A High-throughput Sequencing Screen to Identify Apomixis in Plants

In the past decade, plant biologists have made several major discoveries pertaining to the genetic basis of apomixis (clonal propagation by seed) that have shown promise in preserving high-value hybrid rice and sorghum genotypes. This progress was made possible by foundational gene discovery efforts in model species and natural apomicts, but pleiotropic obstacles still limit its broad agricultural adoption, especially in eudicots. Thus, it follows that investigations of novel apomicts will lead to the development of new molecular tools for plant breeding. The three most common ways to identify clonal seed production are cytoembryological analyses, genome sequencing to compare the DNA sequence of mother and child, and flow-cytometry seed screens. While flow-cytometry has been the dominant method for more than two decades, it requires specialized equipment, expertise, and occasional troubleshooting. Here we developed a method using short-read skim-seq at moderately low sequencing depths (3X and 6X) to screen diverse Malus genotypes maintained in a USDA germplasm collection for clonal seed production. By sequencing 55 genotypes and 1,216 of their embryos, our screening-by-sequencing method found 17 previously undescribed apomictic genotypes. Five additional apomictic genotypes were detected with flow cytometry data and using internal sequencing controls. This skim-seq embryo screening-by-sequencing method is a relatively low-cost, rapid method for detecting apomictic genotypes in diverse plant germplasm, and when used thoughtfully in conjunction with flow cytometry, provides a new way to visualize the genetic outcomes of sexual and asexual reproduction in plants.