Unknown,Transcriptomics,Genomics,Proteomics

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Microarray analysis of Bay-0 x Sha recombinant inbred lines at four seed germination stages

ABSTRACT: Seed germination is characterized by a constant change of gene expression across different time points. These changes are related to specific processes, which eventually determine the onset of seed germination. To get a better understanding on the regulation of gene expression during seed germination, we measured gene expression levels of Arabidopsis thaliana Bay x Sha recombinant inbred lines (RILs) at four important seed germination stages (primary dormant, after-ripened, six-hour after imbibition, and radicle protrusion stage) using. We mapped the eQTL of the gene expression and the result displayed the distinctness of the eQTL landscape for each stage. We found several eQTL hotspots across stages associated with the regulation of expression of a large number of genes. Together, we have revealed that the genetic regulation of gene expression is dynamic along the course of seed germination.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Margi Hartanto

PROVIDER: E-MTAB-9080 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:To address the lack of information on the function of CBC in the ABA signaling pathway during seed germination in barley, we employed TILLING to identify mutants of the genes hvcbp20.ab and hvcbp80.b and subsequently created the double mutant hvcbp20.ab/hvcbp80.b. We found that mutations in both CBC subunits resulted in a unique ABA response that differed from that of single mutants. Through transcriptomic analyses, we identified differential gene expression and alternative splicing patterns. Notably, the hvcbp20.ab/hvcbp80.b double mutant exhibited altered AS regulation and significant changes in brassinosteroid signaling following ABA exposure. Seeds were sterilized in 20% sodium hypochlorite solution for 20 min and washed with sterilized water for 5 min. They were then placed in Petri dishes with three layers of filters and treated with either sterilized water or sterilized water containing 75 μM ABA (cis-trans-abscisic acid; catalog no. 862169; Sigma-Aldrich). The seeds were stratified for 4 d at 4°C and then transferred to a growth chamber. Germination was defined as the visible emergence of the radicle through the seed coat and was assessed on 1 and 7 DAI (Days After Imbibition). The assay was performed in three biological replicates, each comprising 30 seeds of each genotype per petri dish. At 1 DAI, the embryos were isolated from the endosperm using a sharp scalpel blade, placed in microcentrifuge tubes (Eppendorf) containing RNAlater reagent, and stored at 4°C until RNA isolation. RNA was isolated using the mirVana™ Isolation Kit (Ambion, USA) following the manufacturer’s instructions. RNA was isolated in four biological replicates, each consisting of 20 embryos. In total, 32 RNA samples were extracted. RNA concentration and quality were assessed using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, USA) and Agilent Bioanalyzer (Agilent Technologies, Santa Clara, USA). We used grains and embryos of barley TILLING mutants: the hvcbp20.ab, in the CBP20 gene (CAP-BINDING PROTEIN 20; BaRT2v18chr6HG306340; 41,42), and hvcbp80.b, in the CBP80 gene (CAP-BINDING PROTEIN 80; BaRT2v18chr4HG195950), single mutants, the hvcbp20.ab/hvcbp80.b double mutant, and the 'Sebastian' wild-type (WT), all sourced from the HorTILLUS population 91. Single mutants were identified through TILLING in the M2 generation (then backcrossed with WT to clean the genetic background), and the double mutant was isolated from the F2 progeny following crossbreeding of the single mutants

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Microarray analysis of Bay-0 x Sha recombinant inbred lines at four seed germination stages

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