Identification And Mechanism Of Non-Coding RNAs Related To Adventitious Roots In Apple Rootstocks

Adventitious root formation is one of the key factors in the asexual propagation of apple dwarf rootstocks.Non-coding RNA plays an important role in the regulation of adventitious roots(ARs),however,the related research in apple is weak.In this study,the main apple rootstock 'M26',which is widely used in China,was used as the main test material.The whole database was constructed during the critical period of ARs induction,and the whole genome was sequenced to comprehensively identify long non-coding RNAs(lncRNAs)involved in the regulation of ARs.A preliminary analysis of the candidate miRNA160 involved in the function of ARs was carried out.The main results were as follows:1 Three stages of ARs development of apple rootstock 'M26' tissue culture seedlings were identified: AR primordium induction period(0-3 d);AR primordium formation period(3-7 d);AR differentiation formation stage(7-16)d).Endogenous hormone assays at the base of tissue cultured shoots at adventitious root development showed that after IBA treated,auxin(IAA),zeatin(ZR)and jasmonic acid(JA)increased significantly and then decreased on the third day,while Gibberellin(GA3)was significantly decreased during the induction period(3 d).A high-throughput whole-genome sequencing of the stem base zone of the tissue culture seedlings in the control and IBA treatment induction period revealed 855 long non-coding RNAs(lncRNAs),of which 63 were identified as IBA-responsive lncRNAs.Fifteen IBA-responsive lncRNAs were predicted to have a targeted relationship with 94 miRNAs.A total of 2014 lncRNA different expressed target genes(DEGs)were found by matching.The annotation functions of DEGs were mainly related to the biology of plant hormones and sucrose signaling and so on.The ceRNA combined expression pattern was verified by RT-qPCR and 2 ceRNA combinations(CUFF.41325-mdm-miR396b-MdSRP68 and CUFF.41325-mdm-miR396b-MdJIP-23)were finally identified.2 In the apple genome,five family members of miRNA160 were identified,which respectively named mdm-miR160 a,mdm-miR160 b,mdm-miR160 c,mdm-miR160 d and mdm-miR160 e.Four rootstocks with significant differences ability to develop AR include:the easy rooting type 'MP',the medium rooting type 'M26' and 'T337',the hard rooting type'SH6' as the material and the sequence of precursor of five family members of mdm-miR160 cloned in four rootstocks showed that the mdm-miR160 b and mdm-miR160 d sequences differed in four varieties while the other three members were highly conserved.Real-timequantitative PCR showed that the relative expression of mdm-miR160 in the roots of apple rootstock 'M26' at 3 and 7 days after IBA treatment was the highest,and negatively correlated with the target genes(MdARF16 and MdARF17).The functional analysis of transformed with the mdm-miR160 a overexpression vector transformed with SR1 tobacco showed that overexpression of mdm-miR160 significantly inhibited the occurrence of adventitious roots,and exogenous IBA treatment could alleviate this inhibition.