Development of EST-SSR molecular markers in rice (Oryza sativa L.) under salinity stress and identification of key genes

Expressed sequence tags of simple sequence repeats (EST-SSRs) are used to investigate genetic diversity and develop molecular markers for plants under biotic and abiotic stresses. However, there are a limited number of molecular markers based on the ESTs in rice to cope with the abiotic stresses including salinity, for use in breeding programs. Among 8299 ESTs, available in the NCBI database for salinity stress, 525 contigs, and 1139 singleton sequences were obtained. Twenty EST-SSR markers could be introduced for the selection of tolerant varieties to salt stress in rice by analysis of contigs and singletons. In contigs and singletons, significant common gene ontology terms are mainly related to the single-organism cellular process and response to abiotic stresses. The highest percentage of transcription factors for contigs and singletons was related to ERF, Dof, MYB, C2H2, BBR-BPC, bZIP, and WRKY. Moreover, the HSP81-2 (heat shock protein 81-2) and regulators of complement activation were identified as proteins of hub genes that were related to the salt stress tolerance mechanisms. Three uncharacterized hub genes (OS02T0161900-01, OsJ_19443, and OsJ_04035) including EST-SSRs for function identification were investigated by the 3D protein structure homology-modeling. OS02T0161900-01 as tetra ubiquitin, OSj-19443 as a serine/threonine-protein kinase/endoribonuclease, and OSj-04035 as a triosephosphate isomerase were identified. Most of the hub genes were related to environmental stresses and our findings provided candidate genes and transcription factors involved in salinity stress. The development of functional markers associated with abiotic stress tolerance will be helpful to facilitate rice breeding programs. However, before using these markers, laboratory confirmation is necessary.