| Chrysanthemum(C.× morifolium Ramat.)belongs to the Carduoideae Kitam.,there are two types of florets on the capitulum.The bilaterally symmetrical ray florets on the outside and radially symmetrical disc florets on the intermediate side.The genetic backgrounds of the two types of florets are exactly the same,but there are significant differences in morphology,tissue structure,pigment composition and gender.Therefore,the mechanism of the differentiation and development of the two types of florets on the capitulum is the basis for studying the formation mechanism of the capitulum in Asteraceae.The author intended to explore the differences of the two types of florets from the epigenetic perspective.This study used Chrysanthemum lavandulifolium(Fisch.ex Trautv.)Ling et Shih,a related wild species of cultivated chrysanthemum,as experimental material.Starting from the epigenetic differences between the two types of florets in the same genetic background during the flower development,the DNA methylation levels and patterns of the capitulum and the two types of florets at different developmental stages were analyzed by MSAP.The expression levels of the flower development-related genes and the correlation with DNA methylation levels were compared between the two types of florets.DNA methylation maps of the key genes in the two types of florets were constructed to analyze the reasons for genes specific expression.On the other hand,high-throughput sequencing technology was used to perform small RNA sequencing analysis on the two types of florets at different developmental stages,and the transcriptomes databases of the ray and disc florets were used to analysis with the mi RNA databases.The mi RNAs and target genes related to capitulum development were screened.Based on this,the epigenetic differences between the two types of florets on the capitulum of C.lavandulifolium were analyzed.The study obtained the following main results:(1)In this study,DNA methylation level of the capitula was 72.14%～79.13%,and that were73.49%～82.99% and 63.39%～82.85% of ray and disc florets,respectively.The relative DNA methylation levels showed dynamic changes during the development of the capitulum and the two types of florets,and maintained stable high levels.There were significant differences in the relative levels and patterns of DNA methylation between the two types of florets.(2)The expression levels of ABC(D)E genes and Cl CYC2-like genes were dynamically changed with the flower development and showed significant differences between the two types of florets.Correlation analysis revealed that the expression levels of ABC(D)E genes were weakly correlated with DNA methylation levels,while the expression levels of Cl CYC2-like genes were positively correlated with DNA methylation levels in the two types of florets,and it was stronger in the disc florets.It was speculated that the difference in expression levels of Cl CYC2-like genes may be related to DNA methylation during the flower development,and make them as the candidate genes.(3)In order to explain the mechanism of differential expression of genes regulated by DNA methylation in the two types of florets,the single-base resolution DNA methylation analysis was performed on Cl CYC2 c,Cl CYC2 d and Cl CYC2 e that with high expression levels and significant differences.The DNA methylation maps of these three genes in the two types of florets were constructed.The results showed that DNA methylation regulated the differential expression of these three Cl CYC2-like genes between the two types of florets,but the modifications were different.The differences in expression of Cl CYC2 c were regulated by CHH hypermethylation on the intron and the different CG methylation on the gene body.The differences in expression of Cl CYC2 d were regulated by CG methylation on the gene body,and other mechanisms were involved.The differences in expression of Cl CYC2 e might be regulated by the different CHH methylation on the promoter element and the different CG methylation on the gene body.(4)To identify differentially expressed mi RNAs in the two types of florets,12 mi RNA databases of ray and disc florets at two stages were established.A total of 603 mi RNAs were obtained and among which there were 3 known mi RNAs and 600 novel mi RNAs.Combining specific differential expression analysis,gene co-expression analysis and known flower developing-related mi RNA family analysis,16 candidate mi RNAs were finally screened and obtained.They were: novel-mi R258,novel-mi R302,novelmi R578,novel-mi R217,novel-mi R466,novel-mi R243,novel-mi R116,novel-mi R184,novel-mi R199,novel-mi R268,novel-mi R275,novel-mi R421,novel-mi R465,novel-mi R64,novel-mi R188 and novelmi R147.(5)The mi RNA database was combined with the existing transcriptome database of the ray and disc florets at the same stages to explain the function of the mi RNA by functional annotation and functional enrichment of the target genes of differentially expressed mi RNAs.Among the total of 6821 target genes,96.72% obtained annotation information.296 target genes were differentially expressed only in the ray florets,and 1039 were differentially expressed only in the disc florets.And a total of 435 transcription factors were identified in target genes.6 groups of candidate mi RNA-target combinations were screened out,namely mi R167-ARF6/8,mi R167-ARF9,mi R164-NAC21/22,mi R172-AP2,novel-mi R135/546-AP1 和 mi R396-GRF3.The functional enrichment pathways of target genes of differently expressed mi RNAs differed greatly between each sample.GO enrichment analysis showed that the genes regulating developmental processes were significantly different in the development of the ray and disc florets,while KEGG functional enrichment analysis indicated that the genes in BR biosynthesis and signal transduction pathways and auxin signal transduction pathways also played important roles in the two types of florets.Based on the above results,the author proposed that DNA methylation and mi RNA participated in the differentiation and development of the two types of florets in the Asteraceae capitulum by regulating flower developmental homeotic genes.DNA methylation regulated alternative splicing and transcriptional levels of Cl CYC2-like genes in the two types of florets.And mi R159,mi R164,mi R167,mi R172 and mi R396 acted by post-transcriptional regulation of target genes’ expression levels. |
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