Project description:The aim of the present work is to apply microarrays to identify candidate genes associated with the strategies adopted by different Solanum wild species to maintain their metabolism and limit the harm under water stress conditions. Wild genotypes are a rich source of novel genes for introduction into cultivated species to enhance stress tolerance. The mechanisms in which genes act can involve different pathways inside the cell, resulting in the expression of stress responsive genes. We will identify genes whose expression change in the leaves of potato plants subjected to drought stress. We will analyze different Solanum genotypes in six moments of water stress (0,1,3,5,7 & 10 days after initiation of stress). We will selected 4 time-points (days 0,5,7 & 10) and 2 genotypes (with divergence behavior) for microarrays analysis. The candidate genes obtained with this approach will be validated by semi-quantitative northern blot analysis (Typhoon scanner) and/or RT-PCR in each time-point of treatment and with all wild genotypes in study. This will help us to get a better understanding respect the dynamics of expression of a great number of genes in response to stress, as well as to establish functional relations between the involved genes. Research Plan: 5-weeks-old Solanum wild species plants were randomly sorted in 2 groups (25 plants per group). One group was submitted to non-irrigated conditions, withholding water and the other was normally watered. For microarray analysis, leaflets tissue from 4 plants per group of 4 time-points (days 0,5,7, & 10) were pooled, frozen in liquid nitrogen and stored at –80°C.RNA of the pool was extracted following TRIZOL method (Invitrogen). RNA integrity was verified on agarose gel. Hybridizations were performed with n genotypes,t time-points and r repeats. The hybridizations proposed in this experiment to enhance statistical robustness of data will be: 32 slides = n*t*r ; (2 genotypes; 4 time-points; 4 repeats (dye-swap). Keywords: Direct comparison
Project description:For expression profiling analyses of early stages of tuber induction, plants of Solanum tuberosum ssp andigena (7540) were used. This wild subspecies is strictly dependent on photoperiod for tuberisation, such that short days (SD) inductive conditions are required in order to trigger tuber induction in the stolons. Andigena plants were grown in the greenhouse under LD non-inductive conditions until a 10-leaf stage. They were subsequently transferred to inductive SD conditions (8 h light/16 h dark), and sampled at 0, 2, 4, 6 and 8 days after transfer to SDs. Tuber swelling was visible approximately 6-8 days after transfer to inductive conditions. The apical region of the stolons (2 cm) was collected one hour before the beginning of the light period.
Project description:Potato wild relatives (Solanum section Petota) are a source of genetic diversity for improving traits in modern cultivars (S. tuberosum) to meet climate challenges. Potatoes are susceptible to multiple abiotic and biotic stresses and have undergone constant improvement through breeding programs worldwide. The allotetraploid S. acaule Bitter has been used to introgress cold tolerance into potato breeding germplasm. The cold challenged transcriptome of S. acaule was compared with that of autotetraploid S. tuberosum cv. Atlantic, and was found to have fewer differentially expressed genes than the latter. Specifically, subgenome 1 has less downregulated alleles compared to subgenome 2 and S. tuberosum.
Project description:Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from Solanum tuberosum tissues (including leaves, flowers and stolon). The high depth of the resulting datasets enabled us to examine in detail critical small RNA features as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the genome under study.
Project description:The intent was to study, from transcriptome analysis, shade and drought responses in Solanum tuberosum (potato). We performed Illumina 50 bp single-end RNA-seq in tissues of control and treated var. Spunta wild-type plants. Drought experiments also included two independent AtBBX21-overexpressing (BBX21-OE) potato lines.
Project description:St (common potato) is a freezing sensitive species unable to cold acclimate. The close wild relative Sc is freezing tolerant and able to cold acclimate. Here we compare the cold transcriptome of these two species with different levels of freezing tolerance. We also identify the putative CBF regulons by comparing the transcriptomes of wild type plants with that of 35S::AtCBF3 transgenic lines in both species.
Project description:For expression profiling analyses of early stages of tuber induction, plants of Solanum tuberosum ssp andigena (7540) were used. This wild subspecies is strictly dependent on photoperiod for tuberisation, such that short days (SD) inductive conditions are required in order to trigger tuber induction in the stolons. Andigena plants were grown in the greenhouse under LD non-inductive conditions until a 10-leaf stage. They were subsequently transferred to inductive SD conditions (8 h light/16 h dark), and sampled at 0, 2, 4, 6 and 8 days after transfer to SDs. Tuber swelling was visible approximately 6-8 days after transfer to inductive conditions. The apical region of the stolons (2 cm) was collected one hour before the beginning of the light period. All collected stolons (LD; and 2d, 4d, 6d and 8d under SD) were processed as described. Hybridization scheme with a loop design was applied to compare one sample against the rest of the samples in the induction time course.
Project description:Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from Solanum tuberosum tissues (including leaves, flowers and stolon). The high depth of the resulting datasets enabled us to examine in detail critical small RNA features as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the genome under study. Small RNA libraries were derived from leaves, flowers and stolon of Solanum tuberosum. Total RNA was isolated using the Plant RNA Purification Reagent (Invitrogen), and submitted to Illumina (Hayward, CA, http://www.illumina.com) for small RNA library construction using approaches described in (Lu et al., 2007) with minor modifications. The small RNA libraries were sequenced with the Sequencing-By-Synthesis (SBS) technology by Illumina. PERL scripts were designed to remove the adapter sequences and determine the abundance of each distinct small RNA. We thank Barbara Baker for providing the plant material as well as Kan Nobuta and Gayathri Mahalingam for assistance with the computational methods.
Project description:St (common potato) is a freezing sensitive species unable to cold acclimate. The close wild relative Sc is freezing tolerant and able to cold acclimate. Here we compare the cold transcriptome of these two species with different levels of freezing tolerance. We also identify the putative CBF regulons by comparing the transcriptomes of wild type plants with that of 35S::AtCBF3 transgenic lines in both species. Plants were grown in 16:8 photoperiod. Eight hours after dawn, plants were either transfered to cold or kept in the warn. Wild type S. tuberosum and S. commersonii were grown at 2oC for 2h, 24h and 7 days. Wild type plants grown under warm temperatures for 2h was used as control for 2h cold samples; wild type warm grown plants for 24h were used as controls for 24h and 7 days cold samples. Under warm conditions, S. commersonii 35S::AtCBF3 lines were compared to S. commersonii wild type plants (same thing was done for S. tuberosum).