Observation Of Microscopic Morphology And Studies On Transcriptome For Sexual Organ Differentiation In Adiantum Reniforme Var.sinense

The ferns have broad prospects for market application because of their high ornamental and medicinal value.Inducing the gametophytes to form sporophytes efficiently and exploring corresponding regulation mechanism are the key points in the construction of rapid propagation system in the ferns.In present study,Adiantum reniforme var.sinense was used as research materials.Firstly,the culture conditions were screened to construct the systems of gametophytes producing sporophytes efficiently through sexual reproduction and apogamy respectively.The morphogenesis and microscopic characteristics of gametophytes and sporophytes were observed.On this foundation,high-throughput sequencing and bioinformatics were applied to reveal the molecular regulation mechanisms of sexual organ differentiation and efficient apogamy in this fern.It will contribute to high efficiency propagation and massive industrial application for the ferns.The main findings are as follows:1.The culture conditions for gametophytes producing sporophytes efficiently by sexual reproduction and apogamy were constructed.Culturing gametophytes on the medium containing 3.0 mg/L gibberellin can stimulate the differentiation of archegonia and formation of gametophytes with dense archegonia.Transferring the mature gametophytes to water environment could promote the fertilization and rapid formation of sporophytes by sexual reproduction.In other aspect,when culturing on the medium with 40 g/L sucrose and 1/4 MS,the sexual organ differentiation would be inhibited and the gametophytes could produce sporophytes efficiently by apogamy.2.Morphological observation and ploidy identification of sporophytes that formed by different reproductive modes were conducted.It was found that the first frond of sporophytes and surface characteristics of leaves from adult plants under SEM could be used to distinguish the two reproductive modes.The sexual reproductive sporophytes were identified as diploid and the apogamous sporophytes were identified as haploid by flow cytometry.Five pairs of SSR primers were screened out of 200 pairs of SSR primers to distinguish the two reproductive sporophytes and three pairs of primers can be used to construct fingerprints.3.The RNA-seq was used to sequence the gametophytes which could produce sporophytes efficiently by apogamy.4 Gb valid data and 62198 unigenes(>200bp)were generated.The gametophytes at four stages of apogamy were sequenced by DGE profiling,and 20376,20592,22807 and 19750 genes were generated respectively from each stage.Then,a value of FDR?0.001 and foldchange>2 were used as the threshold to judge the significant differences in the gene expressions.Respectively,401,838 and 669 DEGs were identified in Stage1 vs Stage2,Stage2 vs Stage3 and Stage3 vs Stage4.GO and KEGG enrichment analysis of the DEGs indicated that cell wall biosynthesis and plant hormone related pathways might participate in apogamy.In addition,24 transcription factors were identified from the DEGs.The expression patterns of 12 candidate genes were studied at different stages by qRT-PCR.The results suggested that these genes might play a key role in apogamy.4.In order to obtain accurate results of gene expression by qRT-PCR under different experimental conditions for A.reniforme var.sinense,the stabilities of 10 candidate reference genes were evaluated by geNorm,Normfinder and Bestkeeper softwares.The results showed that 40 S,UBQ and H3 were suitable for the gametophytes under sucrose treatment;40s and RPL35 were suitable for gametophytes under gibberellin treatment;40s and the combination of RPL35 and REF2 were suitable for the study of gene expression at different stages of gametophytes.5.The RNA-seq was used to sequence six stages of sexual reproduction and apogamy and generated 264,791 unigenes(>200bp).The analysis of DEGs at different stages showed that the transcriptional regulation was more active in apogamy initiation.Comparative KEGG enrichment analysis of DEGs indicated that pathways regulating the initiation periods were different between the two reproductive modes.Starch and sucrose metabolism pathway and plant hormone signal transduction pathway,which were only significantly enriched in apogamy initiation,might be related to apogamy initiation.SPS?TPS/TPP?GID1 and PP2 C were proposed to play a key role in this course.6.Based on above transcriptome database,2466 DEGs were identified between gametophytes with sexual organ and asexual embryonic cell differentiation by using padj < 0.05 as the criterion.In total of 66 genes homologous to transcription factors related to sexual organ differentiation in angiosperms and 22 methyltransferase genes were screened from the DEGs.The expression patterns of AGL1 and BBM were studied at different stages of two reproductive modes by qRT-PCR.The results suggested that these genes might play a key role in promoting sexual organ and asexual embryonic cell differentiation respectively.