De novo assembly of Citrus limon and target-sequence genotyping toward the detection of genes involved in tolerance to ‘mal secco’ disease

Lemon (Citrus limon (L.) Burm. f.) is one of the most important citrus species, mainly prized for the organoleptic and nutraceutical properties of the juice and the peel. Herein we report on the release of a high-quality reference genome of lemon. The sequencing was carried out combining Illumina and Oxford Nanopore data leading to the definition of a primary and an alternative assembly characterized by a genome size of 312.8 and 324.74 Mb, respectively. Gene annotation allowed the detection of 35,020 and 34,577 predicted transcripts, respectively, while in silico analysis allowed the identification of 2,878 and 2,897 transposable elements (TE) on the primary and alternative assemblies, respectively. For more than a century, ‘mal secco’, a tracheomycosis caused by the fungus Plenodomus tracheiphilus, has presented a major threat to lemon cultivation in many Mediterranean countries, causing the desiccation of twigs and branches, leading to death of the whole plant. To detect candidate genes linked to ‘mal secco’ tolerance, one inter- and two intraspecific segregating populations were obtained. Three tolerant genotypes namely: ‘Interdonato’ lemon, Citrus clementina, and Citrus latipes, were crossed with the highly susceptible ‘Femminello Siracusano 2Kr’. About 300 hybrids were artificially inoculated or subjected to natural infections for two consecutive seasons and symptoms were monitored by visual screening and real-time PCR. Known lemon ancestors (citron, pummelo and sour orange) were also genotyped and phenotyped to achieve a more complete picture of the allelic flow through generations. The genotyping was performed using a single-primer enrichment technology (SPET), leading to the definition of 30,000 SNPs. Two QTL approaches were carried out: a single-family QTL analysis and a pedigree-based approach (PBA), in which all accessions were analysed simultaneously, taking advantage of the availability of pedigree records. Results will shed light on the genetic regulation of the tolerance to ‘mal secco’ and the availability of molecular markers linked to this trait will play a crucial role towards to obtaining novel cultivars with resistance to the disease with optimal fruit quality and productivity.