High‐throughput sequencing of 5S‐IGS in oaks: Exploring intragenomic variation and algorithms to recognize target species in pure and mixed samples

Abstract

Measuring biological diversity is a crucial but difficult undertaking, as exemplified in oaks where complex patterns of morphological, ecological, biogeographical and genetic differentiation collide with traditional taxonomy, which measures biodiversity in number of species (or higher taxa). In this pilot study, we generated high‐throughput sequencing amplicon data of the intergenic spacer of the 5S nuclear ribosomal DNA cistron (5S‐IGS) in oaks, using six mock samples that differ in geographical origin, species composition and pool complexity. The potential of the marker for automated genotaxonomy applications was assessed using a reference data set of 1,770 5S‐IGS cloned sequences, covering the entire taxonomic breadth and distribution range of western Eurasian Quercus, and applying similarity (blast) and evolutionary approaches (maximum‐likelihood trees and Evolutionary Placement Algorithm). Both methods performed equally well, allowing correct identification of species in sections Ilex and Cerris in the pure and mixed samples, and main lineages shared by species of sect. Quercus. Application of different cut‐off thresholds revealed that medium‐ to high‐abundance (>10 or 25) sequences suffice for a net species identification of samples containing one or a few individuals. Lower thresholds identify phylogenetic correspondence with all target species in highly mixed samples (analogous to environmental bulk samples) and include rare variants pointing towards reticulation, incomplete lineage sorting, pseudogenic 5S units and in situ (natural) contamination. Our pipeline is highly promising for future assessments of intraspecific and interpopulation diversity, and of the genetic resources of natural ecosystems, which are fundamental to empower fast and solid biodiversity conservation programmes worldwide.

5S‐IGS rDNA in wind‐pollinated trees (Fagus L.) encapsulates 55 million years of reticulate evolution and hybrid origins of modern species

“…Availability of High-Throughput Sequencing (HTS) approaches is now boosting new efforts into research questions that were previously considered too expensive and labour-intensive (Babik et al, 2009;Glenn, 2011). In a recent study, Piredda et al (2021) generated HTS amplicon data of the nontranscribed intergenic spacers of the 5S rDNA in Quercus (Fagaceae) and demonstrated the great value of this approach to provide phylogenetic information for inspecting range-wide evolutionary patterns while circumventing previous technical limitations (e.g. cloning, abundant samples, special methodological frameworks; Denk and Grimm, 2010;Simeone et al, 2018).…”

5S-IGS rDNA in wind-pollinated trees (FagusL.) encapsulates 55 million years of reticulate evolution and hybrid origins of modern species

“…In F. japonica, the longer, GC-rich O-type and shorter, GC-normal I-type, represent two highly divergent, only distantly related lineages; their divergence (Table 1) parallels that between subgenera of oaks Simeone et al, 2018;Piredda et al, 2021 for first HTS data) and between genera in Betulaceae (Forest et al, 2005). Both types are abundant:…”

“…Amplicon sequencing of loci with high information content (target sequencing) substantially reduces costs, as it makes cloning unnecessary and many individuals can be combined in the same sequencing run (Ekblom & Galindo, 2010). In a recent study, Piredda et al (2021) analysed 5S-IGS HTS amplicon data generated for Quercus (Fagaceae) and demonstrated the great potential of this technique for inspecting range-wide diversity patterns and for assessing interand intra-species relationships. In the present study, we explored the applicability of the same experimental pathway in six geographic samples of the core group of 'subgenus Fagus': the F. crenata -F. sylvatica s.l.…”

“…Availability of High-Throughput Sequencing (HTS) approaches is now boosting new efforts into research questions that were previously considered too expensive and labour-intensive (Babik et al, 2009;Glenn, 2011). In a recent study, Piredda et al (2021) generated HTS amplicon data of the non-transcribed intergenic spacers of the 5S rDNA in Quercus (Fagaceae) and demonstrated the great value of this approach to provide phylogenetic information for inspecting range-wide evolutionary patterns while circumventing previous technical limitations (e.g. cloning, abundant samples, special methodological frameworks; Simeone et al, 2018).…”

Complex Scenarios of Reticulation, Polyploidization, and Species Diversity within Annual Pansies of Subsect. Bracteolatae (Viola Sect. Melanium, Violaceae) in Italy: Insights from 5S-IGS High-Throughput Sequencing and Plastid DNA Variation

“…Several studies have shown that the detected variation may result in the identification of species-specific traits and complex evolutionary signals such as hybridization, introgression, and polyploidization, e.g., [ 50 , 51 , 52 ]. A couple of recent studies have generated HTS amplicon data of the nuclear 5S spacers to improve the efficiency of signal detection and demonstrated the high potential of the technique to inspect diversity patterns and infer evolutive pathways in highly complex species systems such as Fagaceae Dumort [ 53 , 54 ].…”