Construction of a dense SNP map of a highly heterozygous diploid potato population and QTL analysis of tuber shape and eye depth.

KEY MESSAGE: Generation of a dense SNP-based linkage map of a diploid potato population and identification of major QTLs for tuber shape and eye depth on chromosomes 2 and 10. This paper reports the construction of a genetic map of a highly heterozygous full-sib diploid potato population (06H1) based on the use of a set of 8,303 single nucleotide polymorphism (SNP) markers. The map contains 1,355 distinct loci and 2,157 SNPs, 802 of which co-segregate with other markers. We find high levels of collinearity between the 12 chromosomal maps with a recently improved version of the potato genome assembly, with the expected genetic clustering in centromeric regions. The linkage maps are used in combination with highly detailed phenotypic assessments conducted over two growing seasons to perform quantitative trait loci analysis of two important potato traits, tuber shape and eye depth. The major loci segregating for tuber shape in 06H1 map to loci on chromosomes 2 and 10, with smaller effects mapping to three other chromosomes. A major locus for tuber eye depth co-locates with the tuber shape locus on chromosome 10. To assess when tuber shape is established in the developing tuber, we have performed staged observations of tuber formation. Our observations suggest that tuber shape is determined very early in tuber development.

Identification and characterization of barley RNA-directed RNA polymerases.

RNA-directed RNA polymerases (RDRs) play crucial roles in the RNA silencing response of plants by enhancing and maintaining silencing signals. At least two members of the RDR group, namely RDR1 and RDR6, are implicated in defence against plant viruses. RDRs have so far only been characterized in dicot species. In this report, we identified and characterized HvRDR1, HvRDR2 and HvRDR6 genes in the monocot plant barley (Hordeum vulgare). We analysed their expression under various biotic and abiotic stresses including fungal and viral infections, salicylic acid treatment as well as during plant development. The different classes and subclasses of barley RDRs displayed contrasting expression patterns during pathogen challenge and development suggesting their involvement in specific regulatory pathways. Their response to heat and salicylic acid treatment suggests a conserved pattern of expression of these genes between monocot and dicot plant species. The existence of two HvRDR1 and two HvRDR6 genes suggests an evolutionary selection for specialization in response to biotic and abiotic stresses after gene duplication.

Alternative polyadenylation of antisense RNAs and flowering time control.

Flowering time is controlled by precision in gene regulation mediated by different pathways. Two Arabidopsis thaliana components of the autonomous flowering pathway, FCA and FPA, function as genetically independent trans-acting regulators of alternative cleavage and polyadenylation. FCA and FPA directly associate with chromatin at the locus encoding the floral repressor FLC, but appear to control FLC transcription by mediating alternative polyadenylation of embedded non-coding antisense RNAs. These findings prompt the re-examination of how other factors control FLC expression, as it is formally possible that they function primarily to control alternative processing of antisense RNAs. As co-expressed sense and antisense gene pairs are widespread in eukaryotes, alternative processing of antisense RNAs may represent a significant form of gene regulation.

Post-transcriptional Regulation of FLOWERING LOCUS T Modulates Heat-Dependent Source-Sink Development in Potato.

Understanding tuberization in the major crop plant potato (Solanum tuberosum L.) is of importance to secure yield even under changing environmental conditions. Tuber formation is controlled by a homolog of the floral inductor FLOWERING LOCUS T, referred to as SP6A. To gain deeper insights into its function, we created transgenic potato plants overexpressing a codon-optimized version of SP6A, SP6Acop, to avoid silencing effects. These plants exhibited extremely early tuberization at the juvenile stage, hindering green biomass development and indicating a tremendous shift in the source sink balance. The meristem identity was altered in dormant buds of transgenic tubers. This strong phenotype, not being reported so far for plants overexpressing an unmodified SP6A, could be due to post-transcriptional regulation. In fact, a putative SP6A-specific small regulatory RNA was identified in potato. It was effectively repressing SP6A mRNA accumulation in transient assays as well as in leaves of young potato plants prior to tuber formation. SP6A expression is downregulated under heat, preventing tuberization. The molecular mechanism has not been elucidated yet. We showed that this small RNA is strongly upregulated under heat. The importance of the small RNA was demonstrated by overexpression of a target mimicry construct, which led to an increased SP6A expression, enabling tuberization even under continuous heat conditions, which abolished tuber formation in the wild-type. Thus, our study describes an additional regulatory mechanism for SP6A besides the well-known pathway that integrates both developmental and environmental signals to control tuberization and is therefore a promising target for breeding of heat-tolerant potato.

Cold-Dependent Expression and Alternative Splicing of Arabidopsis Long Non-coding RNAs.

Plants re-program their gene expression when responding to changing environmental conditions. Besides differential gene expression, extensive alternative splicing (AS) of pre-mRNAs and changes in expression of long non-coding RNAs (lncRNAs) are associated with stress responses. RNA-sequencing of a diel time-series of the initial response of Arabidopsis thaliana rosettes to low temperature showed massive and rapid waves of both transcriptional and AS activity in protein-coding genes. We exploited the high diversity of transcript isoforms in AtRTD2 to examine regulation and post-transcriptional regulation of lncRNA gene expression in response to cold stress. We identified 135 lncRNA genes with cold-dependent differential expression (DE) and/or differential alternative splicing (DAS) of lncRNAs including natural antisense RNAs, sORF lncRNAs, and precursors of microRNAs (miRNAs) and trans-acting small-interfering RNAs (tasiRNAs). The high resolution (HR) of the time-series allowed the dynamics of changes in transcription and AS to be determined and identified early and adaptive transcriptional and AS changes in the cold response. Some lncRNA genes were regulated only at the level of AS and using plants grown at different temperatures and a HR time-course of the first 3 h of temperature reduction, we demonstrated that the AS of some lncRNAs is highly sensitive to small temperature changes suggesting tight regulation of expression. In particular, a splicing event in TAS1a which removed an intron that contained the miR173 processing and phased siRNAs generation sites was differentially alternatively spliced in response to cold. The cold-induced reduction of the spliced form of TAS1a and of the tasiRNAs suggests that splicing may enhance production of the siRNAs. Our results identify candidate lncRNAs that may contribute to the regulation of expression that determines the physiological processes essential for acclimation and freezing tolerance.

Potato miR828 Is Associated With Purple Tuber Skin and Flesh Color.

Anthocyanins are plant pigments responsible for the colors of many flowers, fruits and storage organs and have roles in abiotic and biotic stress resistance. Anthocyanins and polyphenols are bioactive compounds in plants including potato (Solanum tuberosum L.) which is the most important non-cereal crop in the world, cultivated for its tubers rich in starch and nutrients. The genetic regulation of the flavonoid biosynthetic pathway is relatively well known leading to the formation of anthocyanins. However, our knowledge of post-transcriptional regulation of anthocyanin biosynthesis is limited. There is increasing evidence that micro RNAs (miRNAs) and other small RNAs can regulate the expression level of key factors in anthocyanin production. In this study we have found strong associations between the high levels of miR828, TAS4 D4(-) and purple/red color of tuber skin and flesh. This was confirmed not only in different cultivars but in pigmented and non-pigmented sectors of the same tuber. Phytochemical analyses verified the levels of anthocyanins and polyphenols in different tissues. We showed that miR828 is able to direct cleavage of the RNA originating from Trans-acting siRNA gene 4 (TAS4) and initiate the production of phased small interfering RNAs (siRNAs) whose production depends on RNA-dependent RNA polymerase 6 (RDR6). MYB transcription factors were predicted as potential targets of miR828 and TAS4 D4(-) and their expression was characterized. MYB12 and R2R3-MYB genes showed decreased expression levels in purple skin and flesh in contrast with high levels of small RNAs in the same tissues. Moreover, we confirmed that R2R3-MYB and MYB-36284 are direct targets of the small RNAs. Overall, this study sheds light on the small RNA directed anthocyanin regulation in potato, which is an important member of the Solanaceae family.

Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes.

We describe here a new method for highly efficient detection of microRNAs by northern blot analysis using LNA (locked nucleic acid)-modified oligonucleotides. In order to exploit the improved hybridization properties of LNA with their target RNA molecules, we designed several LNA-modified oligonucleotide probes for detection of different microRNAs in animals and plants. By modifying DNA oligonucleotides with LNAs using a design, in which every third nucleotide position was substituted by LNA, we could use the probes in northern blot analysis employing standard end-labelling techniques and hybridization conditions. The sensitivity in detecting mature microRNAs by northern blots was increased by at least 10-fold compared to DNA probes, while simultaneously being highly specific, as demonstrated by the use of different single and double mismatched LNA probes. Besides being highly efficient as northern probes, the same LNA-modified oligonucleotide probes would also be useful for miRNA in situ hybridization and miRNA expression profiling by LNA oligonucleotide microarrays.

Insight on Genes Affecting Tuber Development in Potato upon Potato spindle tuber viroid (PSTVd) Infection.

Potato (Solanum tuberosum L) is a natural host of Potato spindle tuber viroid (PSTVd) which can cause characteristic symptoms on developing plants including stunting phenotype and distortion of leaves and tubers. PSTVd is the type species of the family Pospiviroidae, and can replicate in the nucleus and move systemically throughout the plant. It is not well understood how the viroid can affect host genes for successful invasion and which genes show altered expression levels upon infection. Our primary focus in this study is the identification of genes which can affect tuber formation since viroid infection can strongly influence tuber development and especially tuber shape. In this study, we used a large-scale method to identify differentially expressed genes in potato. We have identified defence, stress and sugar metabolism related genes having altered expression levels upon infection. Additionally, hormone pathway related genes showed significant up- or down-regulation. DWARF1/DIMINUTO, Gibberellin 7-oxidase and BEL5 transcripts were identified and validated showing differential expression in viroid infected tissues. Our study suggests that gibberellin and brassinosteroid pathways have a possible role in tuber development upon PSTVd infection.

Identification and characterization of miRNA transcriptome in potato by high-throughput sequencing.

Micro RNAs (miRNAs) represent a class of short, non-coding, endogenous RNAs which play important roles in post-transcriptional regulation of gene expression. While the diverse functions of miRNAs in model plants have been well studied, the impact of miRNAs in crop plant biology is poorly understood. Here we used high-throughput sequencing and bioinformatics analysis to analyze miRNAs in the tuber bearing crop potato (Solanum tuberosum). Small RNAs were analysed from leaf and stolon tissues. 28 conserved miRNA families were found and potato-specific miRNAs were identified and validated by RNA gel blot hybridization. The size, origin and predicted targets of conserved and potato specific miRNAs are described. The large number of miRNAs and complex population of small RNAs in potato suggest important roles for these non-coding RNAs in diverse physiological and metabolic pathways.

Direct sequencing of Arabidopsis thaliana RNA reveals patterns of cleavage and polyadenylation.

It has recently been shown that RNA 3'-end formation plays a more widespread role in controlling gene expression than previously thought. To examine the impact of regulated 3'-end formation genome-wide, we applied direct RNA sequencing to A. thaliana. Here we show the authentic transcriptome in unprecedented detail and describe the effects of 3'-end formation on genome organization. We reveal extreme heterogeneity in RNA 3' ends, discover previously unrecognized noncoding RNAs and propose widespread reannotation of the genome. We explain the origin of most poly(A)(+) antisense RNAs and identify cis elements that control 3'-end formation in different registers. These findings are essential to understanding what the genome actually encodes, how it is organized and how regulated 3'-end formation affects these processes.

The spen family protein FPA controls alternative cleavage and polyadenylation of RNA.

The spen family protein FPA is required for flowering time control and has been implicated in RNA silencing. The mechanism by which FPA carries out these functions is unknown. We report the identification of an activity for FPA in controlling mRNA 3' end formation. We show that FPA functions redundantly with FCA, another RNA binding protein that controls flowering and RNA silencing, to control the expression of alternatively polyadenylated antisense RNAs at the locus encoding the floral repressor FLC. In addition, we show that defective 3' end formation at an upstream RNA polymerase II-dependent gene explains the apparent derepression of the AtSN1 retroelement in fpa mutants. Transcript readthrough accounts for the absence of changes in DNA methylation and siRNA abundance at AtSN1 in fpa mutants, and this may explain other examples of epigenetic transitions not associated with chromatin modification.

Ectopic 5' splice sites inhibit gene expression by engaging RNA surveillance and silencing pathways in plants.

The quality control of mRNA maturation is a highly regulated process that surveys pre-mRNA integrity and eliminates improperly matured pre-mRNAs. In nature, certain viruses regulate the expression of their genes by hijacking the endogenous RNA quality control machinery. We demonstrate that the inclusion of 5' splice sites within the 3'-untranslated region of a reporter gene in plants alters the pre-mRNA cleavage and polyadenylation process, resulting in pre-mRNA degradation, exemplifying a regulatory mechanism conserved between kingdoms. Altered pre-mRNA processing was associated with an inhibition of homologous gene expression in trans and the preferential accumulation of 24-nucleotide (nt) short-interfering RNAs (siRNAs) as opposed to 21-nt siRNA subspecies, suggesting that degradation of the aberrant pre-mRNA involves the silencing machinery. However, gene expression was not restored by coexpression of a silencing suppressor or in an RNA-dependent RNA polymerase (RDR6)-deficient background despite reduced 24-nt siRNA accumulation. Our data highlight a complex cross talk between the quality control RNA machinery, 3'-end pre-mRNA maturation, and RNA-silencing pathways capable of discriminating among different types of aberrant RNAs.

RNA interference-mediated repression of sucrose-phosphatase in transgenic potato tubers (Solanum tuberosum) strongly affects the hexose-to-sucrose ratio upon cold storage with only minor effects on total soluble carbohydrate accumulation.

Storage of potato tubers at low temperatures leads to the accumulation of glucose and fructose in a process called 'cold sweetening'. The aim of this work was to investigate the role of sucrose-phosphatase (SPP) in potato tuber carbohydrate metabolism at low temperature (4 degrees C). To this end, RNA interference (RNAi) was used to reduce SPP expression in transgenic potato tubers. Analysis of SPP specific small interfering RNAs (siRNAs), SPP protein accumulation and enzyme activity indicated that SPP silencing in transgenic tubers was stable during the cold treatment. Analysis of soluble carbohydrates showed that in transgenic tubers, cold-induced hexogenesis was inhibited while, despite strongly reduced SPP activity, sucrose levels exceeded wild-type (WT) values four- to fivefold after 34 d of cold treatment. This led to a drastic change in the hexose-to-sucrose ratio from 1.9 in WT tubers to 0.15 to 0.11 in transgenic tubers, while the total amount of soluble sugars was largely unchanged in both genotypes. Sucrose-6(F)-phosphate (Suc6P), the substrate of SPP, accumulated in transgenic tubers in the cold which most likely enables the residual enzyme to operate with maximal catalytic activity in vivo and thus, in the long term, counterbalances reduced SPP activity in the transformants. Northern analysis revealed that cold-induced expression of vacuolar invertase (VI) was blocked in SPP-silenced tubers explaining a reduced sucrose-to-hexose conversion. Suc6P levels were found to negatively correlate with VI expression. A possible role of Suc6P in regulating VI expression is discussed.

Defective interfering RNA hinders the activity of a tombusvirus-encoded posttranscriptional gene silencing suppressor.

Defective interfering (DI) RNAs are subviral replicons originating from the viral genome and are associated with many plant RNA viruses and nearly all animal RNA viruses. The presence of DI RNAs in tombusvirus-infected plants reduces the accumulation of helper virus RNA and results in the development of attenuated symptoms similar to those caused by tombusviruses defective in p19, the posttranscriptional gene silencing (PTGS) suppressor. In situ analysis of infected plants containing DI RNAs revealed that the extent of virus infection was spatially restricted as was found for p19-defective tombusvirus. Previously, p19 was shown to suppress PTGS by sequestering the small interfering RNAs (siRNAs), which act as the specificity determinant for PTGS. Our results demonstrate that DI RNAs dramatically elevate the level of virus-specific siRNAs in viral infections, resulting in the saturation of p19 and the accumulation of unbound siRNAs. Moreover, we showed that, at low temperature, where PTGS is inhibited, DI RNAs are not able to efficiently interfere with virus accumulation and protect the plants. These data show that the activation of PTGS plays a pivotal role in DI RNA-mediated interference. Our data also support a role for 21-nucleotide siRNAs in PTGS signaling.

In situ characterization of Cymbidium Ringspot Tombusvirus infection-induced posttranscriptional gene silencing in Nicotiana benthamiana.

In plants, posttranscriptional gene silencing (PTGS) is an ancient and effective defense mechanism against viral infection. A number of viruses encode proteins that suppress virus-activated PTGS. The p19 protein of tombusviruses is a potent PTGS suppressor which interferes with the onset of PTGS-generated systemic signaling and is not required for viral replication or for viral movement in Nicotiana benthamiana. This unique feature of p19 suppressor allowed us to analyze the mechanism of PTGS-based host defense and its viral suppression without interfering with other viral functions. In contrast to the necrotic symptoms caused by wild-type tombusvirus, the infection of p19-defective mutant virus results in the development of a typical PTGS-associated recovery phenotype in N. benthamiana. In this report we show the effect of PTGS on the viral infection process for N. benthamiana infected with either wild-type Cymbidium Ringspot Tombusvirus (CymRSV) or a p19-defective mutant (Cym19stop). In situ analyses of different virus-derived products revealed that PTGS is not able to reduce accumulation of virus in primary infected cells regardless of the presence of p19 PTGS suppressor. We also showed that both CymRSV and Cym19stop viruses move systemically in the vasculature, with similar efficiencies. However, in contrast to the uniform accumulation of CymRSV throughout systemically infected leaves, the presence of Cym19stop virus was confined to and around the vascular bundles. These results suggest that the role of p19 is to prevent the onset of mobile signal-induced systemic PTGS ahead of the viral infection front, leading to generalized infection.

Short defective interfering RNAs of tombusviruses are not targeted but trigger post-transcriptional gene silencing against their helper virus.

Post-transcriptional gene silencing (PTGS) is a sequence-specific degradation mechanism that operates in almost all eukaryotic cells. In plants, double-stranded RNA triggers PTGS, generating 21- to 25-nucleotide guide RNAs responsible for specific degradation of cognate mRNA. The double stranded RNA intermediates of replicating plant viruses often induce PTGS, leading to symptom attenuation. Here we demonstrate the role of PTGS in defective interfering (DI) RNA-mediated symptom attenuation in plants infected with Cymbidium ringspot tombusvirus (CymRSV). Analysis of 21- to 25-nucleotide RNAs in Nicotiana benthamiana infected with CymRSV indicated that PTGS was not spread homogeneously along the viral genome. The 21- to 25-nucleotide RNAs derived mainly from plus-stranded RNA and likely arose from local basepaired structures. In contrast to helper viral RNA, short DI RNAs were not accessible to helper virus-induced RNA degradation guided by the 21- to 25-nucleotide RNAs. Our results suggest a model in which PTGS plays an important role in the selective accumulation and symptom attenuation mediated by DI RNAs. Because PTGS operates in a wide variety of different organisms, this model is applicable to DI RNA generation and accumulation in both plant and animal cells.

A viral protein suppresses RNA silencing and binds silencing-generated, 21- to 25-nucleotide double-stranded RNAs.

Posttranscriptional gene silencing (PTGS) processes double-stranded (ds) RNAs into 21-25 nucleotide (nt) RNA fragments that direct ribonucleases to target cognate mRNAs. In higher plants, PTGS also generates mobile signals conferring sequence-specific silencing in distant organs. Since PTGS acts as an antiviral system in plants, successful virus infection requires evasion or suppression of gene silencing. Here we report that the 19 kDa protein (p19) of tombusviruses is a potent silencing suppressor that prevents the spread of mobile silencing signal. In vitro, p19 binds PTGS-generated, 21-25 nt dsRNAs and 21-nt synthetic dsRNAs with 2-nt 3' overhanging end(s), while it barely interacts with single-stranded (ss) RNAs, long dsRNAs or blunt-ended 21-nt dsRNAs. We propose that p19 mediates silencing suppression by sequestering the PTGS-generated 21-25 nt dsRNAs, thus depleting the specificity determinants of PTGS effector complexes. Moreover, the observation that p19-expressing transgenic plants show altered leaf morphology might indicate that the p19-targeted PTGS pathway is also important in the regulation of plant development.