Transcriptome Analysis in Cork Oak Using Laser Microdissection and RNA-Seq

Cork from Quercus suber L. is a non-wood forest product with high economic and commercial value, as well social and ecological role. In Portugal, cork represents 33% of all national forestry products placing Portugal as the world leader in cork production, industrial processing, and trade of cork. The impermeability, fire retardancy, and sound insulation properties of cork make it the optimum material for a variety of applications, such as wine bottles stoppers, insulation corkboard, shoe soles, and others fashion purposes [1, 2].

Laser microdissection microscopy (LM) combined with RNA-sequencing are powerful techniques for investigating the transcriptome profile of specific tissues or cell types at a cellular level [3].

In order to identify specific candidate genes linked to secondary growth, a transcriptomic analysis of single-cells isolation of cork oak, by LM technology was performed. Thus, an optimized protocol for single-cell isolation by LM in suber-phellogen and xylem was successfully obtained, followed by RNA isolation and cDNA libraries preparation and RNA sequencing. High-quality reads (MAPQ>20) and a minimum size of 130 bp were obtained, followed by alignment and mapping against the cork oak genome [4]. About 25 to 30 million reads were uniquely mapped to the cork oak genome sequence. The mapping results suggest that single-cell isolation, RNA extraction, and sequencing of Illumina libraries procedures were viable for transcriptomic studies of cork oak tissues. The scRNA-Seq will allow gene expression analysis in individual tissues of oaks, contributing to understand the molecular mechanisms associated with the development processes of secondary growth.

References:

1. Pereira, H. (2007). Cork - Biology, Production and Uses. Elsevier. https://doi.org/https://doi.org/10.1016/B978-0-444-52967-1.X5000-6
2. Graça, J. (2015). Suberin: The biopolyester at the frontier of plants. Frontiers in Chemistry, 3(OCT), 1–11. https://doi.org/10.3389/fchem.2015.00062
3. Gautam, V., & Sarkar, A. K. (2015). Laser Assisted Microdissection, an Efficient Technique to Understand Tissue Specific Gene Expression Patterns and Functional Genomics in Plants. Molecular Biotechnology, 57(4), 299–308. https://doi.org/10.1007/s12033-014-9824-3
4. Ramos, A. M., Usié, A., Barbosa, P., Barros, P. M., Capote, T., Chaves, I., … Gonçalves, S. (2018). The draft genome sequence of cork oak. Scientific Data, 5, 1–12. https://doi.org/10.1038/sdata.2018.69

Acknowledgments: This work was supported by Alentejo2020, through FEDER under Lentidev- “A molecular approach to cork porosity” project (ALT20-03-0145-FEDER-000020). Authors also acknowledge FCT for Contrato – Programa to L. Marum (CEECINST/00131/2018) and FCT for UIDB/05183/2020.