Development and Application of Expressed Sequence Tags (ESTs) for the Improvement of Cotton Crops.

Expressed sequence tags (ESTs), are short cDNA sequences obtained from expressed genes in any plant tissue of Gossypium spp. and represent a suitable approach for studying the transcriptome in the absence of a reference genome. In this study, protocols for EST development were established to explore expressed genes in cotton under diverse biotic and abiotic stress conditions. ESTs were generated by using two approaches: suppression subtractive hybridization (SSH) and next-generation sequencing (NGS) techniques. The process begins with RNA isolation, followed by mRNA purification, cDNA synthesis, and cloning, leading to the generation of single-pass EST libraries. High-quality ESTs were assembled into unigenes using complementary assembly strategies. Functional annotation of the resulting unigenes was carried out to enhance gene representation and to elucidate stress-responsive pathways. This approach supports key objectives in cotton breeding, including improvement of fiber quality and productivity, as well as enhanced resistance to biotic and abiotic stresses.