Development of a low-coverage whole genome sequencing screen for apomixis using a diverse set of Malus germplasm

Abstract

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 should lead to the development of new molecular tools for plant breeding. The two most common ways to identify clonal seed production are flow-cytometry seed screens and genome sequencing to compare the DNA sequences of the maternal parent and progeny, traditionally using low-throughput markers. While flow-cytometry has been the dominant method for more than two decades, it provides indirect information on the genetics of a resulting embryo and can be ineffective in certain species. Here we developed a method using short-read whole-genome sequencing at moderately low coverage (averaging 3X and 6X) to screen diverse Malus genotypes maintained in a USDA germplasm collection for clonal seed production. In total, we sequenced 55 genotypes, 1,216 of their embryos, and identified 17 previously undescribed apomictic genotypes. Several more were detected with the flow cytometry seed screen, which helped resolve certain types of reproduction and sources of noise in low-coverage datasets. This low-pass 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. Author summary: The most established method to determine whether a plant produces clonal seed is the flow cytometry seed screen. However, this method cannot be applied to some species and circumstances and does not directly test for clonality. Therefore, we explored the utility of a high throughput, low-pass whole genome sequencing screen at variable levels of coverage to directly test whether Malus embryos are clones of their maternal genotype. We present a simple way to visualize the outcomes of plant reproduction with low-pass sequencing data, consider the ways in which flow cytometry and sequencing complement one another, and discuss how advancing technologies will enable sequencing to become a more routine method to study reproductive plant biology.

Original publication: PLOS Genetics (2026-09-08). Source. Source DOI: 10.1371/journal.pgen.1012289.