ABSTRACT: Transcription profiling by high throughput sequencing of stem differentiating xylem from Populus trichocarpa transgenics overexpressing PtrMIR397a
Project description:We present an efficient method to genome-wide discover new and drought stress responsive miRNAs in P. euphratica. High throughput sequencing of P. euphratica leaves found 197 conserved miRNAs between P. euphratica and Populus trichocarpa. Meanwhile, 189 new miRNAs which belonged to 120 families were identified, a large increasing to the number of P. euphratica miRNAs. Target prediction and degradome sequencing verification of 22 new and 21 conserved miRNA targets showed these targets were involved in multiple biological processes, including transcription regulation and response to stimulus. Furthermore, comparison of high-throughput sequencing with miRNA microarray profiling data indicated that 104 miRNA sequences were up-regulated, while 27 were down-regulated under drought stress. This preliminary characterization based on our findings provided a framework for future analysis of miRNA genes and their roles in key traits of poplar as stress resistance plant breeding and environment protection usage. Examination of sRNA expression in 2 poplar leaf samples in drought and normal growth conditions.
Project description:PtrHSFB3-1 and PtrMYB092 are xylem specific genes in xylem of P. trichocarpa, and their expression levels are down regulated most significantly in tension wood. These two transcription factors were transiently overexpressed in stem differentiating xylem (SDX) of P. trichocarpa , and transcriptomic sequencing was performed to identify the regulatory effects of the two transcription factors on wood formation related genes.
Project description:We present an efficient method to genome-wide discover new and drought stress responsive miRNAs in P. euphratica. High throughput sequencing of P. euphratica leaves found 197 conserved miRNAs between P. euphratica and Populus trichocarpa. Meanwhile, 189 new miRNAs which belonged to 120 families were identified, a large increasing to the number of P. euphratica miRNAs. Target prediction and degradome sequencing verification of 22 new and 21 conserved miRNA targets showed these targets were involved in multiple biological processes, including transcription regulation and response to stimulus. Furthermore, comparison of high-throughput sequencing with miRNA microarray profiling data indicated that 104 miRNA sequences were up-regulated, while 27 were down-regulated under drought stress. This preliminary characterization based on our findings provided a framework for future analysis of miRNA genes and their roles in key traits of poplar as stress resistance plant breeding and environment protection usage.
Project description:Global gene expression pattern of different poplar tissue types were determined using a Nimblegen microarray based on JGI v1.1 gene models. All tissue except reproductive tissue were obtained from the same clone used for the poplar genome sequencing project (Populus trichocarpa Nisqually-1). Reproductive tissue were from wild Populus trichocarpa trees.
Project description:The purpose of this study was to evaluate a set of 6-7 long oligonucleotide probes developed based on the sequence of the Populus trichocarpa genome, that are optimal for gene expression analysis of P. deltoides and a hybrid of P. deltoides and P. trichocarpa. To evaluate these probes, multiple tissues (differentiating xyle, leaf and whole-root) of a pure P. deltoides and a hybrid (P. deltoides X P. trichocarpa) were transcript profiled for identification of one or more probes that are not biased towards one or the other genotype.
Project description:Stem cuttings of P. trichocarpa (clone 101-74) were rooted in liquid medium without growth regulators (basal medium). The first emerging roots were observed on cuttings 6 days after the start of culture. The highest average root number per cutting (10 ± 2 roots/cutting) was obtained after 14 days. The first macroscopic evidence of root initiation was the appearance of root primordia, as lateral bulges observed at the stem surface 3 to 4 days after transfer to basal medium. Stem cross-sections showed intensely dividing cells forming root primordial. One to two days later the bark split and the organized sequence of cell division and differentiation steps in the primordium led to the establishment of the main root tissues, as well as the vascular connections of the incipient root with the pre-existing stem vasculature. Subsequently, the outgrowth and emergence of the adventitious root occurred. We refer to the dormant cutting as stage 0, the organizing primordium as stage 1, the primordium differentiation as stage 2. To examine changes in gene transcription associated with the development of adventitious roots, we monitored the transcript levels in differentiating primordia using microarrays. cDNA was prepared from replicate sets of P. trichocarpa rooted cuttings harvested at stages 0, 1 and 2. The Populus whole-genome expression array version 2.0 manufactured by NimbleGen Systems Limited (Madison, WI) contains in duplicates three independent, non-identical, 60-mer probes per whole gene model plus control probes and labeling controls. Included in the microarray are 65,965 probe sets corresponding to 55,970 gene models predicted on the P. trichocarpa genome sequence version 1.0 and 9,995 aspen cDNA sequences (Populus tremula, Populus tremuloides, and P. tremula x P. tremuloides). NimbleGen whole genome microarray analyses were performed in triplicate as per manufacturers instructions. We carried out nine hybridizations (NimbleGen) with samples derived from three early developmental stages of P. trichocarpa adventitious roots. cDNA was synthesized using CLONTECH Smart cDNA Synthesis kit containing an amplification step on the cDNA level. All samples were labeled with Cy3.
Project description:We transformed Populus trichocarpa and generated transgenics with knockdown or overexpression of monolignol genes and transcription factors
Project description:Global gene expression pattern of different poplar tissue types were determined using a Nimblegen microarray based on JGI v1.1 gene models. All tissue except reproductive tissue were obtained from the same clone used for the poplar genome sequencing project (Populus trichocarpa Nisqually-1). Reproductive tissue were from wild Populus trichocarpa trees. Replicates: Two biological replicates per tissue type were hybridized to two arrays
Project description:Illumina GAIIx technology was used to generate mRNA profiles from the ectomycorrhizal fungi Laccaria bicolor colonizing roots of Populus trichocarpa. Samples were taken after two, four and 12 weeks of contact in order to identify mycorrhiza-regulated transcripts. 37bp reads were generated and aligned to the Populus trichocarpa (http://www.phytozome.net/poplar.php) and the Laccaria bicolor (http://genome.jgi-psf.org/Lacbi2/Lacbi2.home.html) reference genomes using CLC Genomics Workbench 6. mRNA profiles from Populus trichocarpa roots colonized by Laccaria bicolor for two, four and 12 weeks as well as from control roots and free-living mycelium were generated by using one lane of 37 bp Illumina GAIIx sequencing per sample.