A sequencing-free assay for foliose Ulva species identification, hybrid detection and bulk biomass characterisation

Sea Lettuce (Ulva spp. Ulvophyceae, Ulvaceae) has tremendous ecological and industrial impacts, from the negative effects of green tide events to the industrial production of food, feed, and value-added products. Due to the morphological similarities between Ulva species, their identification requires the use of “barcoding”, which relies on the sequencing of short fragments of DNA and the comparison of the obtained sequence to that of sequences present in public repositories. However, Sanger sequencing can be costly when hundreds of samples need to be analysed. In addition, “barcoding”, which uses Sanger sequencing, cannot be applied directly on bulk biomass, and requires independent assessment of the individuals within, which is often not possible in commercial products. Here, we describe a novel “sequencing-free” method for species identification of foliose Ulva species that by-passes the drawbacks associated with Sanger sequencing. The assay uses restriction digestion enzymes which target species-specific Single Nucleotide Polymorphisms (SNPs) present within the ITS1 sequence. Digestion of the ITS1 PCR product with two enzyme mixtures allows for the discrimination of the main foliose Ulva species, as well as U. compressa, which can have a foliose morphotype. Of the species tested, only U. pseudorotundata and U. arasakii show the same digestion pattern. Since those two Ulva species are allopatric, we expect this issue to be of limited impact for the users. In addition to species identification, we demonstrate that the assay can be used for hybrid detection, which can have interests for Ulva breeding and species delimitation. Importantly, the assay can be used to detect at once the different Ulva species present within a bulk of Ulva biomass, which could allow for traceability and characterisation of the purity of Ulva commercial products. This study provides a new quick and cost-effective method for foliose Ulva species identification that could readily be extended to other species.