Identification of mRNA transfer between citrus rootstock and scion based on characteristic SNP

Abstract:【Objective】Grafting has been an ancient technique widely used to improve and reproduce horticultural crops for at least 1500 years. Citrus plants with a seed-to-seed cycle of 5–15 years, are among the fruit crops that were probably domesticated by grafting. Citrus trees propagated by grafting are composed of scions and rootstocks. Exchange of materials between the rootstocks and scions occur frequently. The interaction directly affects the horticultural characters of citrus, the nutrient absorption of the underground part and the relative resistance of the aboveground part, thus affecting the production and economic benefit of citrus orchards. With the development of molecular biology, a growing number of studies have shown that small molecules such as mRNA can transmit between the rootstocks and the scions and regulate related agronomic traits. This study aimed to screen and validate candidate mRNAs that can be transmitted between the rootstocks and scions in citrus plants.【Methods】The 2- year-old seedlings of Citrus sinensis and Poncirus trifoliata with the same growth potential in the specimen garden of Huazhong Agricultural University were selected for producing nursery trees of C. sinensis/P. trifoliata and P. trifoliata/P. trifoliata. The samples were taken 5 months after removal of grafted plastic wrap and were put in the ice box and were brought to the laboratory. The samples were cleaned and three tissues of each grafting combination were collected, and 3 samples were collected from each part (the sample length was 1 cm). Then the RNA was extracted from the samples. By comparing the transcript sequences between the C. sinensis and the P. trifoliata, we obtained a set of species specific SNP sites. The mRNAs that might be transmitted between the rootstock and the scion were screened, and then the candidate mRNAs were verified by RT-PCR based on the characteristic SNP sites.【Results】The transcriptome sequencing was performed for the grafting interface, rootstock and scion of the grafted citrus plants, and 18 sets of data were obtained. Combined with the previously published transcription data of sweet orange, 254 580 SNP were obtained by analyzing the data of grafting data analysis. Some transmissible candidate mRNAs were detected based on these characteristic SNP sites. The results showed that 24 candidate mRNAs could be transmitted from rootstocks to scions, and 30 candidate mRNAs could be transmitted from scions to rootstocks in the C. sinensis/P. trifoliata grafted nursery trees. The candidate mRNAs were further confirmed by species specific SNP-based PCR amplification and sequencing. The mRNAs of two genes (Pt2g028010.4 and Pt3g020940.1) were found to be surely transferred from the P. trifoliata to the C. sinensis, and the mRNA of one gene (Cs2g16070.1) from the C. sinensis to the P. trifoliata.【Conclusion】By analyzing and comparing the characteristic single nucleotide polymorphisms (SNPs) of sweet orange and trifoliate orange, the mRNAs that could be transmitted between citrus rootstocks and scions were screened out. These mRNAs were further verified by SNP-PCR and three reliable transmissible mRNAs were obtained. The mRNAs transmitted between the rootstocks and scions were identified.