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Timeseries RNA-Seq analysis of low phosphorus-induced adventitious root formation in Populus ussuriensis

ABSTRACT: For RNA-seq, new cuttings from 3-week-old in vivo plants were cultured on the solid Woody Plant Medium (WPM) containing 20 g/L sucrose and 3 g/L Gelrite (Duchefa). After 5 days of culture, the root primordium formed cuttings were transferred to modified liquid WPM containing 10 μM or 200 μM KH2PO4, and harvested the basal ends of stem cuttings containing root primordium with its surrounding tissues using 11# scalpel after 2h, 4h, 8h, 12h and 24h culture. Samples were collected from two biological replicates (50 cuttings each).

ORGANISM(S): Populus ussuriensis

PROVIDER: GSE174726 | GEO | 2022/08/16

REPOSITORIES: GEO

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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 Â± 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:Purpose: The goals of this study was to obtain transcriptome data from five developmental stages of adventitious roots of Pyrus ussuriensis Maxim using transcriptome analysis (RNA-Seq) and qRT-PCR analysis. Mathods: The mRNA profiles of the adventitious root development of Pyrus ussuriensis on day 0, day 3, day 6, day 9, and day 12 were examined by deep sequencing, in triplicate, using Illumina HiSeq 2000. Ultra™ RNA LibraryPrep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and indexcodes were added to attribute sequences to each sample. qRT–PCR validation was performed using TaqMan and SYBR Green assays. Results: The transcriptome regulation analysis of the Pyrus ussuriensis adventitious root formation was performed on this study. And a total of 19,470 DEGs (Differentially Expressed Gene), which were mainly enriched in hormone synthesis and signaling pathways, energy metabolism, synthesis and metabolism of secondary products, were detected. the key regulatory genes of adventitious root formation were screened, mainly including the gene family WOX, LBD and SRS. Auxin, plays a key role in the induction and development of root primordium, its signal transduction pathway-related genes were up-regulated during the induction period of root primordium, and down-regulated during the development of root primordia. Carotenoids is the precursor substance of abscisic acid synthetize. And for the reasons that the genes of carotenoid synthesis and signal transduction pathway of abscisic acid were down-regulated during the period of stable development and the root primordium induction, and up-regulated during the period of root primordium development of adventitious root, it was supposed that abscisic acid was a control substance, that regulated the metabolic network of development of adventitious roots. Conclusions: Our results provide a comprehensive high-resolution characterization of gene expression profiles during the pear root transition process.A number of DEGs were detected from the root primordium to adventitious root growth stages, and these belonged to hormone metabolism pathways.These results provide a valuable resource for studies in other closely related species with similar agricultural and productive value. The differentially expressed genes dataset will also provide useful candidate genes for functional analysis of pear rooting.

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Timeseries RNA-Seq analysis of low phosphorus-induced adventitious root formation in Populus ussuriensis

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