Assessment of Cultivar Distinctness in Alfalfa: A Comparison of Genotyping-by-Sequencing, Simple-Sequence Repeat Marker, and Morphophysiological Observations.

Cultivar registration agencies typically require morphophysiological trait-based distinctness of candidate cultivars. This requirement is difficult to achieve for cultivars of major perennial forages because of their genetic structure and ever-increasing number of registered material, leading to possible rejection of agronomically valuable cultivars. This study aimed to explore the value of molecular markers applied to replicated bulked plants (three bulks of 100 independent plants each per cultivar) to assess alfalfa ( L. subsp. ) cultivar distinctness. We compared genotyping-by-sequencing information based on 2902 polymorphic single-nucleotide polymorphism (SNP) markers (>30 reads per DNA sample) with morphophysiological information based on 11 traits and with simple-sequence repeat (SSR) marker information from 41 polymorphic markers for their ability to distinguish 11 alfalfa landraces representative of the germplasm from northern Italy. Three molecular criteria, one based on cultivar differences for individual SSR bands and two based on overall SNP marker variation assessed either by statistically significant cultivar differences on principal component axes or discriminant analysis, distinctly outperformed the morphophysiological criterion. Combining the morphophysiological criterion with either molecular marker method increased discrimination among cultivars, since morphophysiological diversity was unrelated to SSR marker-based diversity ( = 0.04) and poorly related to SNP marker-based diversity ( = 0.23, < 0.15). The criterion based on statistically significant SNP allele frequency differences was less discriminating than morphophysiological variation. Marker-based distinctness, which can be assessed at low cost and without interactions with testing conditions, could validly substitute for (or complement) morphophysiological distinctness in alfalfa cultivar registration schemes. It also has interest in sui generis registration systems aimed at marketing alfalfa landraces.