Identification Of Exchanged MRNA Between Scion And Rootstock In Grafted Citrus

The economic citrus trees are usually composed with two genetically distinct parts:scion and rootstock.The interaction between scion and rootstock can improve the horticulture traits,physiological activities and resistance of citrus trees.Despite numerous studies on the physiological interaction between scions and rootstocks have been performed in citrus,the molecular mechanisms of the scion-rootstock interaction are remains largely unknown.Previous studies reported that mRNAs can exchange between scion and rootstock in the horticultural plants.However,it is unclear that whether mRNA can also exchange between scion and rootstock in citrus.In this study,we constructed the heterologous grafted citrus plants with the common used scion and rootstock,Citrus sinensis Osbeck and Poncirus trifoliata L.Raf.Based on specific SNP derived from high throughput transcriptome sequencing,we primarily identified the mRNA exchanged between the scion and rootstock in citrus,as well as their possible functions.This study would provide the theoretical basis for understanding the mechanism of scion-rootstock interaction in citrus.The main results of this study are showed as follows:1.Two grafted combinations of sweet orange / trifoliate orange and trifoliate orange/ trifoliate orange was constructed.The scions,rootstocks and junctions of these grafted citrus plants were sampled and subjected to transcriptome sequencing.The 18 transcriptome datas obtained here and previously reported 17 transcriptome datas of sweet orange were analyzed,and 254580 diagnostic SNP of sweet orange and trifoliate orange were yield by comparative analysis.If the mRNA in scion has a diagnostic SNP from rootstock could be detected in the grafted combination of sweet orange / trifoliate orange,this mRNA might be considered to transfer from rootstock to scion.At the same way,the mRNA which transfer from scion to rootstock can also be detected.As a result,30 candidate mRNAs were detected to transfer from sweet orange to trifoliate orange,and 24 candidate mRNAs were detected to transfer from trifoliate orange to sweet orange.2.In the heterologous grafted combinations of sweet orange / trifoliate orange,the mRNAs of sweet orange transcriptome datas were mapped to the sweet orange genome,and the unmatched mRNAs were obtained.Subsequently,the unmatched mRNA were mapped to the trifoliate orange genome,and the well matched mRNAs would be considered to be transferred from rootstock to scion.At the same way,the mRNA which transferred from scion to rootstock can also be detected.As a result,3 candidate mRNAswere identified to transfer from sweet orange to trifoliate orange,and 24 candidate mRNAs were identified to transfer from trifoliate orange to sweet orange.3.The movement of mRNAs based on comparative transcriptome analysis in grafted citrus trees were determined by diagnostic SNP,and RT-PCR and sanger sequencing were used to validate the candidate exchanged mRNAs.The results revealed that Cs2g02150.1 and Cs5g20420.1 could transfer from trifoliate orange to sweet orange,and Cs2g16070.1 could transfer from sweet orange to trifoliate orange.The exchanged mRNAs based on the comparative analysis of genome were validated by PCR,and the results showed that the candidate mRNAs of sweet orange could match to trifoliate orange.The results were against to the prerequisites that mRNAs of sweet orange cannot match to trifoliate orange,so the trifoliate orange genome is incomplete,we cannot get the candidate exchanged mRNAs by comparative analysis of genome.4.The protein consereved domains prediction of three exchanged mRNAs in citrus showed that Cs2g16070.1 encodes a O-methyltransferase,Cs2g02150.1 encodes a ubiquitin protein and Cs5g20420.1 encodes a uridine diphosphate glucosyl transferase.In order to further explore the functions of these mRNAs,over-expression vectors of these genes were constructed.The vectors were then transformed into Arabidopsis thaliana,and the positive lines were obtained.Thus,this study would provide the materials for further verification of fuctions of these exchanged mRNAs.