Screening And Functional Identification Of MiRNAs Related To Petal Coloration Of Pink-flowered Strawberry

The flowers of the genus Fragaria species were white.The pink-flowered strawberry is an intergeneric hybrid with red or pink flowers derived from the cross between the white-flowered Fragaria cultivar(2n=8x=56)and a red-flowered Potentilla palustris species(2n=7x=42),which is an ideal test material to study the mechanism of petal coloration.Pink-flowered strawberry is brightly colored and can be used as a perennial ornamental herbal flower,which is popular because of its high ornamental and edible value.The color of petals,as the most important ornamental character of pink-flowered strawberry,shows different degrees of red petals due to different levels of anthocyanin accumulation.As an important post-transcriptional regulator,mi RNAs play important roles in plants,while it is not clear which mi RNAs are involved in the post-transcriptional regulation of flower development and color change of pink-flowered strawberry.Therefore,in order to clarify the post-transcriptional regulation mechanism of flower color formation in pink-flowered strawberry,the buds of pink-flowered strawberry cultivar ’Sijihong’ in three development stages(young bud stage,coloration beginning stage and big bud stage)were selected for constructing small RNA,transcriptome and mixed degradation libraries of nine petals at different developmental stages and conjoint analysis of s RNAome,transcriptome,and degradome sequencing.The differentially expressed mi RNA-target related to petal color regulation were screened out via multiple bioinformatics methods,which were Fami R858＿R-2-Fa Myb308,Fami R159d-Fa GAMYB,Fami R828a-Fa MYB114,Fami R396e-Fab HLH79 and Fami R156a-Fa SPL13 A respectively.Cloning,overexpression vector construction,bioinformatics analysis,phylogenetic tree construction and function analysis were performed respectively to determine the function of these candidate mi RNAs during flower bud development,and the results will provide a theoretical basisfor further analysis of the molecular mechanism of petals coloration in pink-flowered strawberry.The main results were as follows:1.A total of 739 known mi RNAs and 964 novel mi RNAs were identified via small RNA sequencing,and 8149 differentially expressed target genes were selected via transcriptome sequencing.639 mi RNAs were identified to cleave 2816 target genes based on the degradome data.Additionally,317 differentially expressed mi RNAs among the various stages of flower development were found,which regulated 2134 differentially expressed target genes.These target genes were significantly enriched in the transcriptional regulation,phenylpropanoid biosynthesis,and plant hormone signal transduction pathways.Furthermore,the multi-omics joint analysis suggested that 98 mi RNAs targeted several transcription factors,including MYBs(26),b HLHs(12),NACs(14),and SPLs(19),related to anthocyanin accumulation and no mi RNAs was found to directly regulate the structural genes associated with anthocyanin biosynthesis.In addition,27 differentially expressed mi RNAs might affect anthocyanin biosynthesis by regulating 23 targets involved in the hormone signal transduction pathway.2.According to degradome data and expression profiles of differentially expressed mi RNA-targets,a total of eight Fv MYBs were targeted by different mi RNAs and showed the opposite expression trend.Through constructing phylogenetic tree with At R2R3-MYBs in Arabidopsis thaliana,Fv Myb308 and Fv GAMYB were found to belong to subgroup 4,which inhibited anthocyanin synthesis,Fv MYB114 in subgroup 6 promoted anthocyanin synthesis and Fv MYB4 and Fv MYB5 in subgroup 7 controlled the synthesis of flavonoids in petals of pink-flowered strawberry.Therefore,those corresponding mi RNAs might have important regulatory functions in petal coloring of pink-flowered strawberry including Fami R828a(target Fv MYB114 Fv H4＿2g31020.1),Fami R159d-R-1(target Fv GAMYB Fv H4＿7g04470.1),and Fami R858＿R-2(target Fv Myb308 Fv H4＿2g01320.1).This suggests that Fami R828 a may inhibit anthocyanin accumulation in pink-flowered straeberry petals by targeting Fa MYB114 and Fami R858＿R-2 promote anthocyanin accumulation in pink-flowered straeberry petals by targeting Fv Myb308.3.It was found five Fvb HLHs(Fvb HLH30,Fvb HLH77,Fvb HLH79,Fvb HLH113,and Fvb HLH137)may relate to the anthocyanin synthesis via phylogenetic analysis with Atb HLHs,their corresponding mi RNAs might affect the petal coloration of pink-flowered strawberry including zma-mi R396f-p5(targeting Fvb HLH30),mdm-mi R393a(targeting Fvb HLH77),mdm-mi R396e(targeting Fvb HLH79),nta-mi R172e-p3(targeting Fvb HLH113)and osa-mi R5826＿L-4R-2(targeting Fvb HLH137).Small RNA sequencing results showed that among the five mi RNAs,only mdm-mi R396 e expression level was high,and the rest were low.Transcriptome sequencing showed that the expression of Fvb HLH79 increased,while that of Fami R396 e decreased during flower development.These results indicated that Fami R396 e and Fab HLH79 play the important roles in regulating anthocyanin accumulation.4.mi R156 s positively regulate anthocyanin biosynthesis by targeting SPL transcription factors,and SPLs negatively regulate anthocyanin accumulation through MYB-b HLH-WD40 complex.In this study,eighteen mi R156 s targeted five SPLs,and the expression level of Fami R156 s gradually increased during flower development,with the opposite expression level of their target genes.Five mi RNAs involved in phytohormone signal transduction were identified and the regulatory relationship with their target genes were further verified by q RT-PCR analysis.The results showed that the expression of Fami R160 s gradually decreased with petal development,but the expression of these target gene Fa ARFs increased significantly;Fami R172a＿R+1 accumulated highly in the big bud stage,contrary to Fa ERF(Fv H4＿6g15180.1),Fami R156f-p5 and its target gene Fa CTR1(Fv H4＿3g39380.1)showed the same trend.5.Cloning and screening of mi RNAs-targets associated with the color of pink-flowered strawberry petals,The results show that: the sequence of the precursor of Fami R858＿R-2 was68 bp,and the coding region of the target gene Fa Myb308 was 828 bp,encoding 275 amino acids.The precursor sequence of Fami R159 d was 96 bp,and the coding region of its target gene Fa GAMYB was 1689 bp,encoding 562 amino acids.The precursor sequence of Fami R828 a was 126 bp.The precursor sequence of Fami R396 e was 164 bp,and the coding region of its target gene Fab HLH79 was 804 bp,encoding 267 amino acids.The precursor sequence of Fami R156 a was 104 bp,and the coding region of its target gene Fa SPL13 A was1024 bp,encoding 411 amino acids.In addition,all the phylogenetic trees constructed by mi RNAs-targets are clustered with Rosaceae plants,indicating that they were closely related to Rosaceae plants,similar in functions,and accurate in cloning.6.The p RI-101 AN fused expression vector was constructed for the final selected mi RNA-targets relationship pairs(Fami R858＿R-2-Fa Myb308 、 Fami R828a-Fa MYB114 、Fami R396e-Fab HLH79 and Fami R156a-Fa SPL13A),And overexpression of mi RNAs transiently infected the pink-flowered strawberry cultivar ’Fenyun’.The petals injected with p RI-101 AN were used as the control and observed changes of flower color phenotypes within3-7 days after injection.Through q RT-PCR and anthocyanin content determination.The results show that,injection of Fami R156 a,Fami R396 e,and Fami R858＿R-2 in the ‘Fenyun’increased flower color intensity,and promoted the accumulation of anthocyanin,while transient expression of Fami R828 a decreased flower color intensity and inhibited the accumulation of anthocyanin in ’Fenyun’.