Aerobiological Monitoring and Metabarcoding of Grass Pollen
Abstract
:1. Introduction
2. Results
2.1. Sequencing Results
2.2. Metabarcoding Analysis of Airborne Samples from Moscow and Ryazan
2.3. Comparison of Metabarcoding and Phenological Analysis
3. Discussion
4. Materials and Methods
4.1. Sampling
Phenological Observations
4.2. Statistical Methods
4.3. Optimization of Pollen Wash-off Procedure
4.4. Optimization of Pollen DNA Isolation
- DNA isolation from pollen according to the protocol described in [55]. The approximate time to isolate DNA from six samples is 4 h.
- Enzymatic treatment with lysozyme and zymolysin. First, the enzymatic treatment of pollen was carried out with the addition of 50 μL of lysozyme and 50 μL of zymolysin (1 U each) at a temperature of 37 °C for an hour. Then, 5 µL of proteinase K was added to the solution and incubated at 60 °C for 20 min without the homogenization stage. Next, 800–900 µL of lysis buffer (CTAB, 0.04% SDS) was added up to 1 mL of the final solution and incubated for another hour at a temperature of 60 °C. Then, DNA was extracted by adding a 1× volume of chloroform, stirring, centrifugation at room temperature for 30 min at 13,400 rpm, and collecting the upper aqueous fraction, which was placed in a clean tube. Next, DNA was precipitated by adding an equal volume of isopropanol and 0.1× v/v potassium acetate, followed by centrifugation at 4° C for 30 min at 15,000 rpm. Then, the DNA pellet was washed twice with 700 µL of 70% ethanol and dissolved in 20 µL of nuclease-free water. The approximate time to isolate DNA from six samples is 5 h and 15 min.
- Enzymatic treatment with lysozyme and zymolysin followed theisolation protocol from [55], excluding mechanical treatment (which combines the first and second methods). The approximate time to isolate DNA from six samples is 4 h and 45 min.
4.5. PCR and Sequencing
4.6. Bioinformatics Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Method | C, ng/µL | OD 260/280 | OD 260/230 |
---|---|---|---|
1 | 0.6 ± 0.12 | 1.78 ± 0.28 | 0.24 ± 0.09 |
2 | 0.75 ± 0.52 | 1.62 ± 0.16 | 0.56 ± 0.27 |
3 | 0.71 ± 0.13 | 1.75 ± 0.19 | 0.57 ± 0.48 |
4a | 0.42 ± 0.02 | 1.16 ± 0.67 | 0.68 ± 0.55 |
4b | 0.94 ± 0.69 | 1.77 ± 0.2 | 0.78 ± 0.52 |
4c | 1.13 ± 0.23 | 1.89 ± 0.13 | 2.09 ± 0.75 |
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Krinitsina, A.A.; Omelchenko, D.O.; Kasianov, A.S.; Karaseva, V.S.; Selezneva, Y.M.; Chesnokova, O.V.; Shirobokov, V.A.; Polevova, S.V.; Severova, E.E. Aerobiological Monitoring and Metabarcoding of Grass Pollen. Plants 2023, 12, 2351. https://doi.org/10.3390/plants12122351
Krinitsina AA, Omelchenko DO, Kasianov AS, Karaseva VS, Selezneva YM, Chesnokova OV, Shirobokov VA, Polevova SV, Severova EE. Aerobiological Monitoring and Metabarcoding of Grass Pollen. Plants. 2023; 12(12):2351. https://doi.org/10.3390/plants12122351
Chicago/Turabian StyleKrinitsina, Anastasia A., Denis O. Omelchenko, Artem S. Kasianov, Vera S. Karaseva, Yulia M. Selezneva, Olga V. Chesnokova, Vitaly A. Shirobokov, Svetlana V. Polevova, and Elena E. Severova. 2023. "Aerobiological Monitoring and Metabarcoding of Grass Pollen" Plants 12, no. 12: 2351. https://doi.org/10.3390/plants12122351