DNA Barcoding, Phylogenetic Analysis and Secondary Structure Predictions of Nepenthes ampullaria, Nepenthes gracilis and Nepenthes rafflesiana
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sample Collection
2.2. Genomic DNA Extraction and PCR Amplification
2.3. Bioinformatics Analysis and Phylogenetic Tree
2.4. Secondary Structure Predictions
3. Results
3.1. Amplification, Sequencing, Multiple Sequence Alignment and Species Identification
3.2. Phylogenetic Studies, Intraspecific Variation, Interspecific Divergence and DNA Barcoding Gap
3.3. DNA Barcodes, ITS1 and ITS2 Secondary Structure Predictions
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Steps | No. of Cycles | rbcL 1 | ITS1 2 | ITS2 3 | |||
---|---|---|---|---|---|---|---|
Temperature (°C) | Duration (s) | Temperature (°C) | Duration (s) | Temperature (°C) | Duration (s) | ||
Denaturation | 35 | 98 | 10 | 98 | 10 | 98 | 10 |
Annealing | 53 | 15 | 55 | 15 | 55 | 10 | |
Elongation | 72 | 20 | 72 | 40 | 72 | 30 | |
Hold | 1 | 4 | ∞ | 4 | ∞ | 4 | ∞ |
Region | Primer Name | Primer Sequence (5′ to 3′) | Primer Length (bp) | Reference |
---|---|---|---|---|
rbcL | Rbcla_fwd | ATGTCACCACAAACAGAGACTAAAGC | 26 | [21] |
Rbclb_rvs | GTAAAATCAAGTCCACCRCG | 20 | ||
ITS1 | ITS1_fwd | GGAAGGAGAAGTCGTAACAAGG | 22 | [21] |
ITS1_rvs | AGATATCCGTTGCCGAGAGT | 20 | ||
ITS2 | ITS2_fwd | GGGGCGGATATTGGCCTCCCCTTG | 24 | [22] |
ITS2_rvs | GACGCTTCTCCAGACTACAAT | 21 |
Barcode Genes | DNA Best-Fit Substitution Model |
---|---|
rbcL | Kimura-2-parameter |
ITS1 | Tamura-3-parameter |
ITS2 | Kimura-2-parameter + γ distribution |
Barcode Genes | Sample ID | Scientific Name | Accession Number | E Value | Query Coverage (%) | Percent Identity (%) |
---|---|---|---|---|---|---|
rbcL | NArbcL | N. mirabilis | NC_041271.1 | 0.0 | 100 | 100 |
NGrbcL | N. mirabilis | NC_041271.1 | 0.0 | 100 | 100 | |
NRrbcL | N. mirabilis | NC_041271.1 | 0.0 | 100 | 100 | |
ITS1 | NAits1 | Nepenthes x intermedia | HM204899.1 | 1e-103 | 100 | 98.64 |
NGits1 | N. ventricosa | AB675910.1 | 2e-168 | 100 | 100 | |
NRits1 | Nepenthes x intermedia | HM204899.1 | 2e-116 | 100 | 100 | |
ITS2 | NAits2 | N. gracilis | AB675882.1 | 4e-104 | 100 | 95.85 |
NGits2 | N. gracilis | AB675882.1 | 2e-122 | 100 | 99.20 | |
NRits2 | N. gracilis | AB675882.1 | 5e-113 | 100 | 99.14 |
DNA Barcode | Species | AT (bp) | GC (bp) | Total (bp) | AT (%) | GC (%) |
---|---|---|---|---|---|---|
rbcL | N. gracilis | 280 | 229 | 509 | 55 | 45 |
N. ampullaria | 278 | 224 | 502 | 55.4 | 44.6 | |
N. rafflesiana | 278 | 224 | 502 | 55.4 | 44.6 | |
ITS2 | N. gracilis | 85 | 114 | 250 | 34.0 | 66.0 |
N. ampullaria | 89 | 152 | 241 | 36.9 | 63.1 | |
N. rafflesiana | 78 | 155 | 233 | 33.5 | 66.5 | |
ITS1 | N. gracilis | 111 | 216 | 327 | 33.9 | 66.1 |
N. ampullaria | 69 | 150 | 219 | 31.5 | 68.5 | |
N. rafflesiana | 76 | 157 | 233 | 32.6 | 67.4 |
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Saidon, N.A.; Wagiran, A.; Samad, A.F.A.; Mohd Salleh, F.; Mohamed, F.; Jani, J.; Linatoc, A.C. DNA Barcoding, Phylogenetic Analysis and Secondary Structure Predictions of Nepenthes ampullaria, Nepenthes gracilis and Nepenthes rafflesiana. Genes 2023, 14, 697. https://doi.org/10.3390/genes14030697
Saidon NA, Wagiran A, Samad AFA, Mohd Salleh F, Mohamed F, Jani J, Linatoc AC. DNA Barcoding, Phylogenetic Analysis and Secondary Structure Predictions of Nepenthes ampullaria, Nepenthes gracilis and Nepenthes rafflesiana. Genes. 2023; 14(3):697. https://doi.org/10.3390/genes14030697
Chicago/Turabian StyleSaidon, Nur Azreen, Alina Wagiran, Abdul Fatah A. Samad, Faezah Mohd Salleh, Farhan Mohamed, Jaeyres Jani, and Alona C Linatoc. 2023. "DNA Barcoding, Phylogenetic Analysis and Secondary Structure Predictions of Nepenthes ampullaria, Nepenthes gracilis and Nepenthes rafflesiana" Genes 14, no. 3: 697. https://doi.org/10.3390/genes14030697