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 5′RLM-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.
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:The potato is susceptible to water stress at all stages of development. We examined four clones of tetraploid potato, Cardinal, Desirée, Clone 37 FB and Mije, from the germplasm bank of the National Institute of Agricultural Research (INIA) in Chile. Water stress was applied by suspending irrigation at the beginning of tuberization. Stomatal conductance, tuber and plant fresh and dry weight was used to categorize water stress tolerance. Cardinal had high susceptibility to water stress. Desirée was less suscepetible than Cardinal and had some characteristics of tolerance. Mije had moderate and Clon 37 FB high tolerance. Differential gene expression in leaves from plants with and without water stress were examined using transcriptome sequencing. Water stress susceptible Cardinal had the fewest differentially expressed genes at 101, compared to Desirée at 1867, Clon 37 FB at 1179 and Mije at 1010. Water stress tolerance was associated with up-regulation of expression of transcription factor genes and genes involved in osmolyte and polyamine biosynthesis. Increased expression of genes encoding late embryogenesis abundant (LEA) and dehydrin proteins along with decreased expression of genes involved in nitrate assimilation and amino acid metabolism were found for clones showing water stress tolerance. The results also show that water deficit was associated with reduced biotic stress responses. Additionally, heat shock protein genes were differentially expressed in all clones except for highly susceptible Cardinal. Together the gene expression study demonstrates variation in the molecular pathways and biological processes in response to water stress contributing to tolerance and susceptibility.
Project description:Nitrogen (N) fertilization is an important abiotic factor for the growth of potato (S. tuberosum) because of its potential effects on yield. Because excess N in the soil runs off into water systems and negatively impacts the environment, studies on N use by the plant are key to decrease N-fertilizer use. Three commercial potato cultivars (Shepody, Russet-Burbank and Atlantic) were grown under two different rates of applied N-fertilizer (0 kg N ha-1 and 180 kg N ha-1) to obtain more information on the underlying gene regulation mechanisms associated with N. Plants with no added N had significantly lower concentrations of petiole nitrates, chlorophyll level indices, biomass and yield per hectare. Total mRNA samples were taken at two different time-points during the growth season and used for sequencing. The results for each cultivar and time-point were analysed separately to find differentially expressed genes. In total, thirty genes were found to be over-expressed and nine genes were found to be under-expressed in plants from all potato cultivars when they were grown with added N-fertilizer. The 1000 bp upstream flanking regions of the differentially expressed genes were analysed to find overrepresented motifs using three motif discovery algorithms (Seeder, Weeder and MEME). Nine different motifs were found, indicating potential gene regulatory mechanisms for potato under N-deficiency.