Bjorn Kloosterman

Genome sequence and analysis of the tuber crop potato.

Potato (Solanum tuberosum L.) is the world’s most important non-grain food crop and is central to global food security. It is clonally propagated, highly heterozygous, autotetraploid, and suffers acute inbreeding depression. Here we use a homozygous doubled-monoploid potato clone to sequence and assemble 86% of the 844-megabase genome. We predict 39,031 protein-coding genes and present evidence for at least two genome duplication events indicative of a palaeopolyploid origin. As the first genome sequence of an asterid, the potato genome reveals 2,642 genes specific to this large angiosperm clade. We also sequenced a heterozygous diploid clone and show that gene presence/absence variants and other potentially deleterious mutations occur frequently and are a likely cause of inbreeding depression. Gene family expansion, tissue-specific expression and recruitment of genes to new pathways contributed to the evolution of tuber development. The potato genome sequence provides a platform for genetic improvement of this vital crop.

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.

Genes driving potato tuber initiation and growth: identification based on transcriptional changes using the POCI array

The increasing amount of available expressed gene sequence data makes whole-transcriptome analysis of certain crop species possible. Potato currently has the second largest number of publicly available expressed sequence tag (EST) sequences among the Solanaceae. Most of these ESTs, plus other proprietary sequences, were combined and used to generate a unigene assembly. The set of 246,182 sequences produced 46,345 unigenes, which were used to design a 44K 60-mer oligo array (Potato Oligo Chip Initiative: POCI). In this study, we attempt to identify genes controlling and driving the process of tuber initiation and growth by implementing large-scale transcriptional changes using the newly developed POCI array. Major gene expression profiles could be identified exhibiting differential expression at key developmental stages. These profiles were associated with functional roles in cell division and growth. A subset of genes involved in the regulation of the cell cycle, based on their Gene Ontology classification, exhibit a clear transient upregulation at tuber onset indicating increased cell division during these stages. The POCI array allows the study of potato gene expression on a much broader level than previously possible and will greatly enhance analysis of transcriptional control mechanisms in a wide range of potato research areas. POCI sequence and annotation data are publicly available through the POCI database (http://pgrc.ipk-gatersleben.de/poci).

From QTL to candidate gene: genetical genomics of simple and complex traits in potato using a pooling strategy.

BackgroundUtilization of the natural genetic variation in traditional breeding programs remains a major challenge in crop plants. The identification of candidate genes underlying, or associated with, phenotypic trait QTLs is desired for effective marker assisted breeding. With the advent of high throughput -omics technologies, screening of entire populations for association of gene expression with targeted traits is becoming feasible but remains costly. Here we present the identification of novel candidate genes for different potato tuber quality traits by employing a pooling approach reducing the number of hybridizations needed. Extreme genotypes for a quantitative trait are collected and the RNA from contrasting bulks is then profiled with the aim of finding differentially expressed genes.ResultsWe have successfully implemented the pooling strategy for potato quality traits and identified candidate genes associated with potato tuber flesh color and tuber cooking type. Elevated expression level of a dominant allele of the β-carotene hydroxylase (bch) gene was associated with yellow flesh color through mapping of the gene under a major QTL for flesh color on chromosome 3. For a second trait, a candidate gene with homology to a tyrosine-lysine rich protein (TLRP) was identified based on allele specificity of the probe on the microarray. TLRP was mapped on chromosome 9 in close proximity to a QTL for potato cooking type strengthening its significance as a candidate gene. Furthermore, we have performed a profiling experiment targeting a polygenic trait, by pooling individual genotypes based both on phenotypic and marker data, allowing the identification of candidate genes associated with the two different linkage groups.ConclusionsA pooling approach for RNA-profiling with the aim of identifying novel candidate genes associated with tuber quality traits was successfully implemented. The identified candidate genes for tuber flesh color (bch) and cooking type (tlrp) can provide useful markers for breeding schemes in the future. Strengths and limitations of the approach are discussed.

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.

Identification of alleles of carotenoid pathway genes important for zeaxanthin accumulation in potato tubers

We have investigated the genetics and molecular biology of orange flesh colour in potato (Solanum tuberosum L.). To this end the natural diversity in three genes of the carotenoid pathway was assessed by SNP analyses. Association analysis was performed between SNP haplotypes and flesh colour phenotypes in diploid and tetraploid potato genotypes. We observed that among eleven beta-carotene hydroxylase 2 (Chy2) alleles only one dominant allele has a major effect, changing white into yellow flesh colour. In contrast, none of the lycopene epsilon cyclase (Lcye) alleles seemed to have a large effect on flesh colour. Analysis of zeaxanthin epoxidase (Zep) alleles showed that all (diploid) genotypes with orange tuber flesh were homozygous for one specific Zep allele. This Zep allele showed a reduced level of expression. The complete genomic sequence of the recessive Zep allele, including the promoter, was determined, and compared with the sequence of other Zep alleles. The most striking difference was the presence of a non-LTR retrotransposon sequence in intron 1 of the recessive Zep allele, which was absent in all other Zep alleles investigated. We hypothesise that the presence of this large sequence in intron 1 caused the lower expression level, resulting in reduced Zep activity and accumulation of zeaxanthin. Only genotypes combining presence of the dominant Chy2 allele with homozygosity for the recessive Zep allele produced orange-fleshed tubers that accumulated large amounts of zeaxanthin.

Data integration and network reconstruction with ~omics data using Random Forest regression in potato.

In the post-genomic era, high-throughput technologies have led to data collection in fields like transcriptomics, metabolomics and proteomics and, as a result, large amounts of data have become available. However, the integration of these ~omics data sets in relation to phenotypic traits is still problematic in order to advance crop breeding. We have obtained population-wide gene expression and metabolite (LC-MS) data from tubers of a diploid potato population and present a novel approach to study the various ~omics datasets to allow the construction of networks integrating gene expression, metabolites and phenotypic traits. We used Random Forest regression to select subsets of the metabolites and transcripts which show association with potato tuber flesh color and enzymatic discoloration. Network reconstruction has led to the integration of known and uncharacterized metabolites with genes associated with the carotenoid biosynthesis pathway. We show that this approach enables the construction of meaningful networks with regard to known and unknown components and metabolite pathways.

The PIN family of proteins in potato and their putative role in tuberization

The PIN family of trans-membrane proteins mediates auxin efflux throughout the plant and during various phases of plant development. In Arabidopsis thaliana, the PIN family comprised of 8 members, divided into “short” and “long” PINs according to the length of the hydrophilic domain of the protein. Based on sequence homology using the recently published potato genome sequence (Solanum tuberosum group Phureja) we identified ten annotated potato StPIN genes. Mining the publicly available gene expression data, we constructed a catalog tissue specificity of StPIN gene expression, focusing on the process of tuberization. A total of four StPIN genes exhibited increased expression 4 days after tuber induction, prior to the onset of stolon swelling. For two PIN genes, StPIN4 and StPIN2, promoter sequences were cloned and fused to the GUS reporter protein to study tissue specificity in more detail. StPIN4 promoter driven GUS staining was detected in the flower stigma, in the flower style, below the ovary and petals, in the root tips, in the vascular tissue of the stolons and in the tuber parenchyma cells. StPIN2 promoter driven GUS staining was detected in flower buds, in the vascular tissue of the swelling stolons and in the storage parenchyma of the growing tubers. Based on our results, we postulate a role for the StPINs in redistributing auxin in the swelling stolon during early events in tuber development.

Organ specificity and transcriptional control of metabolic routes revealed by expression QTL profiling of source-sink tissues in a segregating potato population

BackgroundWith the completion of genome sequences belonging to some of the major crop plants, new challenges arise to utilize this data for crop improvement and increased food security. The field of genetical genomics has the potential to identify genes displaying heritable differential expression associated to important phenotypic traits. Here we describe the identification of expression QTLs (eQTLs) in two different potato tissues of a segregating potato population and query the potato genome sequence to differentiate between cis- and trans-acting eQTLs in relation to gene subfunctionalization.ResultsLeaf and tuber samples were analysed and screened for the presence of conserved and tissue dependent eQTLs. Expression QTLs present in both tissues are predominantly cis-acting whilst for tissue specific QTLs, the percentage of trans-acting QTLs increases. Tissue dependent eQTLs were assigned to functional classes and visualized in metabolic pathways. We identified a potential regulatory network on chromosome 10 involving genes crucial for maintaining circadian rhythms and controlling clock output genes. In addition, we show that the type of genetic material screened and sampling strategy applied, can have a high impact on the output of genetical genomics studies.ConclusionsIdentification of tissue dependent regulatory networks based on mapped differential expression not only gives us insight in tissue dependent gene subfunctionalization but brings new insights into key biological processes and delivers targets for future haplotyping and genetic marker development.

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.