Phenotyping and genotyping of a lemon segregating population to mining genetic resistance to biotic stresses

Among citrus species, lemon (Citrus limon (L.) Burm. f.) is one of the most important crops and the most susceptible to a severe tracheomycosis called mal secco (“dry disease” in Italian), which is the most limiting factor for its cultivation in the Mediterranean and Black Sea area (48% of lemon worldwide production). Mal secco is caused by the mitosporic fungus Plenodomus tracheiphilus (Petri) Gruyter, Aveskamp, and Verkley (syn. Phoma tracheiphila (Petri) Kantschaveli and Gikashvili), which induces vein chlorosis of young shoots leaves, followed by phylloptosis, wood discoloration, necrosis, and the progressive desiccation of the whole plant. To date, no agronomic or chemical means have proven to be effective in containing the diffusion of the pathogen, neither breeding programs reached the goals of deciphering the genetic mechanisms of resistance to the pathogen and of developing novel cultivars coupling optimal fruit quality and tolerance to mal secco disease. More recently, modern high-throughput genotyping platforms and bioinformatic analysis were employed for a marker-trait association study aimed at the identification of molecular markers linked to the resistance towards mal secco in a segregating population. To this extent, a lemon segregating population (120 individuals) has been developed by crossing the mal secco-tolerant lemon cultivar ‘Interdonato’ (female parent) with ‘Femminello Siracusano 2Kr’ lemon (male parent), which produces good quality fruits, but is highly susceptible to mal secco. The behaviour of this full-sib population towards mal secco infection has been monitored after artificial inoculation of the pathogen, through an approach including visual and digital assessment of the symptoms, molecular detection of the pathogen spread among infected tissues and histological analysis of host response. Since the lemon segregating population was naturally attacked by the two-spotted spider mite Tetranychus urticae Koch, one of the most damaging polyphagous pests, showing a wide range of symptoms among the different genotypes, the response of the plants to the mite was also considered. To do so, bioassays were developed ad hoc using detached leaves placed in special experimental arenas. A functional genomic study is under progression together with phenotyping. Taking advantage of the recent de novo sequencing of lemon, the full-sib population will be genotyped through the single primer enrichment technology (SPET) approach, and QTL analysis on selected SNPs will lead to the identification of genomic regions significantly associated with resistance to mal secco or to the spider mite. Hopefully, in the near future, robust molecular markers linked to resistance to these biotic stresses would be applicable in marker assisted selection (MAS) and in genetic transformation experiments.