Analysis And Identification Of MiRNAs Related To Flower Color In Red-Flowered Strawberry

The red-flowered strawberry is a new member of strawberry,belonging to a genus hybrid(Fragaria × Potentilla),its red flower trait is derived from the cross of cultivated pineapple strawberry(Fragaria × ananassa Duch.)and a purple-colored marsh cinquefoil relative(Potentilla palustis(L.)Scop.).Compared with traditionally cultivated strawberries,red-flowered strawberry not only has rich nutritional value,but also has good ornamental value.It can be applied to landscaping and potted plants.It is a new type of strawberry with both ornamental and edible characteristics,which has good development potential.Anthocyanin is the material basis for red-flowered strawberry to form red color.The red-flowered strawberry cultivar 'Sijihong' was used as materials,and its three developmental stages with distinct color changes,namely bud stage(PF_L),color change stage(PF_Z),and large bud stage(PF_D)were used sequenceing,nine sample small RNA libraries(PF_L1,PF_L2,PF_L3,PF_Z1,PF_Z2,PF_Z3,PF_D1,PF_D2,PF_D3)and one mixed Degradome libraries(PF)were constructed.We focused on the discovery of mi RNAs that can participate in the regulation of anthocyanin biosynthesis by using high-throughput sequencing technology in the development of red-flowered strawberry petals.Target gene prediction and verification of mi RNAs obtained from small RNA sequencing of red-flowered strawberry using Target Finder software and degradome sequencing technology.In addition,the stable expression of internal reference genes suitable for mi RNA expression in red-flowered strawberry was screened by using ge Norm and Norm Finder software,which laid a foundation for further research on mi RNAs related to flower color formation in red-flowered strawberry.The results were as follows:(1)The original total reads of nine s RNA libraries(PF_L1,PF_L2,PF_L3,PF_Z1,PF_Z2,PF_Z3,PF_D1,PF_D2,PF_D3)obtained by s RNA sequencing technology were 1.70×10~7,1.59×10~7,1.47×10~7,1.27×10~7,1.27×10~7,1.46×10~7,1.18×10~7,1.26×10~7 and 1.32×10~7.A total of 1,703 mi RNAs were identified,of which 665 mi RNAs were expressed in all nine sample s RNA libraries.A total of 317 significantly differentially expressed mi RNAs were identified in the petals of three different developmental stages,and 127 of them had a very significant difference.(2)By degradome sequencing,2.37 × 10~7 original reads were obtained in the PF library.When the 3' adaptors sequences were removed,it was found that the PF degradation group library could match the reads on the strawberry genome to 2.34×10~7.At present,the number of annotated transcripts of strawberries is 39,487,and the number of transcripts of the degradome PF could be matched to 34,202,and the proportion of the total transcripts of the annotated strawberries is 86.26%.Target mi RNA target gene prediction was performed using Target Finder software.The results showed 14,406 target genes corresponding to 1,365 mi RNAs.The degradation sites of the degradome and the transcriptome data were detected by Cleaveland software.A total of 227 target genes corresponding to 166 mi RNAs were detected to be degraded.Further analysis found that more than 80% of mi RNA correspond to multiple target genes,and the number of target genes corresponding to a single mi RNA ranges from 2 to 26.Only 12 mi RNAs were corresponded to the only one target gene.In addition,it had been also found that a single target gene was controlled by multiple mi RNAs.This indicated that there was a complex regulatory network between mi RNAs and their target genes.(3)The stability of the reference genes of eight candidate mi RNAs was evaluated by ge Norm,Norm Finder and Bestkeeper software.The results showed that the mdm-mi R11004 was the most stable candidate reference gene for q RT-PCR of red-flowered strawberry in all conditions.bdi-mi R159 a is the most unstable reference gene expressed in all tissue samples of red-flowered strawberry.(4)Through the combined analysis of sequencing data of mi RNAs,transcriptome and degradome,33 candidate mi RNAs related to flower color formation of red-flowered strawberry and 40 corresponding target genes were predicted.Eight color-related candidate mi RNAs and their targets were selected for q RT-PCR expression analysis in of red-flowered strawberry with different petal colors.Two mi RNAs and their corresponding target genes were initially obtained and probably involved in the regulation of flower color through negative regulation,namely mdm-mi R159 d and its target genes NCL1 and csi-mi R156f-p5 and its corresponding target genes SPL13 and MYB44.These two mi RNAs probably play an important role in the biosynthesis of anthocyanins in red-flowered strawberry.