Gene Discovery in Reproduction: RNA-seq Analysis of Isolated Pistils from Apomictic and Sexual Eragrostis curvula

This protocol describes a comprehensive approach to analyze the differential expression of genes in pistils at distinct developmental stages from sexual and apomictic genotypes of Eragrostis curvula. It integrates the development of reproductive calendars, methods for pistil isolation, and techniques for extracting high-quality RNA from limited tissue quantities. This protocol includes detailed steps for inflorescence collection, measurements and clarification of reproductive tissues, as well as the use of differential interference contrast (DIC) microscopy to ensure precise identification of developmental stages. Additionally, the methodology outlines the bioinformatics pipelines used to analyze gene expression patterns, providing insights into the molecular mechanisms underlying apomixis. This approach addresses key challenges, such as small tissue size and mixed developmental stages, offering a reliable framework for transcriptomic studies in E. curvula.