High-Throughput Sequencing
High-Throughput Sequencing (HTS) has enabled sequencing of thousands to
millions of sequences at one time and has changed our view on Earth’s
biodiversity at all organismal levels (Deiner et al. 2017). Because of
constantly declining costs, HTS technologies put forth new tools to
address questions previously tackled through labor-intensive (and often
less efficient) cloning steps, and amplicon sequencing of loci with high
information content (target sequencing) is probably the most
straightforward application (Ekblom and Galindo 2010). A further key
contribution of HTS lies in the possibility of exploring datasets that
cover wide taxonomic and/or geographic breadths. Good inter- and
intra-specific taxon sampling is usually required to address the
processes underlying speciation, diversification, distribution and
species assembly, especially when taxonomic uncertainties and high
diversity are involved. Hence, the utilization of HTS is especially
cost-effective as many individuals can be combined (multiplexed) in the
same sequencing run and rare variants can be readily detected (Babik et
al. 2009; Glenn 2011).
Studies characterizing the abundance and patterns of intragenomic nrDNA
polymorphisms in different organisms are increasing (Stage and Eickbush
2007; Ganley and Kobayashi 2007; Bik et al. 2013; Straub et al. 2012;
Mahelka et al. 2013; Wang et al. 2016; Symonová 2019). Determination of
the full sequence of the 35S cistrons was successful in different plant
groups (e.g. Malè et al. 2014; Turner et al. 2016; Ji et al. 2019),
although so far with little use to open phylogenetic questions. This is
partly due to limited sampling (usually a single individual per species
or higher taxonomic units). Simon et al. (2012) used a deep sequencing
approach to detect intragenomic ITS polymorphisms among populations ofArabidopsis . However, phylogenetic studies investigating
intragenomic nrDNA polymorphism patterns across many species within the
same genus are still scarce (e.g. Song et al. 2012; Weitemeier et al.
2015), and the full extent of the divergence of the 5S-IGS intra-genomic
variants in plants has not yet been adequately explored (cf. Galián et
al. 2014).
In this pilot study, we generated amplicon data of the intergenic spacer
of the 5S nuclear ribosomal DNA cistron (5S-IGS) using High-Throughput
Sequencing (HTS) from six geographic samples of different composition:
pure samples, including only material of a single target species, and
mixed samples, including all species found at a certain place. The
investigated species cover all common lineages of western Eurasian oaks
(sects Cerris , Ilex and Quercus ). Amplicon data
were analyzed using our clone-sequence data as reference, and the
taxonomic resolution of the 5S-IGS region was assessed comparing the
performance of similarity algorithm (Basic Local Alignment Search
Tool—BLAST ) and evolutionary approaches (Maximum Likelihood
trees—ML ; Evolutionary Placement Algorithm—EPA ).
To our knowledge, this work is the first to thoroughly analyze
intragenomic variation of the nuclear ribosomal 5S region in plants (see
also Heitkam et al. 2015, who identified and developed probes for 5S
genes while screening Illumina-generated sequences for repetitive genome
elements). The potential applications of our approach are manifold and
span from the delineation of oak species, the assessment of intra- and
inter-species diversity, the detection of hybridization/introgression
patterns, the identification of cryptic lineages, to gaining genetic
insights into the structure, assembly, function and evolution of oak
communities.