Assessing Varietal Heterogeneity in Pooled Samples of Cross-Pollinated Crops Using Genetic Markers

Under the regulatory framework of the International Union for the Protection of New Varieties of Plants (UPOV) for plant variety protection, a new variety must be assessed for uniformity, as well as distinctness and stability (DUS). For cross-pollinated crops such as perennial ryegrass (Lolium perenne) and lucerne (Medicago sativa), where cultivars are heterogeneous populations, assessment of uniformity poses particular challenges. These challenges stem from the fact that such evaluations can be costly and require substantial labour and time, as they typically involve the measurement of numerous comparator characteristics across multiple individual plants representative of a candidate cultivar, often over a period exceeding 1 year. Although DUS testing is based on phenotypic characteristics, genetic markers are currently permitted for use in planning of trials, and for distinctness assessment in particular situations. However, genetic markers have not yet been employed for distinctness assessment. Similarly, under OECD Seed Schemes, marketed seed is assessed for varietal purity through the observation of plants in field plots. This study proposes a new measure of genetic heterogeneity based on genetic markers, along with a method to estimate this in pooled samples to reduce costs. The method relies on a system for estimating allele frequencies at SNPs, such as genotyping-by-sequencing or sequence capture. We demonstrate the potential of this approach with example data for perennial ryegrass. The approximation based on pooled samples provided satisfactory estimates of genetic heterogeneity compared with direct assessment based on individual plants, and proved to be sensitive to contamination of samples by other varieties.