Sequence-guided approach to genotyping plant clones and species using polymorphic NB-ARC-related genes

Abstract

Leveraging the heightened levels of polymorphism in NB-ARC-related protein encoding genes in higher plants, a bioinformatic pipeline was created to identify regions in this gene family from sequenced plant genomes that exhibit fragment length or single nucleotide differences in different accessions of the same species. Testing this approach with the aquatic plant Spirodela polyrhiza demonstrated its superior performance in comparison with currently available genotyping technologies based on PCR amplification. Rapid and economical genotyping tools that can reliably distinguish species and intraspecific variations in plants can be powerful tools for biogeographical and ecological studies. Clones of the cosmopolitan duckweed species, Spirodela polyrhiza, are difficult to distinguish morphologically due to their highly abbreviated architecture and inherently low levels of sequence variation. The use of plastidic markers and generic Amplification Fragment Length Polymorphism approaches have met with limited success in resolving clones of S. polyrhiza from diverse geographical locales. Using whole genome sequencing data from nine S. polyrhiza clones as a training set, we created an informatic pipeline to identify and rank polymorphic regions from nuclear-encoded NB-ARC-related genes to design markers for PCR, Sanger sequencing (barcoding), and fragment length analysis. With seven primer sets, we found 21 unique fingerprints from a set of 23 S. polyrhiza clones. However, three of these clones share the same fingerprint and are indistinguishable by these markers. These primer sets can also be used as interspecific barcoding tools to rapidly resolve S. polyrhiza from the closely related S. intermedia species without the need for DNA sequencing. Our work demonstrates a general approach of using hyper-polymorphic loci within genomes as a resource to produce facile tools that can have high resolving power for genotyping applications.

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

“…However, most of the tested molecular markers seem to fail in providing an effective distinction at the clone level (Bog et al, 2019). One possible method capable of addressing this issue was recently presented by Chu et al (2018), using the highly polymorphic regions of NBARC-related genes (nuclearbinding leucine-rich repeat protein) as markers. These authors were successful even in the case of S. polyrhiza, a species with very low genetic variations.…”

Duckweed (Lemnaceae): Its Molecular Taxonomy

“…22%, and the second identified 19 out of 68 clones (28%). An alternative method was recently presented by Chu et al (2018) using the highly polymorphic regions of NB-ARC-related genes (nuclear-binding leucine-rich repeat protein) as markers in Spirodela polyrhiza. These authors could "uniquely" identify 20 out of 23 clones of Spirodela polyrhiza (ca.…”

Studying Whole-Genome Duplication Using Experimental Evolution of Spirodela polyrhiza

“…Alternatively, strains can easily be isolated from the wild. Because of the similarity with S. intermedia (see above), the morphological identification should always be verified using molecular techniques such as barcoding [22,23] or PCR-based methods such as amplified fragment length polymorphism (AFLP) fingerprinting [23], tubulin-based polymorphism (TBP) fingerprinting [24], polymorphic NB-ARC-related genes [25], or microsatellite arrays [26]. Axenicity of fronds isolated from nature can be achieved by submerging the plants for thirty seconds to three minutes in a 10-20% bleach solution with agitation and subsequently washing the fronds twice in demi-water to remove all traces of bleach.…”