Genome-wide transcriptome of rice laser-microdissected crown root primordia reveals genes expressed specifically during the early differentiation stages of crown roots

Crown roots constitute the main part of the rice root system. Several key genes involved in crown root initiation and development have been identified by genetics and functional genomics approaches. Nevertheless these approaches are impaired by gene function redundancy and mutant lethality. To overcome these limitations, genome-wide transcriptome analysis can help to identify genes involved in crown root formation and early development. In this study we aimed to identify the genes speciffically expressed in developing crown root primordia in comparison with adjacent cortex tissue of stem at three different developmental stages before emergence from the stem. For this, we used Laser Capture Microdissection to collect crown root primordia in the stem base of 8-day-old rice seedlings. Affymetrix microarrays were processed in the Microarray Core Facility “Transcriptome“ of the Institute in Regenerative Medicine and Biotherapy, CHU de Montpellier-INSERM-UM Montpellier, http://irmb.chu-montpellier.fr/ . Eight days after sowing, a total of six stem bases from WT cultivar Taichung 65 Oryza sativa, Japonica rice plantlets were fixed and cut into 15 µm sections.These sections were dissected on a LMD 7000 laser dissector (Leica Biosystems). CR primordia belonging to the three successive rings present in plantlets stem bases, corresponding to three primordia developmental stages, were collected and regrouped in three pools corresponding to the three developmental stages. Stem cortex tissues were sampled from the same sections where the CR primordia were collected from. Samples from two stem bases were pooled to perform RNA extractions from the three primordia developmental stages and cortex, that correspond to one independent repetition of the experiment. In total 3 x 2 stem bases were processed that corespond to three intependant repetition of the experiment. RNA was isolated using the Arcturus PicoPure RNA Isolation Kit (Thermo Fisher Scientific).