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Article

Disease Resistant Citrus Breeding Using Newly Developed High Resolution Melting and CAPS Protocols for Alternaria Brown Spot Marker Assisted Selection

1
CREA-Research Centre for Olive, Fruit and Citrus crops—Corso Savoia 190, 95024 Acireale (CT), Italy
2
Department of Agriculture, Food and Environment (Di3A), University of Catania—Via Valdisavoia 5, 95123 Catania, Italy
3
Department of Agriculture and Fisheries, 49 Ashfield Road, Bundaberg, QLD 4670, Australia
4
Centro de Citricultura y Producción Vegetal, Instituto Valenciano de Investigaciones Agrarias (IVIA), Moncada, 46113 Valencia, Spain
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Agronomy 2020, 10(9), 1368; https://doi.org/10.3390/agronomy10091368
Received: 25 July 2020 / Revised: 31 August 2020 / Accepted: 8 September 2020 / Published: 11 September 2020
(This article belongs to the Special Issue Recent Advances in Breeding and Production of Citrus)
Alternaria alternata is a fungus that causes a serious disease in susceptible genotypes of citrus, particularly in mandarins. The Alternaria citri toxin (ACT) produced by the pathogen induces necrotic lesions on young leaves and fruits, defoliation and fruit drop. Here, we describe two methods of marker-assisted selection (MAS) that could be used for the early identification of Alternaria brown spot (ABS)-resistant mandarin hybrids. The first method is based on a nested PCR coupled to high resolution melting (HRM) analysis at the SNP08 locus, which is located at 0.4 cM from the ABS resistance locus, and was previously indicated as the most suitable for the selection of ABS-resistant hybrids. The method was validated on 41 mandarin hybrids of the CREA germplasm collection, and on 862 progenies generated from five crosses involving different susceptible parents. Four out of five populations showed Mendelian segregation at the analyzed locus, while a population involving Murcott tangor as male parent showed distorted segregation toward the susceptible hybrids. The second method is based on a cleaved amplified polymorphic sequences (CAPS) marker that was developed using the same primers as the nested PCR at the SNP08 locus, coupled with BccI restriction enzyme digestion. To verify the reliability of the two genotyping methods, in vitro leaf phenotyping was carried out by inoculating A. alternata spores onto young leaves of 101 hybrids, randomly chosen among the susceptible and resistant progenies. The phenotyping confirmed the SNP08 genotyping results, so the proposed method of selection based on HRM or CAPS genotyping could be routinely used as an alternative to KBioscience competitive allele specific polymerase chain reaction (KASPar) single nucleotide polymorphism (SNP) genotyping system to improve citrus breeding programs. While the study confirmed that the SNP08 marker is a reliable tool for MAS of new citrus hybrids with different genetic backgrounds, it also identified a small group of genotypes where the resistance mechanism requires further investigation. View Full-Text
Keywords: Alternaria alternata; genotyping; mandarins; single nucleotide polymorphisms Alternaria alternata; genotyping; mandarins; single nucleotide polymorphisms
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MDPI and ACS Style

Arlotta, C.; Ciacciulli, A.; Strano, M.C.; Cafaro, V.; Salonia, F.; Caruso, P.; Licciardello, C.; Russo, G.; Smith, M.W.; Cuenca, J.; Aleza, P.; Caruso, M. 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

AMA Style

Arlotta C, Ciacciulli A, Strano MC, Cafaro V, Salonia F, Caruso P, Licciardello C, Russo G, Smith MW, Cuenca J, Aleza P, Caruso M. 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 Style

Arlotta, Carmen, Angelo Ciacciulli, Maria Concetta Strano, Valeria Cafaro, Fabrizio Salonia, Paola Caruso, Concetta Licciardello, Giuseppe Russo, Malcolm Wesley Smith, Jose Cuenca, Pablo Aleza, and Marco Caruso. 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

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