephytinization. Preliminary analysis identified 19 skeletal muscle proteins differentially expressed between male and female pigs (> or = 1.2-fold, p<0.05), but only one of them, GDP-dissociation inhibitor 1, was significant (p<0.05) after false discovery rate correction. Diet dephytinization affected expression of 20 proteins (p<0.05). This study would contribute to an evaluation of the suitability of the pig as a model to study human gender-related differences in gene expression. Transgenic pigs used in this study might also serve as a useful model to understand changes in human physiology resulting from diet dephytinization.

Seed storage protein gene promoters contain conserved DNA motifs in Brassicaceae, Fabaceae and Poaceae.

BACKGROUND: Accurate computational identification of cis-regulatory motifs is difficult, particularly in eukaryotic promoters, which typically contain multiple short and degenerate DNA sequences bound by several interacting factors. Enrichment in combinations of rare motifs in the promoter sequence of functionally or evolutionarily related genes among several species is an indicator of conserved transcriptional regulatory mechanisms. This provides a basis for the computational identification of cis-regulatory motifs. RESULTS: We have used a discriminative seeding DNA motif discovery algorithm for an in-depth analysis of 54 seed storage protein (SSP) gene promoters from three plant families, namely Brassicaceae (mustards), Fabaceae (legumes) and Poaceae (grasses) using backgrounds based on complete sets of promoters from a representative species in each family, namely Arabidopsis (Arabidopsis thaliana (L.) Heynh.), soybean (Glycine max (L.) Merr.) and rice (Oryza sativa L.) respectively. We have identified three conserved motifs (two RY-like and one ACGT-like) in Brassicaceae and Fabaceae SSP gene promoters that are similar to experimentally characterized seed-specific cis-regulatory elements. Fabaceae SSP gene promoter sequences are also enriched in a novel, seed-specific E2Fb-like motif. Conserved motifs identified in Poaceae SSP gene promoters include a GCN4-like motif, two prolamin-box-like motifs and an Skn-1-like motif. Evidence of the presence of a variant of the TATA-box is found in the SSP gene promoters from the three plant families. Motifs discovered in SSP gene promoters were used to score whole-genome sets of promoters from Arabidopsis, soybean and rice. The highest-scoring promoters are associated with genes coding for different subunits or precursors of seed storage proteins. CONCLUSION: Seed storage protein gene promoter motifs are conserved in diverse species, and different plant families are characterized by a distinct combination of conserved motifs. The majority of discovered motifs match experimentally characterized cis-regulatory elements. These results provide a good starting point for further experimental analysis of plant seed-specific promoters and our methodology can be used to unravel more transcriptional regulatory mechanisms in plants and other eukaryotes.

Seeder: discriminative seeding DNA motif discovery.

MOTIVATION: The computational identification of transcription factor binding sites is a major challenge in bioinformatics and an important complement to experimental approaches. RESULTS: We describe a novel, exact discriminative seeding DNA motif discovery algorithm designed for fast and reliable prediction of cis-regulatory elements in eukaryotic promoters. The algorithm is tested on biological benchmark data and shown to perform equally or better than other motif discovery tools. The algorithm is applied to the analysis of plant tissue-specific promoter sequences and successfully identifies key regulatory elements.

SoyXpress: a database for exploring the soybean transcriptome.

BACKGROUND: Experiments using whole transcriptome microarrays produce massive amounts of data. To gain a comprehensive understanding of this gene expression data it needs to be integrated with other available information such as gene function and metabolic pathways. Bioinformatics tools are essential to handle, organize and interpret the results. To date, no database provides whole transcriptome analysis capabilities integrated with terms describing biological functions for soybean (Glycine max (L) Merr.). To this end we have developed SoyXpress, a relational database with a suite of web interfaces to allow users to easily retrieve data and results of the microarray experiment with cross-referenced annotations of expressed sequence tags (EST) and hyperlinks to external public databases. This environment makes it possible to explore differences in gene expression, if any, between for instance transgenic and non-transgenic soybean cultivars and to interpret the results based on gene functional annotations to determine any changes that could potentially alter biological processes. RESULTS: SoyXpress is a database designed for exploring the soybean transcriptome. Currently SoyXpress houses 380,095 soybean Expressed Sequence Tags (EST), linked with metabolic pathways, Gene Ontology terms, SwissProt identifiers and Affymetrix gene expression data. Array data is presently available from an experiment profiling global gene expression of three conventional and two genetically engineered soybean cultivars. The microarray data is linked with the sequence data, for maximum knowledge extraction. SoyXpress is implemented in MySQL and uses a Perl CGI interface. CONCLUSION: SoyXpress is designed for the purpose of exploring potential transcriptome differences in different plant genotypes, including genetically modified crops. Soybean EST sequences, microarray and pathway data as well as searchable and browsable gene ontology are integrated and presented. SoyXpress is publicly accessible at http://soyxpress.agrenv.mcgill.ca.