Evaluation and Refinement of Chinese DUS Test Guidelines Based on Comprehensive Phenotypic Traits: A Case Study of Hawthorn (Crataegus spp.)

Hawthorn (Crataegus spp.), a horticultural crop with significant edible, medicinal, and ornamental values, has garnered increasing attention from breeders to markets, leading to continuous emergence of new varieties. Accurate phenotypic characterization and robust Distinctness, Uniformity, and Stability (DUS) testing are fundamental for plant variety protection and breeding innovation. Employing the Chinese DUS Test Guidelines for Hawthorn (LY/T 3208-2020) as an analytical framework, this study systematically evaluated 197 germplasm accessions from the Chinese National Field Genebank. Results demonstrated: (1) Coefficients of Coincidence (COC) for example varieties and test traits ranged between 0.980 and 1.000, fully meeting the testing requirements; (2) the 22 qualitative (QL) and pseudo-qualitative traits (PQ) were categorized into 2–5 classes with Shannon-Wiener indices of 0.032–1.174, indicating high polymorphism; (3) correlation analysis of 11 quantitative traits (QN) revealed coefficients from 0.006 to 0.922, with 23 trait pairs showing statistical significance, while principal component analysis identified fruit and leaf traits as primary drivers of phenotypic variation; (4) grading schemes were established by using Least Significant Difference (LSD) for eight normally or near-normally distributed traits (5–7 classes) and range-based grading approach for three non-normal traits (5 classes). Based on the above results, this study evaluated China’s current DUS testing guidelines for hawthorn. Subsequently, 197 hawthorn accessions were used to evaluate the QL, PQ, and QN traits, based on which corresponding optimization recommendations were proposed. Ultimately, an optimized testing framework for hawthorn DUS testing was established. The resulting analytical insights and phenotypic grading system will enrich the current descriptor framework and provide a foundational dataset for trait-DNA association and marker-assisted breeding. Notably, this optimization strategy serves as a methodological template for updating DUS guidelines in other taxa. Overall, these insights strengthen the protection, development, and breeding of new hawthorn varieties while advancing the standardization of DUS testing.