Abstract
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.
Highlights
Genetic progress in citrus breeding programs is hampered by the complex reproductive biology, the high level of heterozygosity, large plant size, and the long juvenile period
While the study confirmed that the SNP08 marker is a reliable tool for marker assisted selection (MAS) of new citrus hybrids with different genetic backgrounds, it identified a small group of genotypes where the resistance mechanism requires further investigation
The present study reports the development of two complementary methods based on high resolution melting (HRM) and cleaved amplified polymorphic sequences (CAPS) for the analysis of SNP08 in citrus, with validation on a large number of mandarin cultivars and progenies having different genetic backgrounds
Summary
Genetic progress in citrus breeding programs is hampered by the complex reproductive biology, the high level of heterozygosity, large plant size, and the long juvenile period. Progress has been made in the last few years with the identification of candidate genes or loci associated with important pomological and disease resistance traits, through conventional mapping and Genome-Wide Association Study (GWAS). Markers for assisted selections are available for anthocyanin pigmentation [3], nucellar embryony [4], Alternaria brown spot (ABS) resistance [5] and CTV resistance [6]. ABS is caused by the fungus Alternaria alternata (Fr.) Keissl, whose germinated spores produce an Alternaria citri toxin (ACT) that kills the host cells, so that the pathogen can utilize the released nutrients. The pathogen causes brown-to-black necrotic lesions surrounded by a yellow halo in twigs, young leaves, and fruits [7,8,9], inducing defoliation and fruit drop
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