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.

Evidence for Cardiac Glycosides in Foliage of Colorado Potato Beetle-Resistant Solanum okadae.

Leptinotarsa decemlineata, the Colorado potato beetle (CPB), is a herbivore that primarily feeds on Solanum foliage and is a global pest of the potato agricultural industry. Potato breeding through cross-hybridization with CPB-resistant wild relatives is used for genetic improvement. The wild species Solanum okadae was demonstrated to deter CPB feeding in choice and no choice feeding assays. Liquid chromatography-mass spectrometry (LC-MS) was used for comparative metabolite profiling between S. okadae and CPB-susceptible domesticated potato variety, Solanum tuberosum cv. Shepody. Major foliar metabolites detected were steroidal glycoalkaloids (SGAs) with tomatine and dehydrotomatine produced in S. okadae and solanine and chaconine in S. tuberosum cv. Shepody. Cardiac glycosides were also detected in the foliar metabolite profile of S. okadae but not S. tuberosum cv. Shepody. This class of plant compounds have known insecticidal activity through inhibition of animal Na+/K+ ATPase. Thin-layer chromatography (TLC) separation of foliar extracts also provided evidence for cardiac glycosides in S. okadae. Cardiac glycosides are known inhibitors of Na+/K+ ATPase, and foliar extracts from S. okadae (OKA15), but not S. tuberosum cv. Shepody, were able to inhibit the Na+/K+ ATPase of CPB. These findings suggest a novel mechanism of plant resistance against CPB involving production of cardiac glycosides in S. okadae.

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.

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 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.

Changes in gene expression in potato meristems treated with the sprout suppressor 1,4-dimethylnaphthalene are dependent on tuber age and dormancy status.

Commercial storage of potatoes often relies on the use of sprout inhibitors to prolong storage and reduce spoilage. The compound 1,4-dimethylnaphthalene (DMN) has seen increase application as a sprout inhibitor in the potato industry as older chemistries are being phased out. The mode of action of DMN is poorly understood as is the sensitivity of potato tissues to this new class of inhibitor. During storage potato tubers transition from a state of endo-dormant to eco-dormant and it is not known if the DMN response is consistent across this developmental transition. RNA-seq gene expression profiling was used to establish if stored potato tubers (Solanum tuberosum cv La Chipper) have differential sensitivity to DMN as tubers age. DMN was applied at three different times during storage; just after harvest when tubers are in endo-dormancy, midwinter at early eco-dormancy, and in spring during late eco-dormancy when sprouting was prevented via exposure to cold storage temperatures. Changes in gene expression were lowest during endo-dormancy while midwinter and spring treatments exhibited a greater and more diverse expression response. Functional analysis of differential gene expression demonstrated gene sets associated with DNA replication, cell division, and DNA methylation are suppressed after DMN treatment. However, gene sets associated with salicylic acid, jasmonic acid, abiotic and biotic stress responses are elevated by DMN only after endodormancy terminates. Gene clusters associated with pathogenesis related proteins PR-4 and PR-5 are also upregulated in response to DMN. These results indicate that DMN sensitivity changes as potato tubers age and transition from endo-dormant to eco-dormant in storage and the overall response is a shift in gene classes that regulate growth and response to stress.

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.

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.

Tuber transcriptome profiling of eight potato cultivars with different cold-induced sweetening responses to cold storage.

Tubers are vegetative reproduction organs formed from underground extensions of the plant stem. Potato tubers are harvested and stored for months. Storage under cold temperatures of 2-4 °C is advantageous for supressing sprouting and diseases. However, development of reducing sugars can occur with cold storage through a process called cold-induced sweetening (CIS). CIS is undesirable as it leads to darkened color with fry processing. The purpose of the current study was to find differences in biological responses in eight cultivars with variation in CIS resistance. Transcriptome sequencing was done on tubers before and after cold storage and three approaches were taken for gene expression analysis: 1. Gene expression correlated with end-point glucose after cold storage, 2. Gene expression correlated with increased glucose after cold storage (after-before), and 3. Differential gene expression before and after cold storage. Cultivars with high CIS resistance (low glucose after cold) were found to increase expression of an invertase inhibitor gene and genes involved in DNA replication and repair after cold storage. The cultivars with low CIS resistance (high glucose after cold) showed increased expression of genes involved in abiotic stress response, gene expression, protein turnover and the mitochondria. There was a small number of genes with similar expression patterns for all cultivars including genes involved in cell wall strengthening and phospholipases. It is proposed that the pattern of gene expression is related to chilling-induced DNA damage repair and cold acclimation and that genetic variation in these processes are related to CIS.

Verticillium dahliae Disease Resistance and the Regulatory Pathway for Maturity and Tuberization in Potato.

Kleb. is a pathogenic fungus causing wilting, chlorosis, and early dying in potato ( L.). Genetic mapping of resistance to was done using a diploid population of potato. The major quantitative trait locus (QTL) for resistance was found on chromosome 5. The gene, controlling earliness of maturity and tuberization, was mapped within the interval. Another QTL on chromosome 9 co-localized with the wilt resistance gene marker. Epistasis analysis indicated that the loci on chromosomes 5 and 9 had a highly significant interaction, and that functioned downstream of The alleles were sequenced and found to encode StCDF1.1 and StCDF1.3. Interaction between the resistance allele and the was demonstrated, but not for Genome-wide expression QTL (eQTL) analysis was performed and genes with eQTL at the and loci were both found to have similar functions involving the chloroplast, including photosynthesis, which declines in both maturity and wilt. Among the gene ontology (GO) terms that were specific to genes with eQTL at the , but not the locus, were those associated with fungal defense. These results suggest that controls fungal defense and reduces early dying in wilt through affecting genetic pathway controlling tuberization timing.

Pathogen and Pest Responses Are Altered Due to RNAi-Mediated Knockdown of GLYCOALKALOID METABOLISM 4 in Solanum tuberosum.

Steroidal glycoalkaloids (SGAs) are major secondary metabolites constitutively produced in cultivated potato Solanum tuberosum, and α-solanine and α-chaconine are the most abundant SGAs. SGAs are toxic to humans at high levels but their role in plant protection against pests and pathogens is yet to be established. In this study, levels of SGAs in potato were reduced by RNA interference (RNAi)-mediated silencing of GLYCOALKALOID METABOLISM 4 (GAME4)-a gene encoding cytochrome P450, involved in an oxidation step in the conversion of cholesterol to SGA aglycones. Two GAME4 RNAi lines, T8 and T9, were used to investigate the effects of manipulation of the SGA biosynthetic pathway in potato. Growth and development of an insect pest, Colorado potato beetle (CPB), were affected in these lines. While no effect on CPB leaf consumption or weight gain was observed, early instar larval death and accelerated development of the insect was found while feeding on leaves of GAME4 RNAi lines. Modulation of SGA biosynthetic pathway in GAME4 RNAi plants was associated with a larger alteration to the metabolite profile, including increased levels of one or both the steroidal saponins or phytoecdysteroids, which could affect insect mortality as well as development time. Colonization by Verticillium dahliae on GAME4 RNAi plants was also tested. There were increased pathogen levels in the T8 GAME4 RNAi line but not in the T9. Metabolite differences between T8 and T9 were found and may have contributed to differences in V. dahliae infection. Drought responses created by osmotic stress were not affected by modulation of SGA biosynthetic pathway in potato.

The nitrogen responsive transcriptome in potato (Solanum tuberosum L.) reveals significant gene regulatory motifs.

Nitrogen (N) is the most important nutrient for the growth of potato (Solanum tuberosum L.). Foliar gene expression in potato plants with and without N supplementation at 180 kg N ha(-1) was compared at mid-season. Genes with consistent differences in foliar expression due to N supplementation over three cultivars and two developmental time points were examined. In total, thirty genes were found to be over-expressed and nine genes were found to be under-expressed with supplemented N. Functional relationships between over-expressed genes were found. The main metabolic pathway represented among differentially expressed genes was amino acid metabolism. The 1000 bp upstream flanking regions of the differentially expressed genes were analysed and nine overrepresented motifs were found using three motif discovery algorithms (Seeder, Weeder and MEME). These results point to coordinated gene regulation at the transcriptional level controlling steady state potato responses to N sufficiency.

Comparative metabolite profiling of Solanum tuberosum against six wild Solanum species with Colorado potato beetle resistance.

The Colorado potato beetle Leptinotarsa decemlineata (Say) (CPB) is a coleopteran herbivore that feeds on the foliage on Solanum species, in particular, potato. Six resistant wild Solanum species were identified, and two of these species had low levels of glycoalkaloids. Comparative analysis of the untargeted metabolite profiles of the foliage using UPLC-qTOF-MS was done to find metabolites shared between the wild species but not with Solanum tuberosum (L.) to identify resistance-related metabolites. It was found that only S. tuberosum produced the triose glycoalkaloids solanine and chaconine. Instead, the six wild species produced glycoalkaloids that shared in common tetrose sugar side chains. Additionally, there were non-glycoalkaloid metabolites associated with resistance including hydroxycoumarin and a phenylpropanoid, which were produced in all wild species but not in S. tuberosum.

Decreased defense gene expression in tolerance versus resistance to Verticillium dahliae in potato.

Verticillium dahliae Kleb., a soil-borne fungus that colonizes vascular tissues, induces wilting, chlorosis and early senescence in potato. Difference in senescence timing found in two diploid potato clones, 07506-01 and 12120-03, was studied and genetic variation in response to V. dahliae infection was identified as a causal factor. The clone, 07506-01, was infected with V. dahliae but did not develop symptoms, indicating tolerance to the pathogen. The other diploid clone, 12120-03 had low levels of pathogen with infection and moderate symptoms indicating partial resistance. 07506-01 was found to carry two susceptible alleles of the Ve2 gene and 12120-03 carried one Ve2 resistant and one susceptible allele. Infected leaves of the two clones were compared using gene expression profiling with the Potato Oligonucleotide Chip Initiative (POCI) microrarray. The results provide further evidence for differences in response of the two clones to infection with V. dahliae. Chlorophyll biosynthesis was higher in the tolerant 07506-01 compared to partially resistant 12120-03. On the other hand, expression of fungal defense genes, Ve resistance genes and defense phytohormone biosynthetic enzyme genes was decreased in 07506-01 compared to 12120-03 suggesting defense responses were suppressed in tolerance compared to resistance. Transcription factor gene expression differences pointed to the WRKY family as potential regulators of V. dahliae responses in potato.