On the applicability of the Tubulin-Based Polymorphism (TBP) genotyping method: a comprehensive guide illustrated through the application on different genetic resources in the legume family

Abstract

Background: Plant discrimination is of relevance for taxonomic, evolutionary, breeding and nutritional studies. To this purpose, evidence is reported to demonstrate TBP (Tubulin-Based-Polymorphism) as a DNA-based method suitable for assessing plant diversity. Results: Exploiting one of the most valuable features of TBP, that is the convenient and immediate application of the assay to groups of individuals that may belong to different taxa, we show that the TBP method can successfully discriminate different agricultural species and their crop wild relatives within the Papilionoideae subfamily. Detection of intraspecific variability is demonstrated by the genotyping of 27 different accessions of Phaseolus vulgaris. Conclusions: These data illustrate TBP as a useful and versatile tool for plant genotyping. Since its potential has not yet been fully appreciated by the scientific community, we carefully report all the experimental details of a successful TBP protocol, while describing different applications, so that the method can be replicated in other laboratories.

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

“…Thirty nanograms of genomic DNA was used as a template for each TBP 1st and 2nd intron amplifications. The PCR reaction was performed in 30 μl according to Braglia et al (2020) , and control reactions without DNA template were included in any experiment. Each DNA sample was tested twice.…”

“…The capillary electrophoresis–TBP (CE-TBP) separation samples were prepared using 2 μl of each diluted PCR product. Capillary electrophoresis and data collection were performed according to the parameters defined by Braglia et al (2020) using the Gene Mapper Software v. 5.0 tools (Thermo Fisher Scientific, Inc., Waltham, MA, United States) independently for the two intron regions (1st and 2nd). Raw numerical data concerning the size (in base pairs) and the height (in relative florescence units, RFUs) of each CE-TBP profile peak were converted into Microsoft Office Excel files.…”

New Insights into Interspecific Hybridization in Lemna L. Sect. Lemna (Lemnaceae Martinov)

“…To this end, TBP fingerprinting has been particularly suitable for species delimitation in the genus [20]. This genetic profiling method had been successfully applied to species and subspecies level discrimination in different plant families [28][29][30]. The Landolt Duckweed Collection (LDC, Rotterdam, The Netherlands, http://www.duckweed.ch (accessed on 8 June 2021)) is perhaps the most important historical collection worldwide, with over 500 clones left of the more than 1000 collected and morphologically classified over 70 years by Professor Elias Landolt in Zürich, CH.…”

Strategies for Intraspecific Genotyping of Duckweed: Comparison of Five Orthogonal Methods Applied to the Giant Duckweed Spirodela polyrhiza

“…Overall, 40 polymorphic characters were observed, with 17 based on FLPs and 23 based on SNPs (Table 1). The ambiguous, i.e., non-homozygous differences amounted Here, we used the same clones as those studied by [23] for comparative studies using four additional orthogonal methods: fragment length polymorphisms (FLPs) and/or singlenucleotide polymorphisms (SNPs), tubulin-gene-based polymorphism (TBP; [26]), simple sequence repeat variants (SSR; [27]), multiplexed ISSR genotyping by sequencing (MIGseq; [28]), and genotyping by genome-wide sequencing (GBS; [7]). In all five cases, we calculated dissimilarity trees to evaluate the different methods in terms of their genotyping efficacy and considered the advantages and limitations of each.…”

“…Both the TBP 1st and 2nd introns were scored. Further technical details about the data acquisition and analysis are reported in [26]. The sample 7498 was run in duplicate using the same DNA extract for the error estimation.…”