Disease Resistant Citrus Breeding Using Newly Developed High Resolution Melting and CAPS Protocols for Alternaria Brown Spot Marker Assisted Selection
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Material and Recovery of Hybrids
2.2. DNA Isolation
2.3. Sequencing of the SNP08 Locus
2.4. Bioinformatics Analysis
2.5. Nested-PCR
2.6. HRM Genotyping
2.7. Development of Cleaved Amplified Polymorphic Sequences (CAPS) Marker
2.8. KASPar SNP Genotyping
2.9. Statistical Analysis
2.10. Isolation and Production of A. alternata Inoculum
2.11. Inoculation and Evaluation of Disease Severity in Detached Leaves
3. Results and Discussion
3.1. Sequencing and Analysis of the Locus Flanking SNP08 and Development of the HRM Protocol
3.2. HRM Genotyping of the Germplasm Collection
3.3. HRM and KASPar Genotyping of the Hybrid Populations
3.4. CAPS Assay
3.5. In Vitro Phenotyping
4. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Accession | Description | References |
---|---|---|
50-15A-6 | ‘Clementine’ mandarin × ‘Avana’ mandarin/Unreleased parent of the CREA breeding program | [36] |
Afourer | Possibly ‘Murcott’ tangor × ‘Mandalina’ mandarin | [37,38] |
Avana (Willowleaf) | Old mandarin selection | [39] |
Bower | ‘Clementine’ mandarin × ‘Orlando’ tangelo | [40] |
Cami | ‘50-15A-6′ mandarin × ‘Mapo’ tangelo | [36] |
Carvalhais | Mandarin of unknown parentage | [39] |
Clemapo | ‘Clementine’ mandarin × ‘Mapo’ tangelo/early-maturing hybrid released in the 1990′s by a private breeding program | - |
Daisy | ‘Fortune’ mandarin × ‘Fremont’ mandarin | [41] |
Dancy | Seedling of Moragne tangierine | [39] |
Doppio Sanguigno | Sweet orange clonal selection | [42] |
Ellendale | Tangor of unknown parentage | [39] |
Emperor | ‘Ponkan’ mandarin selection | [38,39] |
Encore | ‘King’ mandarin × ‘Willowleaf’ mandarin | [39] |
Fairchild | ‘Clementine’ mandarin × ‘Orlando’ tangelo | [43] |
Fallglo | ‘Bower’ mandarin ×’ Temple’ tangor | [44] |
Foma107 | ‘Fortune’ mandarin × ‘Mapo’ tangelo/Unreleased parent of the CREA breeding program | - |
Fortune | ‘Clementine’ mandarin × ‘Orlando’ tangelo | [45] |
Fremont | ‘Clementine’ mandarin × ‘Ponkan’ mandarin | [39] |
ISA | Old clementine clone of Italian origin | - |
Kara | ‘King’ mandarin × ‘Owari’ satsuma | [39] |
King | Natural tangor of Asian origin | [39] |
Kinnow | ‘King’ mandarin × ‘Willowleaf’ mandarin | [39] |
Malvasio | Mandarin of unknown parentage | [39] |
Mapo | ‘Avana’ mandarin × ‘Duncan’ grapefruit | [46] |
Michal | ‘Clementine’ mandarin × ‘Dancy’ mandarin | [38] |
Minneola | ‘Duncan’ grapefruit × ‘Dancy’ mandarin | [39] |
Murcott | Tangor of unknown parentage | [39] |
Nova | ‘Clementine’ mandarin × ‘Orlando’ tangelo | [47] |
Okitsu | Satsuma clonal selection | [39] |
Ortanique | Tangor of unknown parentage | [39] |
OTA9 | ‘Oroval’ clementine × ‘Moro’ sweet orange | [48] |
Page | ‘Minneola’ tangelo × ‘Clementine’ mandarin | [39] |
Palazzelli | ‘Clementine’ mandarin × ‘King’ mandarin | [49] |
Ponkan | Old mandarin selection | [39] |
Primosole | ‘Miho’ satsuma × ‘Carvalhais’ mandarin | [50] |
Rubino | Late clementine clone | [51] |
Seedless Kishu | Seedless mutation of ‘Kishu’ mandarin | [52] |
Simeto | ‘Miho’ satsuma × ‘Avana’ mandarin | [50] |
Star Ruby | Grapefruit clonal selection | [53] |
Sunburst | ‘Robinson’ mandarin × ‘Osceola’ mandarin | [54] |
Wilking | ‘King’ tangor × ‘Willowleaf ‘ mandarin | [39] |
Primer Name | Primer Sequence (5′-3′) | Ta (°C) | Product Size (bp) | Position in Chromosome 3 of the Clementine Genome |
---|---|---|---|---|
SNP08_nested PCR | F: AACTCACAAACATGTCTTCAACAA | 57 | 572 | 25861812…25861844 |
R: ATTGTCAATTGGTGGGAAGC | 25862364…25862383 | |||
SNP08_HRM | F: AGCGAATAAATTTGATGCTGAGC | 60 | 110 | 25862022…25862044 |
R: TCTTGTAGGACTAAAATTTCACTTGGA | 25862105…25862131 |
Genotypes | SNP08 | SNP08+18bp | Genotype Code |
---|---|---|---|
Clemapo | TT | CC | 1A |
Fallglo | TT | CC | 1A |
Foma107 | TT | CC | 1A |
ISA | TT | CC | 1A |
Kara | TT | CC | 1A |
Kinnow | TT | CC | 1A |
Mapo | TT | CC | 1A |
Okitsu | TT | CC | 1A |
OTA9 | TT | CC | 1A |
Rubino | TT | CC | 1A |
Simeto | TT | CC | 1A |
Wilking | TT | CC | 1A |
50-15A-6 | TT | CT | 1B |
Avana | TT | CT | 1B |
Cami | TT | CT | 1B |
Carvalhais | TT | CT | 1B |
Encore | TT | CT | 1B |
Fremont | TT | CT | 1B |
King | TT | CT | 1B |
Palazzelli | TT | CT | 1B |
Afourer | TT | TT | 1C |
Bower | GT | CC | 2A |
Daisy | GT | CC | 2A |
Doppio Sanguigno | GT | CC | 2A |
Fairchild | GT | CC | 2A |
Fortune | GT | CC | 2A |
Michal | GT | CC | 2A |
Nova | GT | CC | 2A |
Page | GT | CC | 2A |
Primosole | GT | CC | 2A |
Seedless Kishu | GT | CC | 2A |
Star Ruby | GT | CC | 2A |
Sunburst | GT | CC | 2A |
Ellendale | GT | CT | 2B |
Emperor | GT | CT | 2B |
Murcott | GT | CT | 2B |
Ortanique | GT | CT | 2B |
Ponkan | GT | CT | 2B |
Dancy | GG | CC | 3A |
Malvasio | GG | CC | 3A |
Minneola | GG | CC | 3A |
Parent Combinations | Hybrids Analysed | Resistants | % | Susceptibles | % | Chi-Square | p Value |
---|---|---|---|---|---|---|---|
ISA × Seedless Kishu | 351 | 186 | 53.0 | 165 | 47.0 | 1.256 | 0.262 |
Foma107 × Seedless Kishu | 162 | 74 | 45.7 | 88 | 54.3 | 1.210 | 0.271 |
Cami × Seedless Kishu | 84 | 39 | 46.4 | 45 | 53.6 | 0.429 | 0.513 |
Rubino × Murcott | 148 | 54 | 36.5 | 94 | 63.5 | 10.811 | 0.001 |
Fortune × Ota9 | 117 | 60 | 51.3 | 57 | 48.7 | 0.077 | 0.782 |
Total | 862 | 413 | 47.9 | 449 | 52.1 | 1.503 | 0.220 |
Parent Combinations | Hybrids Analysed | Resistants | % | Susceptibles | % | Chi-Square | p Value |
---|---|---|---|---|---|---|---|
ISA × Seedless Kishu | 14 | 10 | 71.4 | 4 | 28.6 | 2.571 | 0.109 |
Foma107 × Seedless Kishu | 20 | 10 | 50.0 | 10 | 50.0 | 0.000 | 1.000 |
Cami × Seedless Kishu | 20 | 12 | 60.0 | 8 | 40.0 | 0.800 | 0.371 |
Rubino × Murcott | 26 | 8 | 30.8 | 18 | 69.2 | 3.846 | 0.050 |
Fortune × Ota9 | 21 | 13 | 61.9 | 8 | 38.1 | 1.190 | 0.275 |
Total | 101 | 53 | 52.5 | 48 | 47.5 | 0.248 | 0.619 |
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Arlotta, C.; Ciacciulli, A.; Strano, M.C.; Cafaro, V.; Salonia, F.; Caruso, P.; Licciardello, C.; Russo, G.; Smith, M.W.; Cuenca, J.; et al. Disease Resistant Citrus Breeding Using Newly Developed High Resolution Melting and CAPS Protocols for Alternaria Brown Spot Marker Assisted Selection. Agronomy 2020, 10, 1368. https://doi.org/10.3390/agronomy10091368
Arlotta C, Ciacciulli A, Strano MC, Cafaro V, Salonia F, Caruso P, Licciardello C, Russo G, Smith MW, Cuenca J, et al. Disease Resistant Citrus Breeding Using Newly Developed High Resolution Melting and CAPS Protocols for Alternaria Brown Spot Marker Assisted Selection. Agronomy. 2020; 10(9):1368. https://doi.org/10.3390/agronomy10091368
Chicago/Turabian StyleArlotta, Carmen, Angelo Ciacciulli, Maria Concetta Strano, Valeria Cafaro, Fabrizio Salonia, Paola Caruso, Concetta Licciardello, Giuseppe Russo, Malcolm Wesley Smith, Jose Cuenca, and et al. 2020. "Disease Resistant Citrus Breeding Using Newly Developed High Resolution Melting and CAPS Protocols for Alternaria Brown Spot Marker Assisted Selection" Agronomy 10, no. 9: 1368. https://doi.org/10.3390/agronomy10091368