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

Duckweeds (Lemnaceae) are the smallest and fastest-growing angiosperms. This
feature, together with high starch production and good nutritional properties, makes
them suitable for several applications, including wastewater treatment, bioenergy
production, or feed and food supplement. Due to their reduced morphology and
great similarity between diverse species, taxonomic identification of duckweeds is
a challenging issue even for experts. Among molecular genotyping methods, DNA
barcoding is the most useful tool for species identification without a need for cluster
analysis. The combination of two plastid barcoding loci is now considered the gold
standard for duckweed classification. However, not all species can be defined with
confidence by these markers, and a fast identification method able to solve doubtful
cases 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 the
genomic profiling of the genera Spirodela, Landoltia, and Lemna. Ninety-four clones
were analyzed, including at least two representatives of each species of the three
genera, with a special focus on the very heterogeneous species Lemna minor. We
showed that a single PCR amplification with universal primers, followed by agarose gel
analysis, was able to provide distinctive fingerprinting profiles for 10 out of 15 species.
Cluster analysis of capillary electrophoresis-TBP data provided good separation for the
remaining species, although the relationship between L. minor and Lemna japonica was
not fully resolved. However, an accurate comparison of TBP profiles provided evidence
for the unexpected existence of intraspecific hybrids between Lemna turionifera and
L. minor, as further confirmed by amplified fragment length polymorphism and sequence
analysis of a specific b-tubulin locus. Such hybrids could possibly correspond to
L. japonica, as originally suggested by E. Landolt. The discovery of interspecific hybrids
opens a new perspective to understand the speciation mechanisms in the family
of duckweeds.