Project description: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, it can replicate in the nucleus and the viroid RNA moves systemically in infected plants. Its KF440-2 strain can cause severe symptoms in potato. It is not well understood how the viroid can affect host genes for successful invasion and which genes show altered expression levels upon infection. In this study, we used a high-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 pathways connected genes showed up- or down-regulation. Our primary focus is on the identification of genes which can affect tuber formation as the viroid infection can strongly influence tuber development, especially tuber shape is affected. DWARF1/DIMINUTO, Gibberellin 7-oxidase and BEL5 protein were identified and validated which showed differential expression in viroid infected tissues suggesting that gibberellin and brassinosteroid pathways have a possible role in tuber development upon PSTVd infection.
Project description:An in vivo and in vitro potato tuber development gene expression study. For in vitro tuber development expression analysis, RNA was isolated from in vitro microtubers at 2, 5, 10, 20 and 30 days following observed tuber induction. Two microtuber populations were used as biological replicates for the developmental stages. The RNA from all developmental stages was pooled to generate the reference samples. Ten microarray hybridizations were performed. For in vivo tuber development expression analysis, RNA was isolated from tubers growing in growth chamber conditions. Tissues were divided into six group, according to developmental size: stolon (no tuber formation), 1-5 mm tubers, 6-10 mm tubers, 11-15 mm tubers, 16-25 mm tubers, and 26-35 mm tubers. Two biological replicates of ten plants each were grown sequentially in the same growth chamber. The RNA from all developmental stages was pooled to generate the reference samples. Twelve microarray hybridizations were performed. For all experiments, the RNA was labeled using the indirect labeling method with random hexamer primers. Amplified cRNA was used as labeling template for stolons. Total RNA was used as labeling template in all other labeling reactions.
Project description:The potato tuber mitochondrial proteome Three biological replicates, 40 gel sections each (120 files) MS raw files (.raw) and peptide search files [MASCOT (.msf), MSGFBD (.txt), PROLUCID (.sqt) and SEQUEST (_sequest.msf)] Mitochondria were isolated from dormant potato tubers (Solanum tuberosum) and proteins (0.25 mg) resolved by one-dimensional gel electrophoresis , sectioned into 40 equal segments, and tryptic peptides analyzed by liquid chromatography tandem mass spectrometry using an Orbitrap XL. Acquired MS/MS spectra were searched using different algorithms against the potato protein database (http://www.potatogenome.net). To estimate the protein false discovery rate (FDR), randomized sequences were combined with the forward database in a concatenate format. MASCOT server 2.3.01, SEQUEST, MS-GFDB and ProLuCID software were used to interpret the data set using very similar parameters. MASCOT and SEQUEST were used integrated within Proteome Discoverer 1.3 software package (Thermo Scientific). Raw files (Thermo) were converted to ms2 and mzXML files to run ProLuCID and MS-GFDB softwares. All search engines were configured to the following parameters: MW range between 200 and 2,000; 1,000 ppm of precursor ion tolerance; oxidation of methionine and asparagine deamidation as variable modifications and carbamidomethylation of cysteine as a static modification; trypsin; 2 allowed missed cleavages. After searches, the PSMs were filtered out at 5ppm of precursor ion tolerance achieving a false discovery rate (FDR) lower than 1% at the protein level for each biological replicate. Filtered data were uploaded on Protein Herder module within Compass package for protein grouping. At this step, all sets of indistinguishable proteins, which were assigned by the same peptides, are combined into protein groups.
Project description:Tuber bruising in tetraploid potatoes (Solanum tuberosum) is a trait of economical importance, as it affects tubers' fitness for sale. Understanding the genetic components affecting tuber bruising is a key step in developing potato lines with increased resistance to bruising. As the tetraploid setting renders genetic analyses more complex, there is still a lot to learn about this complex phenotype. Here, we used genotype by sequencing data on a panel of half-sibling populations from a breeding programme to perform a genome-wide association analysis for tuber bruising. In addition, we collected transcriptomics data to enrich the GWAS results, by performing a differential expression analysis between samples with high- and low-bruising susceptibility.
Project description:Purpose: MicroRNAs (miRNAs) are ubiquitous components of endogenous plant transcriptome. miRNAs are small, single-stranded and ~21 nt long RNAs which regulate gene expression at the post-transcriptional level and are known to play essential roles in various aspects of plant development and growth. Previously, a number of miRNAs have been identified in potato through in silico analysis and deep sequencing approach. However, identification of miRNAs through deep sequencing approach was limited to a few tissue types and developmental stages. This study reports the identification and characterization of potato miRNAs in three different vegetative tissues and four stages of tuber development by high throughput sequencing. Results: Small RNA libraries were constructed from leaf, stem, root and four early developmental stages of tuberization and subjected to deep sequencing, followed by bioinformatics analysis. A total of 89 conserved miRNAs (belonging to 33 families), 147 potato-specific miRNAs (with star sequence) and 112 candidate potato-specific miRNAs (without star sequence) were identified. The digital expression profiling based on TPM (Transcripts Per Million) and qRT-PCR analysis of conserved and potato-specific miRNAs revealed that some of the miRNAs showed tissue specific expression (leaf, stem and root) while a few demonstrated tuberization stage-specific expressions. Targets were predicted for identified conserved and potato-specific miRNAs, and predicted targets of four conserved miRNAs, miR160, miR164, miR172 and miR171, which are ARF16 (Auxin Response Factor 16), NAM (NO APICAL MERISTEM), RAP1 (Relative to APETALA2 1) and HAIRY MERISTEM (HAM) respectively, were experimentally validated using 5M-bM-^@M-2RLM-RACE (RNA ligase mediated rapid amplification of cDNA ends). Gene ontology (GO) analysis for potato-specific miRNAs was also performed to predict their potential biological functions. Conclusions: We report a comprehensive study of potato miRNAs at genome-wide level by high-throughput sequencing and demonstrate that these miRNAs have tissue and/or developmental stage specific expression profile. Also, predicted targets of conserved miRNAs were experimentally confirmed for the first time in potato. Our findings indicate the existence of extensive and complex small RNA population in this crop and suggest their important role in pathways involved in diverse biological processes, including tuber developmental process. Total seven (Leaf, Root, Stem, Potato Tuber stage 0(PT0),Potato Tuber stage 1(PT1),Potato Tuber stage 2(PT2),Potato Tuber stage 3(PT3) ) small RNA libraries were consctructed and sequenced by deep sequencing using Illumina GAIIx.
Project description:Phosphoproteome was detected by two types of high-throughput mass spectrum in potato tuber. All raw data were analysed by uniform program.