The Formation Mechanism Of Coarse Fruit Of Kuerle Fragrant Pear Based On Transcriptome And Methylation Sequencing

Objectives: Coarse puip and peel and high stone cell content of the coarse fruit affected the development of Korla fragrant pear industry.The differences in the cell microstructure and the transcriptome and genome methylation of between coarse pear and normal pear of the Korla fragrant pear were analyzed by cell biology,transcriptomics,methylation and bioinformatics techniques,in order to explore the formation of the coarse-pear and provide molecular basis for artificial regulation of fruit development and improvement of fruit quality.Methods: The cells microscopic and submicroscopic structure of the pulp in Korla fragrant pear were observe by the paraffin and electron microscopy at 30 d,60 d,90 d,120 dand150 DAFter flowering.The pulp of between CP and NP of Korla fragrant pear sequenced by high-throughput sequencing technology,transcriptome and methylated sequence were analysised by GO,KEGGand other databases for bioinformatics analysis.The effect of methylation on fruit quality was analyzed.The spatiotemporal expression profiles of candidate differential genes were constructed by q RT PCR,and the bioinformatics analysis of candidate differential genes was carried out.Result: 1.The lignin content and stone cell content were extremely significantly higher in CP than in NP of Korla fragrant pear,through the determination of pear lignin and stone cell content.According to the analysis of cell microstructure,a large Number of stone cell clusters were formed in the core of the CP at30 days after anthesis,which mainly composed of stone cells.A large Number of stone cell clusters with large diameter were formed in the core,and began to crack at 60 days after anthesis.The stone cell diameter in the core reached the maxim?m at 90 days after anthesis,and a large number of stone cell clusters in the pulp were cracked.The diameter of stone cell mass in the core decreased greatly and the diameter of stone cell in pericarp continued to increase at 120 day after anthesis.At 150 days after anthesis,the stone cell clusters became smaller and distributed in the whole flesh.The parenchyma cell wall of pulp gradually became thicker from120 dto150 DAFter anthesis.The stone cell mass of NP was composed of sclerenchyma cells.The degree of stone cell mass cracked was higher in NP than in CP,and the diameter of stone cell mass is smaller.The stone cell mass was mainly distributed in the core of fruit at mature stage,and the parenchyma cell wall gradually became thinner.2.The differences transcriptome were analyzed between CP and NP.There were 87.32 g clean bases and101157 unigenes in pear pulp by RNA sequencing,and there were 57% of the reads mapped to Dangshan pear genome.There were 5962 unigenes significantly different between NP and CP,including 2858 upregulated unigenes and 3124 down-regulated unigenes.Through the enrichment analysis of GO and KEGG,There were 30 candidate structural genes related to lignin metabolism,including 16 up-regulated genes and14 down regulated genes.19 candidate genes selected were the expression profile during fruit development period.There were five structural genes correlated with stone cell and lignin content,including c38163_g1and c45761_g2(4CL)?c47829_g2(HCT)?c40485_g1(COMT)?C45001_G1(ccomt),Among them,c47829_g2(HCT)gene reported in Dangshan pear.There were two transcription factors,including c39399_g1(myb)and c28793_g1(b HLH).3.Some candidate genes of 4CL,CCR,POD,MYB,and b HLH families were analysised by bioinformatics.c38163_g1and c45761_g2 genes belonged to 4CL Class I and were involved in lignin metabolism,which.encoded the proteins by 4CL family members.c49561_g2 was involved in cell wall lignin metabolism.The 8 candidate POD genes belonged to POD Class III.C35983_g1 was the same clusters with At POD25 and At POD71,and c38565_g1 belonged to the same clusters with At POD52,which were involved in cell wall lignin metabolism.There were homeopathic elements of light,hormones,and transcription factors.in promoter region of 10 structural genes by the analysis of the structural gene promoter region.There were AC and CACGTG cis-acting elements in promoter region of c45761_g2,which c OUL be recognized by MYB and b HLH.There was highly positive correlated with between transcription factor c39399_g1 and structural genes in NP,and there was significantly positive correlated with between transcription factorc28793_g1and structural genes in CP.There were two structural genes c35983_g1,c38565_g1and transcription factor c39399_g1 related to the delayed development of coarse fruit based on the degree of stone cell cracked.4.There were 164,102,909 reads in pear pulp by methylation sequencing.Compared with the Dangshan pear reference genome,the mapping rate of Korla fragrant pear fruits was no more than 40% by Bismark methylation analysis.There were 4487 DMRs significantly difference in CP with NP by sw DMR software,and there were 3046 DMRs annotated including1640 hyper DMRs and 1406 hypo DMRs.3266 terms in go and 110 in KEGG.The most significant KEGGwas hormone signal transduction pathway.There were23 DMRs in Lignin Metabolism,including 13 hypo DMRs and 10 hyper DMRs.The C site of DNA methylation is mainly distributed in promoter,exon,intron and intron in the whole genome.The results showed that there was 3 genes,including c38995_g1?c49204_g2 and c45761_g2.There were C site of methylations located in the promoter region of c38995_g1 and c49204_g2,and the methylation C site located in the exon and intron regions of c45761_g2.The methylation degrees of these three genes were lower in coarse fruit than in NP.The methylation degree was higher in NP than in CP.of Korla fragrant pear during mature period.Conclusion: The results showed that the lignin content and stone cell content were significantly higher in CP than in normal fruit of Korla fragrant pear,and its development period was delayed.There were significantly 5962 difference genes between CP and NPs,30 genes related to Lignin Metabolism,5 genes significantly related to lignin and stone cell content.The methylation level was higher in NP than in CP.In CP,the high content of sclereids was related to the low degree of stone cell cracked,the thickness of peel may be related to the late sclereids formation of peel,and the coarse pulp may be related to the continuous thickening of parenchyma cell wall during mature stage.