The Involvement Of MiRNAs In The Growth Regulation Of The Sweet Cherry Scion Grafted On Red Bark Rootstock Line

Sweet cherry(Prunus avium L.)is a perennial fruit tree of the genus Prunus in the family Rosaceae.Because of the early maturity and excellent quality of fruit,it has been widely introduced and cultivated in the south of China during the past decade.Grafting is the chief method for plant propagation of sweet cherry.In Guizhou province,it was found that the northern rootstock lines did not well satisfy the southern weather environment,so it is urgent to select suitable local rootstocks.Guizhou province possesses abundant cherry(Cerasus pseudocerasus)germplasms,which provide the original material for the selection and breeding of sweet cherry rootstocks.In previous study,a red bark cherry line was selected from wild cherry,which demonstrated good graft compatibility onto sweet cherries,with increased plant tolerance to waterlogging and showed semi-dwarfism.Previous studies have mostly focused on the cellular structure of the graft union region and the physiological and biochemical level changes of the grafted seedlings after grafting,however the regulatory mechanisms of rootstocks on sweet cherry scions at the molecular level have been rarely reported,particularly,the molecular roles involving miRNAs has not yet been documented so far.To better understand the mechanism of miRNA regulation of scion dwarfism,in the present study,the wild cherry "Red bark" line and its wild type "Cyan bark" were selected as rootstocks,and the terminal buds and leaves of sweet cherry scions grafted on the two rootstocks were selected as materials.The genome-wide identification of sweet cherry miRNAs and its target genes was carried out using smallRNA group and degradome sequencing technologies to investigate the molecular regulatory mechanisms of miRNA-mRNA regulation of sweet cherry scion growth and development,with the aim to provide a theoretical basis for the selection and breeding of sweet cherry rootstocks.The main results were as follows.1.Genome-wide identification of miRNAs and phasiRNA in sweet cherryA total of 12 sRNA libraries from the terminal buds and leaves of sweet cherry scions of two rootstocks were used to identify sweet cherry miRNAs at the genomewide level.A degradome library was constructed from the leaves,and miRNA target genes were identified by degradome sequencing.The results showed that a total of 178 known miRNAs and 39 novel miRNAs were identified,including 48 known miRNA family members and 35 novel miRNA family members.Based on the mature sequence consistency of miRNA families,most of the known miRNA families were highly conserved,except miRN482.Among them,30 known miRNA families(98 members)and 11 novel miRNA families(12 members)were obtained with a total of 170 target genes.A total of 183 phasiRNA loci were obtained in sweet cherry.The analysis of phasiRNAs mediators revealed the presence of phsiRNA pathways conserved in plants,including miR393-TIR/AFB,miR482-NB-LRR,miR858/828-MYB and miR7122-PRR.In addition,the specific phsiRNA pathways of sweet cherry were identified,including miRN3-PHAS85-NAC,miRN10-PHAS42,miRN22-PHAS59,and miRN11-PHAS146.2.Identification of differentially expressed miRNAs in sweet cherry scionsCompared with terminal buds(ZCB)of sweet cherry scion grafted on normal rootstocks,four miRNAs were significantly down-regulated in dwarf rootstock scion terminal buds(YCB),including miR171c-3p,miR2118-3p,miR171h-3p,and miR159a-3p.The significantly up-regulated expression miRNAs mainly included miR166a/b/c/e/d,miR164a-5p,and miR394a/b/c.There were 25 significantly differentially expressed miRNAs between ZCL and YCL,including five novel miRNAs and 20 known miRNAs.The significantly down-regulated miRNAs in dwarf rootstock scion leaves(YCL)included miR171h-3p,miR159b-3p,miR319c-3p and miR398c-3p.The significantly up-regulated expression miRNAs of YCL sample included miR396b-5p,miR393b-5p,miR827-3p,miR858 a,etc.The GO enrichment of differentially expressed miRNA target genes revealed that 18 and 14 GO terms were enriched in the terminal buds and leaves,respectively.The KEGG enrichment results showed that terminal buds and leaves of scion were both enriched in the plant hormone signaling pathway.The target genes of differentially expressed miRNA were classified into four categories,shoot apical meristem-related,stem elongation-related,phytohormone synthesis and signal transduction,and nutrient balance,indicating the complex miRNA regulatory network in the regulation of scion growth by rootstock.3.Functional validation of PavmiR171 h and Pav SCL6The phylogenetic analysis showed that there were great genetic variations in miR171 precursor sequences among species.The PavmiRNA171 of sweet cherry precursors were closely related to the miR171 of peach.A total of forty-five GARS genes were identified from the sweet cherry genome,including three HAM subclass transcription factors.The phylogenetic evolutionary analysis of HAM subclass genes revealed that they could be divided into two subclasses and originated after the separation of gymnosperms and angiosperms,with HAM class I homologs retained in dicotyledons but only HAM class II genes in monocotyledons.The length of stem-loop precursor sequence of PavmiR171 h was 109 bp,and the transgenic lines was significantly taller than the wild type plants.The ORF length of Pav SCL6 gene was2310 bp,encoding 769 aa.The Pav SCL6 gene was localized in the nucleus.The overexpressed-Pav SCL6 transgenic lines caused dwarf plants by inhibiting the expression of gibberellin synthesis genes At GA3OX1,At GA3OX2,At GA20OX1 and At GA20OX2.The yeast one-hybrid assay and dual luciferase assays clarified that the Pav SCL6 gene directly binds to the promoter of Pav GA3OX3 gene and represses gene expression in sweet cherry scions.4.Functional validation of PavmiR159 b and Pav MYB33The mature sequence of miR159 was highly conserved across different species,containing the core sequence "UUGGAUUGAAGGGAGCUC" with a conserved binding sites to target genes.The length of PavmiRNA159 b precursor was 193 bp.Compared with the WT plants,the growth potential of overexpressing PavmiRNA159 b transgenic lines was significantly better than that of the WT.The ORF length of Pav MYB33 gene was 1680 bp,encoding 559 aa.In overexpressed-Pav MYB33 transgenic lines,the Pav MYB33 gene promoted the expression of cell wall hydrolase genes,inhibited vegetative growth and was positively correlated with Pav MYB33 expression.In summary,the present study investigated that the miRNAs affected the development of sweet cherry scions by regulating shoot apical meristem development,phytohormone synthesis and signal transduction,and nutrient uptake and balance.It was demonstrated that PavmiR171h-Pav SCL6 and PavmiR159b-Pav MYB33 modules inhibited plant vegetative growth by regulating gibberellin synthesis and hydrolase gene expression,respectively,and initially revealed the molecular mechanism of miRNA regulation of sweet cherry scion growth.