Molecular Discrimination and Phylogenetic Relationships of Physalis Species Based on ITS2 and rbcL DNA Barcode Sequence
Abstract
:1. Introduction
2. Materials and Methods
2.1. Study Area and Collection of Plant Samples
2.2. Genomic DNA Extraction
2.3. PCR Amplification and Sequencing
2.4. Sequence Alignment, Phylogenetic and Data Analysis
2.5. Analysis of Genetic Divergence
2.6. Determination of Intraspecific and Interspecific Genetic Distance
2.7. Nucleotide Polymorphism and Neutrality Tests
2.8. Barcoding Gap Analysis
3. Results
3.1. Success Rates of PCR Amplification and Sequencing
3.2. Species Discrimnation of Physalis Accessions Using BLASTn Analysis
3.3. Multiple Sequence Alignments
3.4. Species Discrimnation of Physalis Species Based on Phylogenetic Analysis
3.5. Genetic Divergence Analysis between and within Physalis Species Based on ITS2 Sequences
3.5.1. DNA Divergence between Populations Based on ITS2 Sequences
3.5.2. DNA Divergence within Populations Based on ITS2 Sequences
3.6. Genetic Distance between and within Physalis Species Based on ITS2 and rbcL Sequences
3.7. Nucleotide Polymorphism and Neutrality Tests
3.8. Barcoding Gap Analysis
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Barcode Region | Samples Tested (n) | Number of Amplicons Produced | Number of Sequences Produced | Percentage of Amplification Efficiency | Percentage of Sequencing Efficiency | Alignment Length (bp) | Mean Sequence Length (bp) | Mean GC Content (%) |
---|---|---|---|---|---|---|---|---|
ITS2 | 64 | 49 | 32 | 77 | 65 | 841 | 525 | 61.00 |
rbcL | 64 | 54 | 48 | 84 | 89 | 841 | 690 | 43.40 |
Barcode Locus | Base Contents (%) | |||||
---|---|---|---|---|---|---|
A | T | G | C | AT | GC | |
ITS2 | 19.42 | 19.39 | 29.78 | 31.41 | 39.00 | 61.00 |
rbcL | 28.22 | 28.40 | 23.10 | 20.28 | 56.58 | 43.42 |
Population | P. peruviana (P1) | P. cordata (P2) | P. peruviana (P1) | P. purpurea (P2) | P. purpurea (P1) | P. cordata (P2) |
---|---|---|---|---|---|---|
Polymorphic sites in each population | 14 | 21 | 12 | 18 | 2 | 4 |
Total number of polymorphic sites | 35 | 23 | 4 | |||
Average number of nucleotide differences | 17.600 | 6.351 | 0.889 | |||
Nucleotide diversity Pi (t) | 0.33208 | 0.18147 | 0.14821 | |||
Number of fixed differences | 6 | 1 | 0 | |||
Polymorphic mutations in population 1 (P1) but monomorphic ones in population 2 (P2) | 13 | 10 | 2 | |||
Polymorphic mutations in P2 but monomorphic ones in P1 | 28 | 22 | 1 | |||
Shared mutations | 1 | 2 | 2 | |||
Average number of nucleotide differences between populations | 20.625 | 10.158 | 1.477 | |||
Average nucleotide substitution per site between populations (Dxy) | 0.38915 | 0.29026 | 0.24621 | |||
Number of net nucleotide substitutions per site between populations (Da) | 0.12343 | 0.03881 | 0.01299 |
Physalis Species | P. peruviana | P. cordata | P. purpurea |
---|---|---|---|
Total number of sequences | 2 | 4 | 22 |
Number of polymorphic (segregating) sites (S) | 70 | 83 | 20 |
Nucleotide diversity Pi (Total) | 0.31250 | 0.18095 | 0.14898 |
Nucleotide diversity Pi (JC-Total) | 0.40425 | 0.20708 | 0.16609 |
Theta (Total) | 0.31250 | 0.19675 | 0.17396 |
Total number of substitutions | 70 | 101 | 26 |
Groups | P. purpurea | P. peruviana | P. cordata |
---|---|---|---|
P. purpurea | 198.92 | 1589.41 | |
P. peruviana | 9.58 | 357.92 | |
P. cordata | 21.99 | 9.53 |
ITS2 | rbcL | |||||
---|---|---|---|---|---|---|
Polymorphic Sites/Segregation Sites (S) | 4 | Position in the Gene | Variants | 59 | Positions in the Gene | Variants |
Singleton | 1 | 177 | 2 | 48 | 141,272,273,276,280,283,284,293,298, 301,308,309,310,322,325,327,331,334, 335,337,339,340,345,346,347,348,350, 353,357,365,366,373,375,376,386,395, 396,398,413,414,416,419,436,441,447, 457 344,359 | 2 3 |
Parsimony informative sites | 3 | 179 176 178 | 2 3 4 | 11 | 302,336,341,355,358,362,401,430,444 282,363 | 2 3 |
Nucleotide diversity (Pi) | 0.15917 | 0.01632 | ||||
Average number of nucleotide differences (k) | 0.955 | 5.844 | ||||
Sequence length (base pairs) | 532 | 716 | ||||
Number of sequences | 28 | 28 |
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Pere, K.; Mburu, K.; Muge, E.K.; Wagacha, J.M.; Nyaboga, E.N. Molecular Discrimination and Phylogenetic Relationships of Physalis Species Based on ITS2 and rbcL DNA Barcode Sequence. Crops 2023, 3, 302-319. https://doi.org/10.3390/crops3040027
Pere K, Mburu K, Muge EK, Wagacha JM, Nyaboga EN. Molecular Discrimination and Phylogenetic Relationships of Physalis Species Based on ITS2 and rbcL DNA Barcode Sequence. Crops. 2023; 3(4):302-319. https://doi.org/10.3390/crops3040027
Chicago/Turabian StylePere, Katherine, Kenneth Mburu, Edward K. Muge, John Maina Wagacha, and Evans N. Nyaboga. 2023. "Molecular Discrimination and Phylogenetic Relationships of Physalis Species Based on ITS2 and rbcL DNA Barcode Sequence" Crops 3, no. 4: 302-319. https://doi.org/10.3390/crops3040027