Next-Generation Sequencing of Ancient and Recent Fungarium Specimens
Abstract
1. Introduction
2. Materials and Methods
2.1. Taxa Selection
2.2. DNA Extraction, Quantification, and Fragmentation Analysis
2.3. PCR Amplification and Sanger Sequencing
2.4. PCR Amplification and Next-Generation Sequencing
2.5. Next-Generation Sequencing Data Processing, OTU Identification, and Taxonomic Assignment
2.6. Phylogenetic Analyses
3. Results and Discussion
3.1. Taxa Sampled
3.2. DNA Extraction, Quantification, and Fragmentation
3.3. Sanger Sequencing
3.4. Next-Generation Sequencing
3.5. Contamination
3.6. Phylogenetic Analyses
- Gyromitra arctica
- Gyromitra korshinskii
- Gyromitra leucoxantha
- Gyromitra ussuriensis
3.7. Costs
3.8. Comparisons with Other Studies
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Taxonomic Name | Fungarium Number | Year |
---|---|---|
Discina repanda (Wahlenb.) Sacc. | CUP-A-030055 | 1898 |
Gyromitra arctica Vassilkov (HOLOTYPE) | LE 179562 | 1960 |
Gyromitra korshinskii Jacz. (HOLOTYPE) | LE 179630 | 1886 |
Gyromitra leucoxantha (Bres.) Harmaja (HOLOTYPE) | S-F-11771 | 1880s? |
Gyromitra perlata (Fr.) Harmaja (NEOTYPE) | UPS-F-144599 | 1863? |
Gyromitra ussuriensis Lj.N. Vassiljeva (NEOTYPE) | TAAM 060483 | 1961 |
Gyromitra ussuriensis | LE 179636 | 1960s? |
Lasiosphaeria lanuginosa (P. Crouan and H. Crouan) A.N. Mill. and Huhndorf (+control) | ILLS 00176154 | 2021 |
Propolis sp. | HUH 00941116 | 1974 |
Stictis cylindrocarpa Peck | ILLS 00169337 | 1935 |
Stictis fulva Peck (ISOTYPE) | HUH 00941122 | 1879 |
Stictis fulva | HUH 00941117 | 1897 |
Xerotrema megalospora Sherwood and Coppins | TRH L-13532 | 2009 |
Xerotrema megalospora | UBC L-63079 | 2005 |
Xerotrema megalospora | ASU L572580 | 1993 |
Xerotrema megalospora | E 00948846 | 1999 |
Xerotrema megalospora | E 00278634 | 2000 |
Xerotrema quercicola Coppins and Aptroot | E 00817833 | 2002 |
Xerotrema quercicola | E 00817832 | 2002 |
Xerotrema quercicola (HOLOTYPE) | E 00278636 | 2006 |
Taxonomic Name | Top BLASTn Hit | DNA Extraction | Qubit (ng/µL) | Length | Sequences per Sample | Sequences of Target (%) | Sanger Sequences | GenBank Number |
---|---|---|---|---|---|---|---|---|
Discina repandaX | inconclusive | NaOH + EZNA | 1.65 | 329–355 | 9050 | 0 | no | N/A |
Gyromitra arcticaX (HOLOTYPE) | G. infula | EZNA | 3.08 | 329 | 21406 | 20821 (97%) | yes (ITS2) | OP265173 |
Gyromitra korshinskiiX (HOLOTYPE) | G. sphaerospora | EZNA | too low | 362–363 | 16467 | 14400 (87%) | yes (ITS) | OP265174 |
Gyromitra leucoxantha (HOLOTYPE) | G. leucoxantha | NaOH * | too low | 339 | 12780 | 5934 (46%) | no | OP265175 |
Gyromitra perlata (NEOTYPE) | no PCR amplification | NaOH * | 6.56 | N/A | N/A | N/A | no | N/A |
Gyromitra ussuriensisX (NEOTYPE) | G. gigas | EZNA | 2.02 | 349 | 11830 | 2769 (23%) | no | ON527922 |
Gyromitra ussuriensisX | G. perlata | EZNA | too low | 337 | 12784 | 6699 (52%) | no | OP265176 |
Lasiosphaeria lanuginosa (+control) | L. lanuginosa | EZNA | too low | 288 | 11826 | 11814 (99%) | yes (ITS) | OP265177 |
Propolis sp. | inconclusive | EZNA * | too low | N/A | 13513 | 0 | no | N/A |
Stictis cylindrocarpaX | inconclusive | EZNA * | too low | N/A | 12985 | 0 | no | N/A |
Stictis fulvaX (ISOTYPE) | inconclusive | EZNA * | too low | N/A | 17124 | 0 | no | N/A |
Stictis fulvaX | inconclusive | EZNA * | too low | N/A | 16022 | 0 | no | N/A |
Xerotrema megalosporaX | inconclusive | EZNA * | too low | N/A | 6716 | 0 | no | N/A |
Xerotrema megalosporaX | inconclusive | EZNA * | too low | N/A | 14458 | 0 | no | N/A |
Xerotrema megalosporaX | inconclusive | EZNA * | too low | N/A | 17815 | 0 | no | N/A |
Xerotrema megalosporaX | inconclusive | EZNA * | too low | N/A | 20123 | 0 | no | N/A |
Xerotrema megalosporaX | inconclusive | EZNA * | too low | N/A | 32862 | 0 | no | N/A |
Xerotrema quercicolaX | inconclusive | EZNA * | too low | N/A | 13786 | 0 | no | N/A |
Xerotrema quercicolaX | inconclusive | EZNA * | too low | N/A | 35694 | 0 | no | N/A |
Xerotrema quercicolaX (HOLOTYPE) | inconclusive | EZNA * | too low | N/A | 15442 | 0 | no | N/A |
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Miller, A.N.; Karakehian, J.; Raudabaugh, D.B. Next-Generation Sequencing of Ancient and Recent Fungarium Specimens. J. Fungi 2022, 8, 932. https://doi.org/10.3390/jof8090932
Miller AN, Karakehian J, Raudabaugh DB. Next-Generation Sequencing of Ancient and Recent Fungarium Specimens. Journal of Fungi. 2022; 8(9):932. https://doi.org/10.3390/jof8090932
Chicago/Turabian StyleMiller, Andrew N., Jason Karakehian, and Daniel B. Raudabaugh. 2022. "Next-Generation Sequencing of Ancient and Recent Fungarium Specimens" Journal of Fungi 8, no. 9: 932. https://doi.org/10.3390/jof8090932
APA StyleMiller, A. N., Karakehian, J., & Raudabaugh, D. B. (2022). Next-Generation Sequencing of Ancient and Recent Fungarium Specimens. Journal of Fungi, 8(9), 932. https://doi.org/10.3390/jof8090932