Phenotyping and genotyping of a lemon segregating population to mining genetic resistance to the two-spotted spider mite attack

The two-spotted spider mite (Tetranychus urticae Koch) is a polyphagous mite, which represents a serious adversity for a huge number of open-field and protected crops, including citrus. Its control is extremely difficult since it easily develops resistance against acaricides, due to its short life cycle, abundant progeny and the arrhenotokous parthenocarpy reproduction. Mites penetrate plant cells with their mouthparts, mainly on the underside of leaves, causing spot discoloration and photosynthesis impairment. Among citrus species, lemon (Citrus limon Burm. f.) is one of the most affected by T. urticae and in severe cases rust symptoms are visible on leaves and notably on fruits, negatively affecting their marketability. Furthermore, leaf fall will negatively affect the production in the following year. Even though chemical control and/or the use of natural enemies are efficient strategies to control T. urticae, little is known about the genetic bases of plant-pest interaction. An interspecific segregating population of lemon (120 individuals) was established crossing ‘Interdonato’ (♀) and ‘Femminello Siracusano 2kr’ (♂) for a market-trait association analysis. Offspring were naturally attacked by the two-spotted spider mites and showed a wide range of symptoms, from no symptoms to others that were severely attacked. In light of this field observation, an in vitro assay was performed employing detached leaves placed in a modified Huffaker cell and 4 spider mite females were released on the leaf abaxial surface. Damage percentage was digitally assessed, confirming the wide range of damage on the different genotypes. Based on these results, an untargeted metabolomic approach employing PTR-Tof_MS will be employed to characterize the production of volatile organic compounds (VOCs) of genotypes with contrasting responses to mite infestation. In parallel, the 120 accessions will be genotyped employing the single primer enrichment technology (SPET), taking advantage of the recent de novo sequencing of lemon. Afterwards, a quantitative trait locus (QTL) analysis will be performed for the detection of SNPs (thus specific genomic regions) significantly associated with resistance to two-spotted spider mite attack. Robust molecular markers linked to the trait of interest would be applicable in marker assisted selection (MAS) and in candidate genes identification.