RNA Expression and Post-transcriptional Editing Analyses of Cucumber Plastids Reveals Genetic Differences Associated with Chilling Tolerance.

Tolerance to chilling injury in cucumber (Cucumis sativus L.) is associated with three plastomic single nucleotide polymorphisms (ptSNPs) at bp positions 4,813, 56,561, and 126,349 that are co-inherited. An understanding of the genetic expression of these ptSNPs as a response to chilling is critical for the development of elite germplasm with chilling tolerance. Therefore, a study was designed to elucidate the genotypic and transcriptional effects of these ptSNPs. Sequenom® MassARRAY technology was utilized to characterize heteroplasty and the degree of post-transcriptional editing in 159 individuals from exact reciprocal advanced backcross (ABL: BC_1-5) and inbred-backcross (IBL: BC₂S_1-3) lines grown under greenhouse conditions without chilling. The parents of these populations were ‘Chipper’ (chilling tolerant plastid) and line M29 (chilling susceptible plastid). Additionally, qPCR and RT-qPCR were used to quantify nuclear to plastomic DNA (ptDNA) ratios and ascertain RNA expressional changes across the ptSNPs in ‘Chipper’ and M29 after chilling stress (5.5 hours at 4 ºC under 270 µmol•s-2•m-2 fluorescent lighting). While ‘Chipper’ was homoplastic and monomorphic for all ptSNPs, M29 was heteroplastic and polymorphic for sites 4,813 and 56,561. Plastidic constitutions were constant in the reciprocal ABL and IBL populations throughout backcross generations. Cucumber line NC-76, which possesses the nuclear chilling gene Ch, was heteroplastic, monomorphic for ptSNP 4,813, and polymorphic for sites 56,561 and 126,349. Although the ptSNPs examined were not edited in ‘Chipper’ mRNA transcripts, ptSNP sites 4,813 and 56,561 were partially and fully edited in M29 transcripts, respectively, restoring the sequence to the predominantly susceptible nucleotide. Data indicate that ptDNA quantity and RNA transcription are similar and consistent in both chilling-response types. Quantitative comparison between ptDNA and a single-copy nuclear marker (CsCYP1: cyclophilin) indicated that the relative ratio of ptDNA to nuclear DNA was approximately 1000:1. This study provides the first known quantification of ptDNA in cucumber, and indicates the absence of ptDNA degradation under these chilling conditions. These results provide increased understanding for more efficient introgression of plastidic chilling tolerance in cucumber.