Screening The Important Genes Of 'Hongzaosu' Pear Color Variation And Molecular Mechanism Of Color Formation

Red-skins pears are widely accepted by consumers because of its beautiful appearance and enrichment in health-promoting anthocyanins.However,it is rare of red-skinned pears with high quality,and the colour of the red pears was unstable,uneven,less pigments and other problems.Many studies have been performed on the physiological and molecular mechanisms of red-skins pears.Yet,the regulation network of the pear skin color is not clear.In this study,the key gene PyMYB114 was identified from the F1 generation of'Bayuehong' and 'Dangshansuli',which could regulate the fruit skins color by interacting with PyERF3.In addition,the molecular mechanism of'Hongzaosu' and its green mutant strains were discussed from DNA level(whole genome DNA methylation analysis and SNPs analysis)and RNA level(mRNA expression profile and smallRNA expression profile).The results are as follows:1.Identification of R2R3 MYB gene PyMYB114 by QTL-mapping on LG5.The co-expression of AP2/ERF gene PyERF3 with PyMYB114 by RNA-seq data and showed a significant positive correlation with pear skins coloration.Overexpression of PyMYB114 was identified by transient transformation of tobacco,strawberry and pear can promote anthocyanin biosynthesis,suppression the expression of PyMYB114 can inhibit anthocyanin biosynthesis in red pears.PyERF3 interacted with PyMYB114 and PybHLH3,as shown using dual luciferase reporter and yeast two-hybrid(Y2H)assays,bond to the promoter sequences of PyDFR,PyANS,PyUFGT.A model of the PyMYB114 interaction with PyERF3 and PybHLH3 to co-regulate anthocyanin biosynthesis is proposed.2.The stems,leaves,flowers and fruits of 'Hongzaosu' and its green mutant as materials were used to screen the key genes which could regulate the pear skin color by transcriptome analysis.A total of 14.03G clear reads were obtained from 20 cDNA libraries.Approximately 79.05-80.87%clear reads can be successfully compared to the pear reference genome.A total of 7,650 differentially expressed genes DEGs were identified from the tissues of‘Hongzaosu' and its green mutant.DEGs were enriched in 3,799 GO terms and 1,110 KEGG pathways.Among of them,48 genes involve in flavonoid biosynthesis and 16 genes involve in carotenoid biosynthesis.In addition,there were 6 co-expression DEGs among the four tissues.12 transcription factors were co-expressed in stems,leaves and fruits,including MYB,AP2/ERF,NAC,bZIP and TCP gene family members.Transcriptome data were verified from 12 candidate genes by RT-qPCR analysis,the correlation coefficients were from 0.719 to 0.974.In particularly,the co-expression of PyGST F12-like involved in anthocyanin transport with transcription factors PyMYB114 and PyHY5.The gene PyMYB114 interacted with PyHY5 were identified by the Y2H and dual luciferase reporter system,which largely activated the promoter sequence of PyGST F12-like from the fruit skins of 'Hongzaosu' and the activity reduced from the green mutant of 'Hongzaosu'.Transient transformation strawberry receptacle with PyHY5 and PyMYB114 can promote anthocyanin biosynthesis.3.In this study,the DNA methylation of the stems,leaves,flowers and fruits of'Hongzaosu' and its green mutant was analyzed by whole genome bisulfite sequencing.Hiseq2000 sequencing platform obtained 17?25 G of clear reads,73.14?78.25%of clear reads can be successfully mapping to pear reference genome.The bisulfite conversion efficiency was from 99.54 to 99.61%,and the effective coverage rate of cytosine was 75.79%.Genome methylation level on chromosome distribution of CG,CHG and CHH motifs were 47.81%,32.90%and 5.75%,respectively.In the different transposable element(TE),intron was the highest,followed by mRNA,3'-UTR and CDS,5'-UTR was the lowest.CG and CHG methylation from different organs,the proportion of leaves was highest(40%?41%),followed by stems(34%?37%)and fruit skins(33%?36%),the lowest proportion was flowers(33%).Yet,the highest is fruit skins,followed by flowers,stems and leaves in CHH methylation.Hyper-methylation is closely related to CG and CHG motifs,while hypo-methylation is associated with CHH motif.The trend of methylation mainly distributed in the transposition interval and upstream and downstream 2 kb region,the gene body was a low level.The highest density distribution of methylation was in CGI,followed by mRNA and upstream 2k promoter region,and the methylation of the downstream 2 kb promoter was the lowest.Based on the requirement of at least 6 CG methylation per 100 bp window sliding,135 differential methylation regions(DMR)-related genes were screened,there were two genes involved in flavonoid metabolic pathways and DMR were at the promoter region,and the methylation level of the green skin was much higher than the red skins pears.The CG motif was 0.92,followed by CHH(0.907)and CHG(0.85).4.MiRNA sequencing was used to analysis the fruit skins of 'Hongzaosu' and its green mutant at different development stages.128 known miRNAs and 449 novel miRNAs were identified from four miRNA libraries.The results showed that 36 miRNAs were different expression at different developmental stages by the comparison.Further analysis,there were 250 predicted target genes from differentially expressed miRNA.In addition,the known transcription families,WRKY,SPL,ERF/AP2 gene family genes,there were new transcription factors encoding DELLA protein and Ankyrin repeat domain involved in anthocyanin biosynthesis in pear skins.