Duckweed Species Genotyping and Interspecific Hybrid Discovery by Tubulin-Based Polymorphism Fingerprinting

Duckweeds (Lemnaceae) are the smallest and fastest-growing angiosperms. Thisfeature, together with high starch production and good nutritional properties, makesthem suitable for several applications, including wastewater treatment, bioenergyproduction, or feed and food supplement. Due to their reduced morphology andgreat similarity between diverse species, taxonomic identification of duckweeds isa challenging issue even for experts. Among molecular genotyping methods, DNAbarcoding is the most useful tool for species identification without a need for clusteranalysis. The combination of two plastid barcoding loci is now considered the goldstandard for duckweed classification. However, not all species can be defined withconfidence by these markers, and a fast identification method able to solve doubtfulcases is missing. Here we show the potential of tubulin-based polymorphism (TBP),a molecular marker based on the intron length polymorphisms of b-tubulin loci, in thegenomic profiling of the genera Spirodela, Landoltia, and Lemna. Ninety-four cloneswere analyzed, including at least two representatives of each species of the threegenera, with a special focus on the very heterogeneous species Lemna minor. Weshowed that a single PCR amplification with universal primers, followed by agarose gelanalysis, was able to provide distinctive fingerprinting profiles for 10 out of 15 species.Cluster analysis of capillary electrophoresis-TBP data provided good separation for theremaining species, although the relationship between L. minor and Lemna japonica wasnot fully resolved. However, an accurate comparison of TBP profiles provided evidencefor the unexpected existence of intraspecific hybrids between Lemna turionifera andL. minor, as further confirmed by amplified fragment length polymorphism and sequenceanalysis of a specific b-tubulin locus. Such hybrids could possibly correspond toL. japonica, as originally suggested by E. Landolt. The discovery of interspecific hybridsopens a new perspective to understand the speciation mechanisms in the familyof duckweeds.