Deep resequencing unveils novel SNPs, InDels, and large structural variants for the clonal fingerprinting of sweet orange [Citrus sinensis (L.) Osbeck]

The large phenotypic variability characterizing the sweet orange [Citrus sinensis (L.) Osbeck] germplasm arose from spontaneous somatic mutations and led to the diversification of major groups (common, acidless, Navel, and pigmented). Substantial divergence also occurred within each varietal group. The genetic basis of such variability (i.e., ripening time, fruit shape, color, acidity, and sugar content) is largely uncharacterized, and therefore not exploitable for molecular breeding. Moreover, the clonal nature of all sweet orange accessions hinders the traceability of propagation material and fruit juice using low-density molecular markers. To build a catalog of somatic mutations in Italian varieties, 20 accessions were sequenced at high coverage. This allowed the identification of single nucleotide polymorphisms (SNPs), structural variants (SVs), and large hemizygous deletions, specific to clones or varietal groups. A panel of 239 SNPs was successfully used for genotyping 221 sweet orange accessions, allowing them to be clustered into varietal groups. Furthermore, genotyping of SNPs and SVs was extended to leaf and juice samples of commercial varieties belonging to two varietal groups (Moro and Tarocco) collected from 26 sites in Southern Italy, confirming the usefulness of the identified markers for the identification of specific clones. Interestingly, we found that the insertion of the transposable element VANDAL in the gene exons significantly affected the level of allelic-specific expression. Finally, the markers developed in the present work contribute to unraveling the origin and diversification of sweet oranges, representing a reliable and efficient molecular tool for the unambiguous fingerprint of somatic mutants and an asset for the traceability of orange plant material and fruit juice.