Next-generation sequencing technologies and genome editing to investigate the genetic bases of self-incompatibility in citrus

Self-incompatibility (SI) is a system used by flowering plants to prevent self-fertilization and promote outbreeding. Among citrus species, several pummelo, mandarin, and mandarin-like accessions show a gametophytic SI system based on Sribonucleases (S-RNases) able to impair self-pollen tube growth in the style. In these species, SI coupled with a variable degree of parthenocarpy ensures the production of seedless fruits, highly appreciated by consumers. Several S-RNases have been identified including a mutated S-RNase (Sm-RNase) responsible for the loss of SI in different citrus species. In the present study, a reference genome of the self-compatible (SC) ‘Monreal’ clementine (Citrus clementina Hort. ex Tan.), a mutant of the SI ‘Comune’ clementine, is presented for the first time, and employed for molecular characterization of the SI/SC mechanism. The analysis clarified that both genotypes are characterized by the presence of S7- and S11-RNAases. RNA-seq analysis of unpollinated pistils at the mature stage revealed the lack of expression of S7-RNase in ‘Monreal’ indicating its involvement in the loss of SI response. The alignment of the RNA-seq reads against the reference genome of ‘Comune’ clementine led to the identification of 165 DEGs characterized by the presence of polymorphisms predicted to induce mutations with a relevant effect on their amino acid sequence. Most of the identified genes include several transcription factors and genes associated with flowering, such as SQUAMOSA, Agamous-like MADS-box proteins, MIB 111, WRKY and WUSCHEL, suggesting their involvement in the regulation of SI. Furthermore, CRISPR/Cas9 approach is underway in ‘Doppio Sanguigno’ sweet orange (SC) by targeting the Sm-RNase with the aim to revert its mutation and consequently restore the SI. The results will help to understand the genetic basis of SI in citrus and will allow to plan breeding programs to obtain new seedless genotypes.