RNA-seq, miRNA-seq, and degradome-seq analysis of taproots and lateral roots of Quercus robur seedlings under well-watered and drought stress conditions [miRNA-seq]

Identification of genes involved in the regulation of taproot and lateral root growth in Quercus robur seedlings under drought stress and well-watered conditions. Genes involved in the regulation of taproot and lateral root growth in Quercus robur seedlings were identified using RNA-seq, miRNA-seq, and degradome-seq. The analysis focused on the gene expression, miRNA regulation, and mRNA degradation profiles of taproots and lateral roots under both drought stress and well-watered conditions. Key genes and their regulatory miRNAs were identified, along with the role of mRNA degradation pathways in response to stress, providing insights into the molecular mechanisms controlling root growth and development in oak seedlings under varying water availability. The study employed a two-factor completely randomized design comparing container-grown versus rhizotron-sown Quercus robur seedlings under two watering regimes (optimal irrigation vs. 75% water reduction for 4 weeks), with sampling of both taproot and lateral root meristematic zones following an 8-week acclimatization period in standardized growth conditions (peat:perlite 5:1 substrate with controlled fertilization in a greenhouse environment). All plants were monitored for soil moisture (at 15 cm and 45 cm depths) and temperature throughout the experiment, with physiological (chlorophyll fluorescence measurements of PSII efficiency parameters) and molecular (transcriptome/miRNA) endpoints collected concurrently after drought treatment confirmation. The design incorporated triplicate biological replicates (10-15 root tips per replicate) and technical replication in sequencing (minimum 20 million 150bp paired-end reads od 50bp single-end reads per sample on Illumina NovaSeq), enabling statistically powered comparisons (α=0.05, 80% power to detect 1.5-fold expression changes) between cultivation methods, watering treatments, and root types while controlling for potential environmental variability through automated irrigation systems and continuous environmental monitoring. Key features included synchronization of molecular and physiological measurements, standardized transplantation protocols for container-grown seedlings, and integrated bioinformatics pipelines for multi-omics data analysis, providing a comprehensive framework to evaluate both nursery practice legacy effects and organ-specific drought responses.