Multilocus Sequence Analysis of Selected Housekeeping- and Pathogenicity-Related Genes in Venturia inaequalis
Abstract
:1. Introduction
2. Results
2.1. Phylogenetic Analyses
2.2. Intragene Polymorphisms
2.3. DNA Divergence and Differentiation between Strains of Rvi6-Virulent and -Avirulent Populations
2.4. Recombination and Linkage Disequilibrium Analysis
3. Discussion
3.1. Phylogenetic Relationships between Rvi6-Virulent and Rvi6-Avirulent Strains
3.2. Factors Influencing the Genetic Diversity and Divergence among Rvi6-Virulent and Rvi6-Avirulent Strains
3.3. Recombination between Rvi6-Virulent and Rvi6-Avirulent Strains
4. Materials and Methods
4.1. Collection of Fungal Samples
4.2. Designing Primers for PCR and Amplification of Pathogenicity-Related Genes
4.3. Amplification of Housekeeping Genes
4.4. Polymorphism and Intragene Differences Analyses
4.5. Structure Analyses
4.6. Model Selection and Phylogenetic Analysis
4.7. Detection of Recombination
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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R Gene of Host | Orchard Location, Cultivar | Control Type | Population Names | No. of Strains Used |
---|---|---|---|---|
0 | Dabrowice, F1 seedling of Malus x zumi var. “Colocarpa” | organic | OZD | 5 |
0 | Lublin, Gala | organic | MGL | 5 |
0 | Lublin, Paulared | organic | PAL | 5 |
0 | Nowy Dwor, Ligolina | organic | LND | 5 |
0 | Nowy Dwor, Delbard Jubile | organic | DND | 5 |
Rvi1 | Brzezna, Golden Delicious | chemical | GDB | 5 |
Rvi1 | Jajkowice, Golden Delicious | chemical | GDJ | 5 |
Rvi1 | Milobadz, Golden Delicious | chemical | GDM | 5 |
Rvi6 | Nowy Dwor, Enterprise | organic | END | 5 |
Rvi6 | Nowy Dwor, Rajka | organic | RND | 5 |
Rvi6 | Brzeziny, Rubinola | organic | RUB | 5 |
Rvi6 | Brzeziny, Topaz | organic | TOB | 5 |
Rvi6 | Jeziorsko, Topaz | organic | TOJ | 5 |
Rvi6 | Jeziorsko, Biogolden | organic | BGJ | 5 |
Rvi6 | Lublin, Ariwa | organic | ARL | 5 |
Rvi6 | Lublin, Gold Milenium | organic | GML | 5 |
Rvi17 | Brzezna, Antonovka | organic | ABR | 5 |
Rvi17 | Siedlce, Antonovka | organic | ASI | 5 |
(a) | ||||||||||||
Gene Fragment | Length in Basepairs | G/C Content | No. of Polymorphic/Segregating Sites [S] | Polymorphic Sites in % | % of Haplotypes Per locus | No. of Parsimony Informative Sites | Haplotype [Gene] Diversity [Hd] | Nucleotide Diversity [π] | Theta per Site from S [θ-W] | Tajima’s Test D | Fu and Li’s D Test for Neutrality of Mutations | Average Percentage of Sequence Similarity between Strains |
EF-1α | 336 | 0.505 | 103 | 30.7 | 49 | 64 | 0.869 | 0.02109 | 0.05921 | −2.29955 * | −2.58706 * | 93.3 |
β-tubulin | 384 | 0.529 | 23 | 6 | 22 | 12 | 0.800 | 0.00813 | 0.01160 | −1.13774 | −2.92669 * | 98.45 |
mannosidase | 784 | 0.481 | 27 | 3.4 | 25 | 21 | 0.741 | 0.00994 | 0.00665 | 1.33244 | −0.18782 | 98.9 |
glucosidase | 533 | 0.569 | 66 | 12.38 | 41 | 49 | 0.862 | 0.02321 | 0.02514 | −0.42071 | −0.90093 | 95.8 |
(b) | ||||||||||||
Gene fragment | Length in basepairs | G/C content | No. of polymorphic/segregating sites [S] | Polymorphic sites in % | % of haplotypes per population | No. of parsimony informative sites | Haplotype [gene] diversity [Hd] | Nucleotide diversity [π] | Theta per site from S [θ-W] | Tajima’s test D | Fu and Li’s D test for neutrality of mutations | |
all strains, concatened EF-1α and β-tubulin | 720 | 0.518 | 126 | 17.5 | 67 | 76 | 0.974 | 0.1416 | 0.03385 | −2.12766 * | −2.95196 * | |
all strains, concatened mannosidase and glucosidase | 1317 | 0.556 | 93 | 7.06 | 61 | 70 | 0.955 | 0.01515 | 0.01466 | 0.1097 | −0.74098 | |
(c) | ||||||||||||
Gene fragment | Length in basepairs | G/C content | No. of polymorphic/segregating sites [S] | Polymorphic sites in % | % of haplotypes per population | No. of parsimony informative sites | Haplotype [gene] diversity [Hd] | Nucleotide diversity [π] | Theta per site from S [θ-W] | Tajima’s test D | Fu and Li’s D test for neutrality of mutations | |
EF-1α Rvi6-virulent | 336 | 0.504 | 75 | 22.3 | 60 | 46 | 0.89359 | 0.02859 | 0.05248 | −1.87014 * | −1.25818 | |
EF-1α Rvi6-avirulent | 336 | 0.505 | 64 | 19 | 47 | 27 | 0.85367 | 0.01530 | 0.04049 | −2.24047 * | −3.11016 * | |
β-tubulin Rvi6-virulent | 384 | 0.528 | 18 | 4.7 | 22.5 | 7 | 0.67949 | 0.00957 | 0.01105 | −0.43613 | −2.49531 | |
β-tubulin Rvi6-avirulent | 384 | 0.529 | 17 | 4.43 | 25 | 11 | 0.70226 | 0.00467 | 0.00952 | −1.72921 | −1.15951 | |
mannosidase Rvi6-virulent | 784 | 0.48 | 24 | 3.06 | 35 | 20 | 0.72740 | 0.00849 | 0.00720 | 0.39781 | 0.29570 | |
mannosidase Rvi6-avirulent | 784 | 0.481 | 23 | 2.93 | 22 | 19 | 0.72655 | 0.01067 | 0.00629 | 1.98139 | 0.07949 | |
glucosidase Rvi6-virulent | 533 | 0.571 | 24 | 4.5 | 35 | 18 | 0.767 | 0.0181 | 0.01152 | 1.93341 | −0.27958 | |
glucosidase Rvi6-avirulent | 533 | 0.568 | 64 | 12 | 55 | 48 | 0.9240 | 0.02697 | 0.02876 | −0.21363 | −0.45163 |
Gene Region. | Neutral | Non-Neutral | Total |
---|---|---|---|
EF-1α | 12 | 22 | 34 |
β-tubulin | 17 | 17 | 34 |
mannosidase | 26 | 3 | 29 |
glucosidase | 23 | 48 | 71 |
DNA Divergence between Rvi6-Virulent vs. Rvi6-Avirulent Populations | ||||
---|---|---|---|---|
Gene Flow Estimates of Nei 1973 | Average No. of Nucleotide Differentiations between Pops | Average Number of Nucleotide Substitutions per Site, between Pops Dxy | Minimum Number of Recombination Events [Rm] | |
EF-1α | Gst = 0.00025 Nm = 990.68 | 7.699 | 0.02291 | 20 |
β-tubulin | Gst = 0.13228 Nm = 1.64 | 3.912 | 0.01021 | 3 |
1,2-alpha-D-mannosidase | Gst = 0.01172 Nm = 21.08 | 7.792 | 0.00994 | 11 |
glucan-1,3-beta-glucosidase | Gst = 0.01324 Nm = 18.64 | 11.354 | 0.02239 | 12 |
concatened EF-1α and β-tubulin | Gst = 0.01068 Nm = 23.15 | 11.601 | 0.01614 | 23 |
concatenated mannosidase and glucosidase | Gst = 0.00344 Nm = 72.33 | 19.128 | 0.01482 | 23 |
DNA Region and Groups of Populations | Among Groups | Among Populations within Groups | Among Individuals within Populations | FST |
---|---|---|---|---|
EF-1α/Rvi6-virulent vs. Rvi6-avirulent | 3.2 | 20.2 | 76.7 | 0.23 |
β-tubulin/Rvi6-virulent vs. Rvi6-avirulent | 31.9 | 2.5 | 65.6 | 0.34 |
1,2-alpha-D-mannosidase/Rvi6-virulent vs. Rvi6-avirulent | 2.6 | 13.5 | 83.9 | 0.16 |
glucan-1,3-beta-glucosidase/Rvi6-virulent vs. Rvi6-avirulent | 0.6 | 7.7 | 91.7 | 0.08 |
EF-1α/Rvi6-virulent | 23.0 | 77.0 | 0.23 | |
EF-1α/Rvi6-avirulent | 18.0 | 82.0 | 0.18 | |
β-tubulin/Rvi6-virulent | 10.2 | 89.8 | 0.10 | |
β-tubulin/Rvi6-avirulent | 22.0 | 78.1 | 0.22 | |
1,2-alpha-D-mannosidase/Rvi6-virulent | 19.6 | 80.4 | 0.20 | |
1,2-alpha-D-mannosidase/Rvi6-avirulent | 10.8 | 89.2 | 0.11 | |
glucan-1,3-beta-glucosidase/Rvi6-virulent | 17.8 | 82.2 | 0.18 | |
glucan-1,3-beta-glucosidase/Rvi6-avirulent | 3.4 | 96.6 | 0.03 |
DNA Region and Compared Groups of Strains | Index of Association [rBARd] |
---|---|
EF-1α all strains/two groups | 0.15 * |
β-tubulin all strains/two groups | 0.3 * |
1,2-alpha-D-mannosidase all strains/two groups | 0.5 * |
glucan-1,3-beta-glucosidase all strains/two groups | 0.17 * |
concatened EF-1α and β-tubulin, all strains/two groups | 0.11 |
concatened mannosidase and glucosidase, all strains/two groups | 0.13 * |
Name of the Primer Set (Target Region) | Sequence of the Primer in 5′->3′ Orientation | Sequence of the Primer in 5′->3′ Orientation | Product Length in Basepairs | Reference |
---|---|---|---|---|
VNEFI-f/VNEFI-r (EF-1α) | ACTTGATCTACAAGTGCGGTG | AGGAGTCTCGAACTTCCAGAG | 385 | [14] |
C/D (β-tubulin) | GAGGAATTCCCAGACCGTATGATG | GCTGGATCCTATTCTTTGGGTCGAACAT | 436 | [39] |
cont189/675 (β-tubulin) | CACGGAAGATAGCGGAGCAAGTAA | GAGGAATTCCCAGACCGTATGATG | 487 | this study |
VinManno1/ VinManno2 | TTGCTCGCGTAACGGCTCCAGA | TTCGCTCATCGCAAATCCCCATAC | 858 | this study |
VinGluco1/VinGluco2 | TAAGCACGGCCATCACAACCTACG | CGCAGGCCCTCTAAATTCCAAACT | 1006 | this study |
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Michalecka, M.; Puławska, J. Multilocus Sequence Analysis of Selected Housekeeping- and Pathogenicity-Related Genes in Venturia inaequalis. Pathogens 2021, 10, 447. https://doi.org/10.3390/pathogens10040447
Michalecka M, Puławska J. Multilocus Sequence Analysis of Selected Housekeeping- and Pathogenicity-Related Genes in Venturia inaequalis. Pathogens. 2021; 10(4):447. https://doi.org/10.3390/pathogens10040447
Chicago/Turabian StyleMichalecka, Monika, and Joanna Puławska. 2021. "Multilocus Sequence Analysis of Selected Housekeeping- and Pathogenicity-Related Genes in Venturia inaequalis" Pathogens 10, no. 4: 447. https://doi.org/10.3390/pathogens10040447
APA StyleMichalecka, M., & Puławska, J. (2021). Multilocus Sequence Analysis of Selected Housekeeping- and Pathogenicity-Related Genes in Venturia inaequalis. Pathogens, 10(4), 447. https://doi.org/10.3390/pathogens10040447