Abstract
While recent sequencing technologies (third generation sequencing) can successfully sequence all copies of nuclear ribosomal DNA (rDNA) markers present within a genome and offer insights into the intragenomic variation of these markers, high intragenomic variation can be a source of confusion for high-throughput species identification using such technologies. High-throughput (HT) amplicon sequencing via PacBio SEQUEL I was used to evaluate the intragenomic variation of the ITS region and D1–D2 LSU domains in nine Cordyceps species, and the accuracy of such technology to identify these species based on molecular phylogenies was also assessed. PacBio sequences within strains showed variable level of intragenomic variation among the studied Cordyceps species with C. blackwelliae showing greater variation than the others. Some variants from a mix of species clustered together outside their respective species of origin, indicative of intragenomic variation that escaped concerted evolution shared between species. Proper selection of consensus sequences from HT amplicon sequencing is a challenge for interpretation of correct species identification. PacBio consensus sequences with the highest number of reads represent the major variants within a genome and gave the best results in terms of species identification.
Highlights
After the discovery of polymerase chain reaction (PCR) technique and Sanger sequencing in the early 90s [1], nucleotide states became important characters in fungal taxonomy and molecular markers have been continuously applied in this field [2,3]
A total number of 22 strains belonging to nine Cordyceps species (Figure 1) were se3 of 19 lected from the BIOTEC Culture Collection (BCC) with deposited specimens in the BIOTEC Bangkok Herbarium (BBH); duplicate cultures exist at the Thailand Bioresource
HT-amplicon sequencing could generate barcodes allowing accurate species identification, which is similar to the objective of our study, where we evaluated the performance in identifying Cordyceps species from our culture collections
Summary
Received: 9 August 2021Accepted: 10 September 2021Published: 16 September 2021Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.Licensee MDPI, Basel, Switzerland.Attribution (CC BY) license (https://creativecommons.org/licenses/by/ 4.0/).After the discovery of polymerase chain reaction (PCR) technique and Sanger sequencing in the early 90s [1], nucleotide states became important characters in fungal taxonomy and molecular markers have been continuously applied in this field [2,3]. Nowadays, molecular markers are regularly used for delimiting novel species, identification, and the inference of phylogenetic relationship. The nuclear internal transcribed spacer (nrITS)
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