Allelic variation in the autotetraploid potato: genes involved in starch and steroidal glycoalkaloid metabolism as a case study.

BACKGROUND: Tuber starch and steroidal glycoalkaloid (SGA)-related traits have been consistently prioritized in potato breeding, while allelic variation pattern of genes that underlie these traits is less explored. RESULTS: Here, we focused on the genes involved in two important metabolic pathways in the potato: starch metabolism and SGA biosynthesis. We identified 119 genes consisting of 81 involved in starch metabolism and 38 in the biosynthesis of steroidal glycoalkaloids, and discovered 96,166 allelic variants among 2,169 gene haplotypes in six autotetraploid potato genomes. Comparative analyses revealed an uneven distribution of allelic variants among gene haplotypes and that the vast majority of deleterious mutations in these genes are retained in heterozygous state in the autotetraploid potato genomes. Leveraging full-length cDNA sequencing data, we find that approximately 70% of haplotypes of the 119 genes are transcribable. Population genetic analyses identify starch and SGA biosynthetic genes that are potentially conserved or diverged between potato varieties with varying starch or SGA content. CONCLUSIONS: These results deepen the understanding of haplotypic diversity within functionally important genes in autotetraploid genomes and may facilitate functional characterization of genes or haplotypes contributing to traits related to starch and SGA in potato.

Genome architecture and tetrasomic inheritance of autotetraploid potato.

Potato (Solanum tuberosum) is the most consumed non-cereal food crop. Most commercial potato cultivars are autotetraploids with highly heterozygous genomes, severely hampering genetic analyses and improvement. By leveraging the state-of-the-art sequencing technologies and polyploid graph binning, we achieved a chromosome-scale, haplotype-resolved genome assembly of a cultivated potato, Cooperation-88 (C88). Intra-haplotype comparative analyses revealed extensive sequence and expression differences in this tetraploid genome. We identified haplotype-specific pericentromeres on chromosomes, suggesting a distinct evolutionary trajectory of potato homologous centromeres. Furthermore, we detected double reduction events that are unevenly distributed on haplotypes in 1021 of 1034 selfing progeny, a feature of autopolyploid inheritance. By distinguishing maternal and paternal haplotype sets in C88, we simulated the origin of heterosis in cultivated tetraploid with a survey of 3110 tetra-allelic loci with deleterious mutations, which were masked in the heterozygous condition by two parents. This study provides insights into the genomic architecture of autopolyploids and will guide their 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.

Neofunctionalisation of the Sli gene leads to self-compatibility and facilitates precision breeding in potato.

Genetic gain in potato is hampered by the heterozygous tetraploid genome of cultivated potato. Converting potato into a diploid inbred-line based F1-hybrid crop provides a promising route towards increased genetic gain. The introduction of a dominant S-locus inhibitor (Sli) gene into diploid potato germplasm allows efficient generation of self-fertilized seeds and thus the development of potato inbred lines. Little is known about the structure and function of the Sli locus. Here we describe the mapping of Sli to a 12.6 kb interval on chromosome 12 using a recombinant screen approach. One of two candidate genes present in this interval shows a unique sequence that is exclusively present in self-compatible lines. We describe an expression vector that converts self-incompatible genotypes into self-compatible and a CRISPR-Cas9 vector that converts SC genotypes into SI. The Sli gene encodes an F-box protein that is specifically expressed in pollen from self-compatible plants. A 533 bp insertion in the promotor of that gene leads to a gain of function mutation, which overcomes self-pollen rejection.

Extracting knowledge networks from plant scientific literature: potato tuber flesh color as an exemplary trait.

BACKGROUND: Scientific literature carries a wealth of information crucial for research, but only a fraction of it is present as structured information in databases and therefore can be analyzed using traditional data analysis tools. Natural language processing (NLP) is often and successfully employed to support humans by distilling relevant information from large corpora of free text and structuring it in a way that lends itself to further computational analyses. For this pilot, we developed a pipeline that uses NLP on biological literature to produce knowledge networks. We focused on the flesh color of potato, a well-studied trait with known associations, and we investigated whether these knowledge networks can assist us in formulating new hypotheses on the underlying biological processes. RESULTS: We trained an NLP model based on a manually annotated corpus of 34 full-text potato articles, to recognize relevant biological entities and relationships between them in text (genes, proteins, metabolites and traits). This model detected the number of biological entities with a precision of 97.65% and a recall of 88.91% on the training set. We conducted a time series analysis on 4023 PubMed abstract of plant genetics-based articles which focus on 4 major Solanaceous crops (tomato, potato, eggplant and capsicum), to determine that the networks contained both previously known and contemporaneously unknown leads to subsequently discovered biological phenomena relating to flesh color. A novel time-based analysis of these networks indicates a connection between our trait and a candidate gene (zeaxanthin epoxidase) already two years prior to explicit statements of that connection in the literature. CONCLUSIONS: Our time-based analysis indicates that network-assisted hypothesis generation shows promise for knowledge discovery, data integration and hypothesis generation in scientific research.

Potato CYCLING DOF FACTOR 1 and its lncRNA counterpart StFLORE link tuber development and drought response.

Plants regulate their reproductive cycles under the influence of environmental cues, such as day length, temperature and water availability. In Solanum tuberosum (potato), vegetative reproduction via tuberization is known to be regulated by photoperiod, in a very similar way to flowering. The central clock output transcription factor CYCLING DOF FACTOR 1 (StCDF1) was shown to regulate tuberization. We now show that StCDF1, together with a long non-coding RNA (lncRNA) counterpart, named StFLORE, also regulates water loss through affecting stomatal growth and diurnal opening. Both natural and CRISPR-Cas9 mutations in the StFLORE transcript produce plants with increased sensitivity to water-limiting conditions. Conversely, elevated expression of StFLORE, both by the overexpression of StFLORE or by the downregulation of StCDF1, results in an increased tolerance to drought through reducing water loss. Although StFLORE appears to act as a natural antisense transcript, it is in turn regulated by the StCDF1 transcription factor. We further show that StCDF1 is a non-redundant regulator of tuberization that affects the expression of two other members of the potato StCDF gene family, as well as StCO genes, through binding to a canonical sequence motif. Taken together, we demonstrate that the StCDF1-StFLORE locus is important for vegetative reproduction and water homeostasis, both of which are important traits for potato plant breeding.

Modelling the physiological relevance of sucrose export repression by an Flowering Time homolog in the long-distance phloem of potato.

Yield of harvestable plant organs depends on photosynthetic assimilate production in source leaves, long-distance sucrose transport and sink-strength. While photosynthesis optimization has received considerable interest for optimizing plant yield, the potential for improving long-distance sucrose transport has received far less attention. Interestingly, a recent potato study demonstrates that the tuberigen StSP6A binds to and reduces activity of the StSWEET11 sucrose exporter. While the study suggested that reducing phloem sucrose efflux may enhance tuber yield, the precise mechanism and physiological relevance of this effect remained an open question. Here, we develop the first mechanistic model for sucrose transport, parameterized for potato plants. The model incorporates SWEET-mediated sucrose export, SUT-mediated sucrose retrieval from the apoplast and StSP6A-StSWEET11 interactions. Using this model, we were able to substantiate the physiological relevance of the StSP6A-StSWEET11 interaction in the long-distance phloem for potato tuber yield, as well as to show the non-linear nature of this effect.

Haplotype-resolved genome analyses of a heterozygous diploid potato.

Potato (Solanum tuberosum L.) is the most important tuber crop worldwide. Efforts are underway to transform the crop from a clonally propagated tetraploid into a seed-propagated, inbred-line-based hybrid, but this process requires a better understanding of potato genome. Here, we report the 1.67-Gb haplotype-resolved assembly of a diploid potato, RH89-039-16, using a combination of multiple sequencing strategies, including circular consensus sequencing. Comparison of the two haplotypes revealed ~2.1% intragenomic diversity, including 22,134 predicted deleterious mutations in 10,642 annotated genes. In 20,583 pairs of allelic genes, 16.6% and 30.8% exhibited differential expression and methylation between alleles, respectively. Deleterious mutations and differentially expressed alleles were dispersed throughout both haplotypes, complicating strategies to eradicate deleterious alleles or stack beneficial alleles via meiotic recombination. This study offers a holistic view of the genome organization of a clonally propagated diploid species and provides insights into technological evolution in resolving complex genomes.

Source-Sink Regulation Is Mediated by Interaction of an FT Homolog with a SWEET Protein in Potato.

Potato plants form tuberous storage organs on underground modified stems called stolons. Tubers are rich in starch, proteins, and other important nutrients, making potato one of the most important staple food crops. The timing of tuber development in wild potato is regulated by day length through a mechanism that is closely related to floral transition [1, 2]. Tuberization is also known to be regulated by the availability of assimilates, in particular sucrose, the transported form of sugar, required for starch synthesis. During the onset of tuber development, the mode of sucrose unloading switches from apoplastic to symplastic [3]. Here, we show that this switch may be mediated by the interaction between the tuberization-specific FT homolog StSP6A and the sucrose efflux transporter StSWEET11 [4]. The binding of StSP6A to StSWEET11 blocked the leakage of sucrose to the apoplast, and is therefore likely to promote symplastic sucrose transport. The direct physical interaction between StSWEET11 and StSP6A proteins represents a link between the sugar and photoperiodic pathways for the regulation of potato tuber formation. Our data suggest that a previously undiscovered function for the FT family of proteins extends their role as mobile signals to mediators of source-sink partitioning, opening the possibility for modifying source-sink interactions.

The tuberization signal StSP6A represses flower bud development in potato.

Potato (Solanum tuberosum L.) can reproduce sexually through flowering and asexually through tuberization. While tuberization has been thoroughly studied, little research has been done on potato flowering. Flower bud development in the strictly short-day tuberizing S. tuberosum group Andigena is impaired under short-day conditions. This impaired development may indicate that tuberization negatively influences flowering. Here, we determine how tuberization affects flower bud development. To find out whether the absence of tubers improves flowering, we prevented tuberization by: (i) grafting potato scions onto wild potato rootstocks, which were unable to form tubers; (ii) removing stolons, the underground structures on which tubers form; and (iii) using plants that were silenced in the tuberization signal StSP6A. Additionally, transgenic plants with increased StSP6A expression were used to determine if flower bud development was impaired. The absence of a tuber sink alone did not accelerate flower bud development, nor did it allow more plants to reach anthesis (open flowering stage) or have more open flowers. Interestingly, reducing StSP6A expression improved flower bud development, and increasing expression impaired it. Our results show that flower bud development in potato is repressed by the tuberization signal StSP6A, and not by competition with the underground tuber sink.

Coincidence of potato CONSTANS (StCOL1) expression and light cannot explain night-break repression of tuberization.

In the obligate short-day potato Solanum tuberosum group Andigena (Solanum andigena), short days, or actually long nights, induce tuberization. Applying a night break in the middle of this long night represses tuberization. However, it is not yet understood how this repression takes place. We suggest a coincidence model, similar to the model explaining photoperiodic flowering in Arabidopsis. We hypothesize that potato CONSTANS (StCOL1), expressed in the night of a short day, is stabilized by the light of the night break. This allows for StCOL1 to repress tuberization through induction of StSP5G, which represses the tuberization signal StSP6A. We grew S. andigena plants in short days, with night breaks applied at different time points during the dark period, either coinciding with StCOL1 expression or not. StCOL1 protein presence, StCOL1 expression and expression of downstream targets StSP5G and StSP6A were measured during a 24-h time course. Our results show that a night break applied during peak StCOL1 expression is unable to delay tuberization, while coincidence with low or no StCOL1 expression leads to severely repressed tuberization. These results imply that coincidence between StCOL1 expression and light does not explain why a night break represses tuberization in short days. Furthermore, stable StCOL1 did not always induce StSP5G, and upregulated StSP5G did not always lead to fully repressed StSP6A. Our findings suggest there is a yet unknown level of control between StCOL1, StSP5G and StSP6A expression, which determines whether a plant tuberizes.

QTLTableMiner++: semantic mining of QTL tables in scientific articles.

BACKGROUND: A quantitative trait locus (QTL) is a genomic region that correlates with a phenotype. Most of the experimental information about QTL mapping studies is described in tables of scientific publications. Traditional text mining techniques aim to extract information from unstructured text rather than from tables. We present QTLTableMiner++ (QTM), a table mining tool that extracts and semantically annotates QTL information buried in (heterogeneous) tables of plant science literature. QTM is a command line tool written in the Java programming language. This tool takes scientific articles from the Europe PMC repository as input, extracts QTL tables using keyword matching and ontology-based concept identification. The tables are further normalized using rules derived from table properties such as captions, column headers and table footers. Furthermore, table columns are classified into three categories namely column descriptors, properties and values based on column headers and data types of cell entries. Abbreviations found in the tables are expanded using the Schwartz and Hearst algorithm. Finally, the content of QTL tables is semantically enriched with domain-specific ontologies (e.g. Crop Ontology, Plant Ontology and Trait Ontology) using the Apache Solr search platform and the results are stored in a relational database and a text file. RESULTS: The performance of the QTM tool was assessed by precision and recall based on the information retrieved from two manually annotated corpora of open access articles, i.e. QTL mapping studies in tomato (Solanum lycopersicum) and in potato (S. tuberosum). In summary, QTM detected QTL statements in tomato with 74.53% precision and 92.56% recall and in potato with 82.82% precision and 98.94% recall. CONCLUSION: QTM is a unique tool that aids in providing QTL information in machine-readable and semantically interoperable formats.

Homologues of potato chromosome 5 show variable collinearity in the euchromatin, but dramatic absence of sequence similarity in the pericentromeric heterochromatin.

BACKGROUND: In flowering plants it has been shown that de novo genome assemblies of different species and genera show a significant drop in the proportion of alignable sequence. Within a plant species, however, it is assumed that different haplotypes of the same chromosome align well. In this paper we have compared three de novo assemblies of potato chromosome 5 and report on the sequence variation and the proportion of sequence that can be aligned. RESULTS: For the diploid potato clone RH89-039-16 (RH) we produced two linkage phase controlled and haplotype-specific assemblies of chromosome 5 based on BAC-by-BAC sequencing, which were aligned to each other and compared to the 52 Mb chromosome 5 reference sequence of the doubled monoploid clone DM 1-3 516 R44 (DM). We identified 17.0 Mb of non-redundant sequence scaffolds derived from euchromatic regions of RH and 38.4 Mb from the pericentromeric heterochromatin. For 32.7 Mb of the RH sequences the correct position and order on chromosome 5 was determined, using genetic markers, fluorescence in situ hybridisation and alignment to the DM reference genome. This ordered fraction of the RH sequences is situated in the euchromatic arms and in the heterochromatin borders. In the euchromatic regions, the sequence collinearity between the three chromosomal homologs is good, but interruption of collinearity occurs at nine gene clusters. Towards and into the heterochromatin borders, absence of collinearity due to structural variation was more extensive and was caused by hemizygous and poorly aligning regions of up to 450 kb in length. In the most central heterochromatin, a total of 22.7 Mb sequence from both RH haplotypes remained unordered. These RH sequences have very few syntenic regions and represent a non-alignable region between the RH and DM heterochromatin haplotypes of chromosome 5. CONCLUSIONS: Our results show that among homologous potato chromosomes large regions are present with dramatic loss of sequence collinearity. This stresses the need for more de novo reference assemblies in order to capture genome diversity in this crop. The discovery of three highly diverged pericentric heterochromatin haplotypes within one species is a novelty in plant genome analysis. The possible origin and cytogenetic implication of this heterochromatin haplotype diversity are discussed.

Chromosomal organizations of major repeat families on potato (Solanum tuberosum) and further exploring in its sequenced genome.

One of the most powerful technologies in unraveling the organization of a eukaryotic plant genome is high-resolution Fluorescent in situ hybridization of repeats and single copy DNA sequences on pachytene chromosomes. This technology allows the integration of physical mapping information with chromosomal positions, including centromeres, telomeres, nucleolar-organizing region, and euchromatin and heterochromatin. In this report, we established chromosomal positions of different repeat fractions of the potato genomic DNA (Cot100, Cot500 and Cot1000) on the chromosomes. We also analysed various repeat elements that are unique to potato including the moderately repetitive P5 and REP2 elements, where the REP2 is part of a larger Gypsy-type LTR retrotransposon and cover most chromosome regions, with some brighter fluorescing spots in the heterochromatin. The most abundant tandem repeat is the potato genomic repeat 1 that covers subtelomeric regions of most chromosome arms. Extensive multiple alignments of these repetitive sequences in the assembled RH89-039-16 potato BACs and the draft assembly of the DM1-3 516 R44 genome shed light on the conservation of these repeats within the potato genome. The consensus sequences thus obtained revealed the native complete transposable elements from which they were derived.

Construction of reference chromosome-scale pseudomolecules for potato: integrating the potato genome with genetic and physical maps.

The genome of potato, a major global food crop, was recently sequenced. The work presented here details the integration of the potato reference genome (DM) with a new sequence-tagged site marker-based linkage map and other physical and genetic maps of potato and the closely related species tomato. Primary anchoring of the DM genome assembly was accomplished by the use of a diploid segregating population, which was genotyped with several types of molecular genetic markers to construct a new ~936 cM linkage map comprising 2469 marker loci. In silico anchoring approaches used genetic and physical maps from the diploid potato genotype RH89-039-16 (RH) and tomato. This combined approach has allowed 951 superscaffolds to be ordered into pseudomolecules corresponding to the 12 potato chromosomes. These pseudomolecules represent 674 Mb (~93%) of the 723 Mb genome assembly and 37,482 (~96%) of the 39,031 predicted genes. The superscaffold order and orientation within the pseudomolecules are closely collinear with independently constructed high density linkage maps. Comparisons between marker distribution and physical location reveal regions of greater and lesser recombination, as well as regions exhibiting significant segregation distortion. The work presented here has led to a greatly improved ordering of the potato reference genome superscaffolds into chromosomal "pseudomolecules".

Down regulation of StGA3ox genes in potato results in altered GA content and affect plant and tuber growth characteristics.

GA biosynthesis and catabolism has been shown to play an important role in regulating tuberization in potato. Active GAs are inactivated in the stolon tips shortly after induction to tuberization. Overexpression of a GA inactivation gene results in an earlier tuberization phenotype, while reducing expression of the same gene results in delayed tuberization. In addition, overexpression of genes involved in GA biosynthesis results in delayed tuberization, while decreased expression of those genes results in earlied tuberization. The final step in GA biosynthesis is catalysed by StGA3ox1 and StGA3ox2 activity, that convert inactive forms of GA into active GA1 and GA4. In this study we cloned StGA3ox2 gene in an RNAi construct and used this construct to transform potato plants. The StGA3ox2 silenced plants were smaller and had shorter internodes. In addition, we assayed the concentrations of various GAs in the transgenic plants and showed an altered GA content. No difference was observed on the time point of tuber initiation. However, the transgenic clones had increased number of tubers with the same yield, resulting in smaller average tuber weight. In addition, we cloned the promoter of StGA3ox2 to direct expression of the GUS reporter gene to visualize the sites of GA biosynthesis in the potato plant. Finally, we discuss how changes of several GA levels can have an impact on shoot, stolon and tuber development, as well as the possible mechanisms that mediate feed-forward and feed-back regulation loops in the GA biosynthetic pathway in potato.

Naturally occurring allele diversity allows potato cultivation in northern latitudes.

Potato (Solanum tuberosum L.) originates from the Andes and evolved short-day-dependent tuber formation as a vegetative propagation strategy. Here we describe the identification of a central regulator underlying a major-effect quantitative trait locus for plant maturity and initiation of tuber development. We show that this gene belongs to the family of DOF (DNA-binding with one finger) transcription factors and regulates tuberization and plant life cycle length, by acting as a mediator between the circadian clock and the StSP6A mobile tuberization signal. We also show that natural allelic variants evade post-translational light regulation, allowing cultivation outside the geographical centre of origin of potato. Potato is a member of the Solanaceae family and is one of the world's most important food crops. This annual plant originates from the Andean regions of South America. Potato develops tubers from underground stems called stolons. Its equatorial origin makes potato essentially short-day dependent for tuberization and potato will not make tubers in the long-day conditions of spring and summer in the northern latitudes. When introduced in temperate zones, wild material will form tubers in the course of the autumnal shortening of day-length. Thus, one of the first selected traits in potato leading to a European potato type is likely to have been long-day acclimation for tuberization. Potato breeders can exploit the naturally occurring variation in tuberization onset and life cycle length, allowing varietal breeding for different latitudes, harvest times and markets.

A crosstalk of auxin and GA during tuber development.

Several hormones have been studied for their effect on tuber initiation and development. Until recently, the hormone with the most prominent role in tuber initiation was attributed to GA. Genes involved in GA degradation do exhibit an upregulated profile during early stages of tuber development, leading to a rapid decrease of active GA content, thereby facilitating stolon-tip swelling. While GA is known to be involved in shoot and stolon elongation, the development of the new tuberorgan requires changes in meristem identity and the reorientation ofthe plane of cell division. In other developmental processes, such as embryo patterning, flower development and lateral root initiation auxin plays a key role. Recent evidence on the involvement of auxin in tuber formation was providedby the measurement of auxin content in swelling stolons. Auxin content in the stolon tips increased several fold prior to tuber swelling. In vitro tuberisation experiments with auxin applications support the role of auxin during tuber initiation. Taken together, it is becoming clear that the initiation and induction of tubers in potato is a developmental process that appears to be regulated by a crosstalk between GA and auxin.

The effects of auxin and strigolactones on tuber initiation and stolon architecture in potato.

Various transcriptional networks and plant hormones have been implicated in controlling different aspects of potato tuber formation. Due to its broad impact on many plant developmental processes, a role for auxin in tuber initiation has been suggested but never fully resolved. Here, auxin concentrations were measured throughout the plant prior to and during the process of tuber formation. Auxin levels increase dramatically in the stolon prior to tuberization and remain relatively high during subsequent tuber growth, suggesting a promoting role for auxin in tuber formation. Furthermore, in vitro tuberization experiments showed higher levels of tuber formation from axillary buds of explants where the auxin source (stolon tip) had been removed. This phenotype could be rescued by application of auxin on the ablated stolon tips. In addition, a synthetic strigolactone analogue applied on the basal part of the stolon resulted in fewer tubers. The experiments indicate that a system for the production and directional transport of auxin exists in stolons and acts synergistically with strigolactones to control the outgrowth of the axillary stolon buds, similar to the control of above-ground shoot branching.