Detection of natural and induced mutations from next generation sequencing data in sweet orange bud sports

Somatic mutations are a cause of intraspecific diversity in many fruit crops, including citrus. In the case of sweet orange [Citrus sinensis (L.) Osbeck], intraspecific variability is determined only by somatic mutations. Tools for clonal fingerprinting are required by breeders and nurserymen and have important implications for traceability. With the aim of studying genomic variability and identifying mutational events responsible for varietal diversification, we deep-resequenced 22 accessions including navel, common and blood oranges using an Illumina platform. We also resequenced an induced mutant of Tarocco 'Scire D2062' obtained using two cycles of gamma ray irradiation at 40 Gy. A robust and reliable set of single nucleotide polymorphisms (SNPs), structural variants (SVs) and indels, specific of each accession or common to varietal groups, was identified both in natural and induced mutants. A subset of SNPs, transposable element insertions and small indels (2 to 18 base pairs) was validated by Sanger sequencing, PCR amplification and high resolution melting analysis, confirming the results of the bioinformatics analysis. Moreover, to identify a reliable marker set for traceability of specific cultivars, we collected leaf and juice samples from many Italian citrus growing areas and used a KASP platform for their fingerprinting. These tools will be useful to prove true-to-typeness of specific sweet orange cultivars.